25th LACE - FGUA · Daniel Rojas Tizón ... José Alberto Fracassi da Silva. 25th LACE Symposium Alcalá de Henares, Madrid, Spain, September 29th - October 2nd, 2019 15 16:10 - 16:30

ORGANIZED BY:

BOOK OF ABSTRACTS

LACE2019

th25

www.lace2019.com

29SEP/2OCT/ ALCALÁ DE HENARESMADRID. SPAIN

th25 Latin-American Symposium on Biotechnology, Biomedical, Biopharmaceutical, and Industrial Applications of Capillary Electrophoresis and Microchip Technology

25th Latin-American Symposium on Biotechnology, Biomedical,

Biopharmaceutical, and Industrial Applications of Capillary Electrophoresis and Microchip Technology

September 29th - October 2nd 2019 Alcalá de Henares, Madrid, Spain

Book of Abstracts

Published by: Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering University of Alcalá, Alcalá de Henares, Madrid, Spain

Book coordinators: Norberto A. Guzman, Alberto Escarpa, Agustín G. Crevillén, Robert Weinberger Design and layout: Ronda Vázquez Martí

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25th LACE Symposium

Alcalá de Henares, Madrid, Spain, September 29th - October 2nd, 2019 5

Table of Content

Symposium Welcoming Remarks ..........................................................................7Organizers, Collaborators and Sponsors ................................................................8Committees ............................................................................................................9Previous meetings ................................................................................................12Scientific Program ...............................................................................................13Index of Abtracts ..................................................................................................19Plenary Lectures ..................................................................................................31Keynote Lectures .................................................................................................39Oral Presentations ...............................................................................................57Flash Communications ......................................................................................117Poster Presentations ..........................................................................................155List of Authors ...................................................................................................225Authors Index ....................................................................................................235

25th LACE Symposium


Symposium Welcoming RemarksThis year, the Latin-American Symposium on Biotechnology, Biomedical, Biopharmaceutical, and Industrial Applications of Capillary Electrophoresis and Microchip Technology, LACE 2019, celebrates its 25th anniversary and the Organizing Committee wishes to honor and acknowledge Professor Norberto A. Guzman, founder and Emeritus Chair, for his extraordinary efforts to successfully sustain this symposium series for a quarter of a century. Coincidently, this year also commemorates Professor Ziad El-Rassi 25 years as Editor-in-Chief of the journal Electrophoresis, and he will be delivering the LACE closing plenary lecture. Opening the symposium is Professor Robert Kennedy, Associate Editor of the journal Analytical Chemistry. No question that this is an excellent start and finish!

The symposium is structured into a four days conference beginning with the traditional pre-symposium postgraduate course, followed by plenary and keynote lectures and oral communications, as well as poster sessions, vendor seminars and an instrument exposition. For this silver anniversary of LACE, founded by our dearest Norberto, the last session of each day will feature flash communications and poster presentations to provide PhD and junior Post-Doctoral students a more effective interaction and utmost visibility with prestigious experts in CE.

Sessions will include omics (proteomics, glycomics, foodomics, metabolomics), genetic analysis, DNA sequencing and separation, bioanalysis, biotechnology, multidimensional separation approaches, hyphenated techniques, mass spectrometry, novel instrumentation, sample treatment, miniaturization, microfluidics, microchip electrophoresis, paper based analytical devices, (bio)pharmaceutical, food, bioanalytical and environmental applications, and much more.

It is our sincere wish that your experience this year in Alcalá de Henares be both enjoyable and professionally rewarding. In addition, it will give you an extraordinary opportunity to visit our beautiful and unique World Heritage Places declared by UNESCO in 1998 such as the University and Historic Town.

Welcome to Alcalá, the birthplace of the renowned Miguel de Cervantes, author of the fa-mous Spanish novel Don Quixote of La Mancha!

Our warmest regards,

Alberto Escarpa and Agustín G. Crevillén Chairs of LACE 2019

Norberto A. Guzman Founder and Emeritus Chair

25th LACE Symposium

8 Alcalá de Henares, Madrid, Spain, September 29th - October 2nd, 2019

Organizers, Collaborators and Sponsors

Organized by:

With the accreditation of:

Sponsored by:

25th LACE Symposium


Organizing CommitteeChairs:

Alberto Escarpa University of Alcalá

Agustín G. Crevillén UNED

Committee members:

María Cristina González Martín University of Alcalá

Miguel Ángel López Gil University of Alcalá

Víctor de la Asunción Nadal University of Alcalá

María Moreno Guzmán Complutense University of Madrid

Juan Víctor Perales-Rondón University of Burgos and University of Alcalá

Laura García Carmona University of Alcalá

Roberto María Hormigos University of Alcalá

Águeda Molinero Fernández University of Alcalá

Marta Pacheco Jerez University of Alcalá

Daniel Rojas Tizón University of Alcalá

Tania Sierra Gómez University of Alcalá

Silvia Dortez Herranz University of Alcalá

Kaisong Yuan University of Alcalá

Juan Francisco Hernández Rodríguez University of Alcalá

Beatriz Jurado Sánchez University of Alcalá

Scientific Committee

Founder and Emeritus Chair:

Norberto A. Guzman Princeton Biochemicals Inc., U.S.A.

Committee members:

Carme Aguilar Tarragona, Spain

Lourdes Arce Córdoba, Spain

Coral Barbas Madrid, Spain

Fernando Benavente Barcelona, Spain

25th LACE Symposium


Ana Mª García Campaña Granada, Spain

Alegría Carrasco Pancorbo Granada, Spain

Mario Castaño Álvarez Oviedo, Spain

Alejandro Cifuentes Madrid, Spain

Jeff Chapman Brea, U.S.A.

Agustín G. Crevillén Madrid, Spain

Flavio Della Pelle Teramo, Italy

José Carlos Diez-Masa Madrid, Spain

Claudimir L. do Lago São Paulo, Brazil

Ziad El-Rassi Oklahoma, U.S.A.

Alberto Escarpa Madrid, Spain

Mª Teresa Fernández Abedul Oviedo, Spain

Frantisek Foret BRNO, Czech Republic

José Alberto Fracassi da Silva Campinas, Brazil

Mercedes de Frutos Madrid, Spain

Carlos D. García Clemson, U.S.A.

María Cristina González Madrid, Spain

Andras Guttman Veszprem, U.S.A.

Peter Hauser Basel, Switzerland

Charles S. Henry Fort Collins, U.S.A.

Maykel Hernández Mesa Nantes, France

José Manuel Herrero Martínez Valencia, Spain

Lisa A. Holland Morgantown, U.S.A.

Beatriz Jurado Sánchez Madrid, Spain

Vaclav Kasicka Prague, Czech Republic

Robert Kennedy Ann Arbor, MI, U.S.A.

Alexander Kuhn Burdeaux, France

James P. Landers Charlottesville, U.S.A.

Herbert H. Lindner Innsbruck, Austria

Miguel Ángel López Madrid, Spain

Susan M. Lunte Lawrence, U.S.A.

Mª Luisa Marina Alegre Madrid, Spain

María José Medina Hernández Valencia, Spain

Andrew deMello Zurich, Switzerland

Bruce R. McCord Miami, U.S.A.

Linda B. McGown Troy, U.S.A.

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Martin Pumera Prague, Czech Republic

Vincent T. Remcho Corvallis, U.S.A.

Marja-Liisa Riekkola Helsinki, Finland

Ángel Ríos Ciudad Real, Spain

Andreas Rizzi Vienna, Austria

Virginia A. Robinson-Fuentes Morelia, Mexico

Encarnación Rodríguez Gonzalo Salamanca, Spain

Alberto Sánchez Arribas Madrid, Spain

Bettina Sarg Innsbruck, Austria

María Fernanda Silva Mendoza, Argentina

Ana Valéria C. Simionato Campinas, Brazil

Carolina Simó Ruiz Madrid, Spain

Govert W. Somsen Amsterdam, The Netherlands

Marina F.M. Tavares São Paulo, Brazil

Nora M. Vizioli Buenos Aires, Argentina

Robert Weinberger Chappaqua, U.S.A.

25th LACE Symposium


Previous meetings

Chairperson/s Location Year

María Fernanda Silva & Nora M. Vizioli Mendoza, Argentina 2018V.A. Robinson-Fuentes & V.H. Abrego-Reyes Mexico City, Mexico 2017N. Vizioli, M. Tavares, E. Carrilho & M.A. Valenzuela Santiago, Chile 2016N. Vizioli, M. Tavares & E. Carrilho Cartagena, Colombia 2015M. Tavares & E. Carrilho Natal, Brazil 2014M. Tavares Lima, Peru 2013N. Vizioli & M. Tavares Buenos Aires, Argentina 2012B. McCord, C. Garcia & M. Tavares Miami, U.S.A. 2011E. Carrilho & M. Tavares Florianopolis, Brazil 2010C. Barbas & M. Tavares Sevilla, Spain 2009N. A. Guzman & A. González Puerto Vallarta, Mexico 2008N. A. Guzman & M.A. Valenzuela Santiago, Chile 2007N. A. Guzman, B. Vallejo & A. Gonzalez Queretaro, Mexico 2006N. A. Guzman, M. Tavares & E. Carrilho Guaruja, Brazil 2005N. A. Guzman & A. Rios Toledo, Spain 2004N. A. Guzman, A. Revilla & G. Vargas Mexico City, Mexico 2003N. A. Guzman & N. Vizioli Mar del Plata, Argentina 2002N. A. Guzman & M.A. Valenzuela Santiago, Chile 2001N. A. Guzman Punta del Este, Uruguay 2000N. A. Guzman, B. Vallejo & A. González Acapulco, Mexico 1999N. A. Guzman, M. Tavares & E. Carrilho São Paulo, Brazil 1998N. A. Guzman, C. Carducci & J. Castagnino Buenos Aires, Argentina 1997N. A. Guzman, C. Calvo & E. Guerrero Santiago, Chile 1996N. A. Guzman Santiago, Chile 1995

25th LACE Symposium


Scientific Program

Sunday, September 29th, 2019

08:00 Pre-symposium Course

Registration

08:30 - 10:00 Introduction to capillary electrophoresis Robert Weinberger

10:00 - 11:00 Clinical capillary electrophoresis Norberto A. Guzman

11:00 - 11:30 Break

11:30 - 13:00 New advances in CE-MS for metabolomics and biomarker discovery: expanding throughput and metabolome coverage with quality control Philip Britz-McKibbin

13:00 - 14:00 Microchip-electrophoresis coupled to nanomaterial-based electrochemical detection in food analysis Flavio Della Pelle

14:00 Lunch (pre-symposium course)

Inaugural session15:00 - 16:30 Registration Poster set up (all posters)

16:30 - 16:45 Welcoming remarks

16:45 - 17:15 PL-1 An insider’s view of the evolution of clinical science through 25 years of annual LACE Symposiums Norberto A. Guzman

17:15 - 18:00 PL-2 High throughput protein assays using microchip electrophoresis Robert Kennedy

20:00 Welcome Cocktail

Monday, September 30th, 2019

08:00 Registration

Session I Chairperson: Fernando Benavente (UB)

09:00 - 09:30 KL-1 Fabrication of structured magnetic metallic nano-platelets for bioanalytical applications Frantisek Foret

25th LACE Symposium


09:30 - 09:50 OP-1 Aptamers and evolution Linda B. McGown

09:50 - 10:10 OP-2 New approaches based on electrochemical immunosensing microfluidic platforms for neonatal sepsis biomarkers determination Águeda Molinero-Fernández

10:10 - 10:30 OP-3 Preconcentration strategies for the determination of cathinones in biological samples Alberto Pérez-Alcaraz

10:30 - 10:50 OP-4 Development of an aptamer functionalized polymer monolith for the selective detection of concanavalin A by on-line solid-phase extraction capillary electrophoresis José Manuel Herrero Martínez

11:00 Break

Session II Chairperson: José Carlos Diez-Masa (IQOG, CSIC)

11:30 - 12:00 KL-2 12 year’s experience of low-cost analytical device based on capillary electrophoresis for falsified or sub-standard drug quality control Serge Rudaz

12:00 - 12:20 OP-5 Multiplexed CE-MS for biomarker discovery in metabolomics: Improved screening of cystic fibrosis in newborns without genetic testing? Philip Britz-McKibbin

12:20 - 12:40 OP-6 Sheathless CE-MS for the in-depth characterization of IgGs and IgG subdomains Elena Domínguez Vega

12:40 - 13:00 OP-7 LC-MS for the discovery of potential diagnostic and prognostic biomarkers of non-Hodgkin Lymphoma Ana Valéria C. Simionato

13:00 - 13:20 OP-8 CE-MS for metabolomics evaluation of the mitochondrial response to ketamine-related compounds Francisco Javier Rupérez

13:30 Lunch

Session III Chairperson: Ana Mª García Campaña (UGR) 15:00 - 15:30 KL-3 Development of capillary electrophoresis hardware for implementation on

spaceflight missions of exploration María Fernanda Mora

15:30 - 15:50 OP-9 Lab-on-paper electroanalytical devices: towards the fully integration of steps Mª Teresa Fernández Abedul

15:50 - 16:10 OP-10 Direct preparation of microfluidic devices using 3D printing for applications on nanoparticles synthesis and microchip electrophoresis José Alberto Fracassi da Silva

25th LACE Symposium


16:10 - 16:30 OP-11 E pluribus unum: Unraveling the molecular heterogeneity of pro-B-type natriuretic peptides and its consequences as biomarker for heart failure Herbert H. Lindner

16:30 - 17:15 Cafe seminar TC-1 Recent Advances in the use of capillary electrophoresis for biopharmaceutical analysis Walter Belloni, SCIEX

17:15 - 17:30 Coffee break courtesy of Sciex

Session IV Chairperson: María Castro-Puyana (UAH)

17:30 - 18:30 Flash communications from FC-1 to FC-7 and poster sessions

19:00 - 20:00 Tour around Old University and Chapel

Tuesday, October 1st, 2019

08:00 Registration

Session V Chairperson: José Manuel Herrero Martínez (UV)

09:00 - 09:30 KL-4 Ultra-high-throughput multi-parametric imaging flow cytometry Andrew deMello

09:30 - 09:50 OP-12 Capillary electrophoresis of therapeutic RNAs Robert Weinberger

09:50 - 10:10 OP-13 Electrokinetic separations applied to simple and complex biological samples Virginia A. Robinson-Fuentes

10:10 - 10:30 OP-14 Immunoaffinity capillary electrophoresis for the analysis of biomarkers in blood Nora M. Vizioli

10:30 - 10:50 OP-15 Screening gut microbiota for the identification of bacteria with harmful metabolic traits for cardiovascular health Virginia García-Cañas

11:00 - 11:30 Break

Session VI Chairperson: Lourdes Arce (UCO) 11:30 - 12:00 KL-5 Purpose made CE instruments

Peter C. Hauser

12:00 - 12:20 OP-16 Bipolar electrochemistry/fluorescence detector for microchip electrophoresis Susan M. Lunte

25th LACE Symposium


12:20 - 12:40 OP-17 Carbon black based electrochemical transducers for microfluidic detection Flavio Della Pelle

12:40 - 13:00 OP-18 Fluorescence detection of proteins in capillary and microchip electrophoresis José Carlos Diez-Masa

13:00 - 13:20 OP-19 Determination of glycoproteins by microchip electrophoresis and electrochemical detection Agustín G. Crevillén

13:30 - 15:00 Lunch

Session VII Chairperson: Mª Luisa Marina (UAH)

15:00 - 15:30 KL-6 Capillary electrophoresis and isotachophoresis applied for analysis and characterization of antimicrobial peptides Vaclav Kasicka

15:30 - 15:50 OP-20 Analytical metrology for nanomaterials: the role of CE and related techniques Ángel Ríos

15:50 - 16:10 OP-21 Chiral ionic liquids as synergistic selectors for enantiomeric separations by capillary electrophoresis María Castro-Puyana

16:10 - 16:30 OP-22 Enantioselective study of drug biodegradation by chiral capillary electrophoresis Yolanda Martín Biosca

16:30 - 16:45 OP-23 Use of whole electrophoretic profiles and chemometric tools for the differentiation of three olive oil qualities using the compounds extracted by liquid-liquid extraction and supercritical fluid extraction Lourdes Arce

16:45 - 17:05 OP-24 Assessing chirality and conformation by gas-phase electrophoresis Govert W. Somsen

17:05 - 17:30 Break

Session VIII Chairperson: Beatriz Jurado Sánchez (UAH)

17:30 - 19:00 Flash communications from FC-8 to FC-15 and poster sessions

19:00 Poster removal

20:00 Concert and Gala Dinner

25th LACE Symposium


Wednesday, October 2nd, 2019

08:00 Registration

Session IX Chairperson: Ángel Ríos (UCLM)

09:00 - 09:30 KL-7 Ideal-filter capillary electrophoresis Sergey N. Krylov

09:30 - 09:50 OP-25 Integrating nucleic acid extraction with microscale analysis: One step closer to democratizing molecular biology Jeff Chapman

09:50 - 10:10 OP-26 CE-MS as an useful tool in the control of chemical hazards in foods: some selected examples Ana García-Campaña

10:10 - 10:30 OP-27 Aptamer affinity sorbents for the analysis of protein biomarkers in biological fluids by on-line solid-phase extraction capillary electrophoresis-mass spectrometry Fernando Benavente

10:30 - 10:50 OP-28 Deep N-glycomic analysis of human serum by capillary electrophoresis and CESI-MS: the challenge of sample preparation Andras Guttman

10:50 - 11:10 OP-29 Foodomics: ten years on the road Alejandro Cifuentes

11:20 - 11:50 Break

Symposium Closing Session11:50 - 12:35 PL-3 Nanoparticle-based separation media for electro- and liquid phase-separation

techniques Ziad El-Rassi

12:40 - 13:30 Closing ceremony at Historic Paraninfo Closing remarks and poster awards. Mercedes de Frutos, head of the awards committee Announcement for LACE 2020 and photo session




Index of Abstracts

25th LACE Symposium


Plenary Lectures[PL-1] An insider’s view of the evolution of clinical science through 25 years

of annual LACE Symposiums .......................................................................33Norberto A. Guzman

[PL-2] High throughput protein assays using microchip electrophoresis...............34Robert T. Kennedy, Claire Ouimet and Natalie Cleaveland

[PL-3] Nanoparticle-based separation media for electro- and liquid phase-separation techniques .......................................................................36Ziad El Rassi, Sarah Alharthi, Nisansala Ganewatta and Cemil Aydogan

Keynote Lectures[KL-1] Fabrication of structured magnetic metallic nano-platelets for

bio-analytical applications ..........................................................................41Jakub Novotny, Petra Juskova , Rudolf Kupcik , Zuzana Bilkova and Frantisek Foret

[KL-2] 12 year’s experience of low-cost analytical device based on capillary electrophoresis for falsified or sub-standard drug quality control .............43Serge Rudaz, J. Schappler, J.-L. Veuthey, P. Bonnabry, C. Rohrbasser, S.O. Sarr, S. Roth and O. Vorlet

[KL-3] Development of capillary electrophoresis hardware for implementation on spaceflight missions of exploration ...............................45M. Fernanda Mora, F. Kehl, E. Tavares da Costa, N. Bramall and P. Willis

[KL-4] Ultra-high-throughput multi-parametric imaging flow cytometry ..............46Andrew J. deMello, Gregor Holzner and Stavros Stavrakis

[KL-5] Purpose made CE instruments ....................................................................49Peter C. Hauser and Jasmine S. Furter

[KL-6] Capillary electrophoresis and isotachophoresis applied for analysis and characterization of antimicrobial peptides ...........................................51Vaclav Kasicka, Tereza Tumova, Lenka Monincova and Vaclav Cerovsky

[KL-7] Ideal-filter capillary electrophoresis ...........................................................54Sergey N. Krylov, Svetlana M. Krylova, An T. H. Le, Stanislav S. Beloborodov and Tong Ye Wang

25th LACE Symposium


Oral Presentations[OP-1] Aptamers and evolution ...............................................................................59

Linda B. McGown

[OP-2] New approaches based on electrochemical immunosensing microfluidic platforms for neonatal sepsis biomarkers determination ........60Águeda Molinero-Fernández, María Moreno-Guzmán, Miguel Ángel López and Alberto Escarpa

[OP-3] Preconcentration strategies for the determination of cathinones in biological samples ...................................................................................61Alberto Pérez-Alcáraz, Francesc Borrull, Marta Calull and Carme Aguilar

[OP-4] Development of an aptamer functionalized polymer monolith for the selective detection of concanavalin A by on-line solid-phase extraction capillary electrophoresis ............................................................63María Vergara-Barberán, Ancuta Moga, Ernesto Francisco Simó-Alfonso, Fernando Benavente and José Manuel Herrero-Martínez

[OP-5] Multiplexed CE-MS for biomarker discovery in metabolomics: improved screening of cystic fibrosis in newborns without genetic testing? ............................................................................................65Philip Britz-McKibbin, Alicia DiBattista Osama Aldirbashi and Pranesh Chakraborty

[OP-6] Sheathless CE-MS for the in-depth characterization of IgGs and IgG subdomains ....................................................................................67Elena Domínguez-Vega, C. Gstöttner, T. Senard, D. Falck and M. Wuhrer

[OP-7] LC-MS for the discovery of potential diagnostic and prognostic biomarkers of non-Hodgkin Lymphoma ......................................................69Ana Valéria Colnaghi-Simionato, Anna Maria Alves de Piloto-Fernandes, Patricia de Oliveira-Carvalho3,Victor Piana-de Andrade, Jayr Schmidt-Filho, Gustavo Henrique Bueno-Duarte

[OP-8] CE-MS for metabolomics evaluation of the mitochondrial response to ketamine-related compounds ...................................................................71Francisco J. Rupérez, Andrea Faccio, Nagendra S. Singh, Santiago Angulo, Marina F. Tavares, Michel Bernier, Coral Barbas and Irving W. Wainer

[OP-9] Lab-on-paper electroanalytical devices: towards the fully integration of steps ......................................................................................73E. Costa-Rama, E. Núñez-Bajo, A. González-López, O. Amor-Gutiérrez, L. Blanco-Covián, M.C. Blanco-López, H.P.A. Nouws, C. Delerue-Matos, P.I. Nanni, R.E. Madrid and M.T. Fernández-Abedul

25th LACE Symposium


[OP-10] Direct preparation of microfluidic devices using 3D printing for applications on nanoparticles synthesis and microchip electrophoresis ....76José Alberto Fracassi da Silva, Lucas P. Bressan, Brenda María de Castro Costa, Taíssa Moreira da Silva Lima, Aline Guadalupe Coelho, Michael Beauchamp and Adam T. Woolley

[OP-11] E pluribus unum: unraveling the molecular heterogeneity of pro-B-type natriuretic peptides and its consequences as biomarker for heart failure .....................................................................79Herbert H. Lindner, Benno Amplatz and Klaus Faserl

[OP-12] Capillary electrophoresis of therapeutic RNAs ...........................................81Robert Weinberger and James Timmins

[OP-13] Electrokinetic separations applied to simple and complex biological samples ......................................................................................82Virginia A. Robinson-Fuentes, Xiomara Zavala-Sánchez, Ma. Soledad Vázquez-Garcidueñas and Gerardo Vázquez-Marrufo

[OP-14] Immunoaffinity capillary electrophoresis for the analysis of biomarkers in blood .................................................................................84Nora M. Vizioli

[OP-15] Screening gut microbiota for the identification of bacteria with harmful metabolic traits for cardiovascular health ....................................85Carolina Simó and Virginia García-Cañas

[OP-16] Bipolar electrochemistry/fluorescence detector for microchip electrophoresis .............................................................................................87Susan M. Lunte, Manjula Wijesinghe, Dulan Gunasekara and Indika Warnakula

[OP-17] Carbon black based electrochemical transducers for microfluidic detection .............................................................................89Flavio Della Pelle, Daniel Rojas, Michele Del Carlo, Luis Vázquez, Dario Compagnone and Alberto Escarpa

[OP-18] Fluorescence detection of proteins in capillary and microchip electrophoresis .............................................................................................92Ángel Puerta, Mercedes de Frutos and José Carlos Díez-Masa

[OP-19] Determination of glycoproteins by microchip electrophoresis with electrochemical detection ....................................................................94Agustín G. Crevillen, Tania Sierra and Alberto Escarpa

[OP-20] Analytical metrology for nanomaterials: the role of capillary electrophoresis and related techniques .......................................................97Ángel Ríos

25th LACE Symposium


[OP-21] Chiral ionic liquids as synergistic selectors for enantiomeric separations by capillary electrophoresis .....................................................99María Castro-Puyana, Maider Greño, Sandra Salido-Fortuna

and María Luisa Marina

[OP-22] Enantioselective study of drug biodegradation by chiral capillary electrophoresis ...........................................................................................101Yolanda Martín-Biosca, L. Escuder-Gilabert, S. Sagrado and M.J. Medina-Hernández

[OP-23] Use of whole electrophoretic profiles and chemometric tools for the differentiation of three olive oil qualities using the compounds extracted by liquid-liquid extraction and supercritical fluid extraction ....103Natividad Jurado-Campos, Laura Valbuena, Rocío Rodríguez-Gómez, Natalia Arroyo-Manzanares and Lourdes Arce

[OP-24] Assessing chirality and conformation by gas-phase electrophoresis ........106Govert W. Somsen, Raquel Pérez-Míguez, Robert L.C. Voeten, María Castro-Puyana, María Luisa Marina, Elena Domínguez-Vega

and Rob Haselberg

[OP-25] Integrating nucleic acid extraction with microscale analysis: One step closer to democratizing molecular biology ...............................108Jeff Chapman, David Saul and Jo Stanton

[OP-26] CE-MS as a useful tool in the control of chemical hazards in foods: some selected examples .............................................................................110Ana M. García-Campaña, Francisco J. Lara, David Moreno-González, Carmen Tejada-Casado, Maykel Hernández-Mesa, Laura Gámiz-Gracia and Monsalud del Olmo-Iruela

[OP-27] Aptamer affinity sorbents for the analysis of protein biomarkers in biological fluids by on-line solid-phase extraction capillary electrophoresis-mass spectrometry ...........................................................112Fernando Benavente, Roger Pero-Gascon, Maxim V. Berezovski and Victoria Sanz-Nebot

[OP-28] Deep N-glycomic analysis of human serum by capillary electrophoresis and CESI-MS: The challenge of sample preparation ................................114Andras Guttman and Marton Szigeti

[OP-29] Foodomics: ten years on the road .............................................................115Gerardo Álvarez-Rivera, Diego Ballesteros-Vivas, Alberto Valdés, Zully Suárez-Montenegro, J. David Sánchez-Martínez, José A. Mendiola, Miguel Herrero, Elena Ibáñez and Alejandro Cifuentes

25th LACE Symposium


Flash Communications[FC-1] Development of a self-pumping microfluidic herringbone device

for pesticide analysis .................................................................................119Daniel B. Carrão, Ruth F. Menger, Wei Wang, Rob B. Channon, Anderson R. M. de Oliveira, Arun K. Kota and Charles S. Henry

[FC-2] Low-cost and accessible rapid-prototyping of microfluidic devices ...............................................................................122Juan F. Hernández-Rodríguez, Daniel Rojas and Alberto Escarpa

[FC-3] Determination of ammonium in the vitreous humour based on capillary electrophoresis: preliminary application in thanatochemistry ...................................................................................124Covadonga Palacio, Rossella Gottardo and Franco Tagliaro

[FC-4] In situ synthesis of gold nanoparticles on a thread based microfluidic device and their application in surface-enhanced raman scattering ........127Dosil P. de Jesus, Cristina B. Adamo, Ayandra S. Junger, Lucas P. Bressan, José Alberto F. da Silva and Ronei J. Poppi

[FC-5] Combination of off-line sample treatments and on- line preconcentration for the analysis of 5- nitroimidazoles residues in food, environmental and biological samples by capillary electrophoresis.......................................................................130Maykel Hernández-Mesa, Diego Airado-Rodríguez, Carmen Cruces-Blanco and Ana M. García-Campaña

[FC-6] Determination of ofloxacin based on fluorimetric detection coupled to capillary electrophoresis using graphene quantum dots as fluorescent enhancer ......................................................................133Samah Lahouidak, M. Laura Soriano, R. Salghi, Mohammed Zougagh and Ángel Ríos

[FC-7] Tunable and pH-independent electroosmotic flow for capillary electrophoresis separations .......................................................................136Dušan Koval, Veronika Šolínová and Renáta Konášová

[FC-8] Microchip capillary electrophoresis (MCE) for analysis of different samples including pharmaceuticals in body fluids .................138Ángel Puerta, Mercedes de Frutos and José Carlos Diez-Masa

[FC-9] Analysis of circulating microRNA biomarkers in serum by on-line solid-phase extraction capillary electrophoresis-mass spectrometry .......140Roger Pero-Gascon, Maxim V. Berezovski, José Barbosa, Victoria Sanz-Nebot and Fernando Benavente

25th LACE Symposium


[FC-10] Development of a capillary electrophoresis methodology for the stereoselective separation of tetrametrin .................................................142M. Greño, M.A. García, Maria Luisa Marina and María Castro-Puyana

[FC-11] Enantiodetermination of cathinones in urine by field-amplified sample injection-capillary electrophoresis and electrokinetic supercharging-capillary electrophoresis ...................................................144Albert Pérez-Alcaraz, Francesc Borrull, Carme Aguilar and Marta Calull

[FC-12] Ion Analysis Strategies for Habitability Assessment in Planetary Science ..................................................................................146Elizabeth Jaramillo, Aaron Noell, M. Fernanda Mora and Peter Willis

[FC-13] Magneto-catalytic Janus micromotor in labs-on-a-chip applications: mobile sensors and microreactors .............................................................148Marta Pacheco, Beatriz Jurado-Sánchez and Alberto Escarpa

[FC-14] Metabolomic study of the ex vivo effect of kynurenic acid after ischemia-reperfusion in rats ............................................................151Douglas Oscar Ceolin Mariano, Daniel Carvalho Pimenta, Juliana Mozer Sciani, Mario Angelo Claudino, Aline Gonçalves Mora, Sabrina Janussi, Coral Barbas and Francisco Javier Rupérez

[FC-15] Development of a CE-MS metabolomics approach to study in vitro high glucose-induced changes in HK-2 cells ............................................153Samuel Bernardo-Bermejo, Elena Sánchez-López, María Castro-Puyana, Selma Benito, Francisco Javier Lucio-Cazaña and María Luisa Marina

Poster Presentations[PP-1] Indirect Calibration in Capillary Electrophoresis

with Conductivity Detection ......................................................................157Michele Alves Santana and Claudimir Lucio do Lago.

[PP-2] A proposal for the sensitive determination of seven neonicotinoids in soils and enviromental waters by micellar electrokinetic chromatography .........................................................................................160Laura Carbonell-Rozas, Francisco J. Lara, Monsalud del Olmo-Iruela and Ana M. García-Campaña

[PP-3] A chiral analytical methodology for colchicine determination using nano-liquid chromatography and an amylose-based stationary phase .....163Natalia Casado, María Ángeles García, Zhengjin Jiang and María Luisa Marina

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[PP-4] Modeling the enantiomeric separation of phenoxy acid herbicides by CE using a dual cyclodextrin system ....................................................165Natalia Casado, José María Saz, María Ángeles García and María Luisa Marina

[PP-5] Determination of primary aromatic amines in kitchenware by capillary electrophoresis –tandem mass spectrometry ............................................167Mary Ângela Favaro Perez, Marisa Padula, Carla Beatriz Grespan Bottoli, Claudimir Lucio do Lago and Daniela Daniel

[PP-6] Chiral capillary electrohoresis for the separation of ivabradine enantiomers. Quantitative analysis in pharmaceutical formulations .......170Natalia Casado, Antonio Salgado, María Castro-Puyana, María Ángeles García and María Luisa Marina

[PP-7] Enantioseparation of amino acids using an O-[2- (methacryloyloxy)-ethylcarbamoyl]-10,11- dihydroquinidine-silica hybrid monolithic column by nano-LC. Determination of norvaline and tryptophan in food supplements ..........................................................172Dongsheng Xu, Elena Sánchez-López, Qiqin Wang, Zhengjin Jiang and María Luisa Marina,

[PP-8] Glycopeptide analysis by on-line TiO2 solid-phase extraction capillary electrophoresis-mass spectrometry ............................................174Estela Giménez, Montserrat Mancera-Arteu, Nejsi Lleshi, Fernando Benavente and Victoria Sanz-Nebot

[PP-9] Dispersive micro solid phase extraction using modified magnetite nanoparticles with ionic surfactants .........................................................176Y. Martín-Biosca, M. Pérez-Baeza, R. Muñoz-Espí, L. Escuder-Gilabert, S. Sagrado and M.J. Medina-Hernández

[PP-10] Assessment of a HPLC method with in-line diode-array, fluorescence and electrochemical detectors for the simultaneous and reliable determination of parabens in cosmetic products .......................................178L. Abad , S. Lucas, M.T. Sevilla, M.J. Gismera and J.R. Procopio

[PP-11] Enantiomeric determination of the agrochemical prothioconazole by chiral capillary electrohoresis .............................................................181Sara Jiménez-Jiménez, María Castro-Puyana, María Ángeles García and María Luisa Marina.

[PP-12] Study of combined toxicity of duloxetine and econazole on non-target organisms using chiral capillary electrophoresis .....................................183Jesús Valimaña-Traverso, Georgiana Amariei, Karina Boltes, María Angeles García and María Luisa Marina

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[PP-13] In-source fragmentation for identification of modified lysines by CE-ESI(+)-TOF-MS in biological matrices .........................................185Maricruz Mamani-Huanca, Ana Gradillas, Ángeles López-Gonzálvez and Coral Barbas

[PP-14] Enantiomeric determination of econazole and sulconazole in pharmaceutical formulations by CE using hydroxypropyl- β-CD combined with chiral ionic liquids .................................................187Sandra Salido-Fortuna, María Castro-Puyana and María Luisa Marina

[PP-15] Evaluation of selective polyphenol indexes in wines based on capillary electrophoresis with electrochemical detection .........................189Alberto Sánchez Arribas, Mónica Moreno, Noelia Blázquez, Eugenia Alonso, Esperanza Bermejo, Antonio Zapardiel and Manuel Chicharro

[PP-16] A novel approach to screen for natural extracts with inhibitory activity against microbial carnitine monooxygenase using capillary electrophoresis ..........................................................................192Júlia Boldú, Carolina Simó and Virginia García-Cañas

[PP-17] First proposal based on capillary electrophoresis for the analysis of fipronil and metabolites. Application to egg samples ............................194M. Mar Aparicio-Muriana, Ivona Lhotská, Francisco J. Lara

and Ana M. García-Campaña

[PP-18] PSA from urine: challenges for CE analysis of its isoforms .....................197Diana Navarro-Calderon, Raquel Saez-Brox, Sergio Lopez-Duque, Ángel Puerta, José Carlos Diez-Masa and Mercedes de Frutos

[PP-19] Paper-based electrochemical platforms for emerging contaminant analysis: preconcentration and detection of diclofenac and azithromycin .......................................................................................199E. Costa-Rama, T. Magalhães, O. Amor-Gutiérrez, H.P.A. Nouws, M.C. Blanco-López, C. Delerue-Matos and M.T. Fernández-Abedul

[PP-20] Ionic liquid and multi-walled carbon nanotubes for the extraction of carbamate pesticides from water samples prior to their determination by capillary electrophoresis ...............................................202Jihane Ben Attig, Latifa Latrous, Mohammed Zougagh and Ángel Ríos

[PP-21] Development of a CE-UV method for monitoring markers of spoilage in apple juice caused by yeast and bacteria .............................................205Fabiana Amaral da Silva and Ana Valéria Colnaghi Simionato

[PP-22] Lab-on-a-chip systems based on active transport using graphdiyne micromotors ...........................................................................208Víctor de la Asunción-Nadal, Kaisong Yuan, Beatriz Jurado-Sánchez and Alberto Escarpa

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[PP-23] Lab-on-a-chip operations performed by magnetic carbon-based micromotors ...................................................211Roberto María-Hormigos, Beatriz Jurado-Sánchez and Alberto Escarpa

[PP-24] Electrochemical microfluidic chip coupled to magnetoimmunoassay for procalcitonin detection in clinical samples. ........................................214María Moreno-Guzmán, Águeda Molinero-Fernández, Miguel Ángel López and Alberto Escarpa

[PP-25] Metal-organic frameworks in polymer monoliths for on-line solid-phase extraction capillary electrophoresis of polycyclic aromatic hydrocarbon derivatives .............................................................217Héctor Martínez Pérez-Cejuela, Fernando Benavente, Ernesto Francisco Simo-Alfonso and Jose Manuel Herrero-Martinez

[PP-26] CE-MS metabolomics study of H9C2 cardiac cells submitted to oxidative stress ......................................................................................219Mónica Forca Lima, Aline Mara dos Santos, Coral Barbas, Ana Valeria Colnaghi Simionato and Francisco Javier Rupérez

[PP-27] Comparative characterization of the FC domain N-Glycosylation in monoclonal antibody and fusion protein therapeutics by CGE-LIF and UHPLC-FL ....................................................................221Andras Guttman, Marton Szigeti, Akos Szekrenyes, Marcin Moczulski, Jean Charles Berliet and Stephen Lock

[PP-28] Development of isolation techniques and CESI-MS methods for the characterisation of nanomaterial protein coronas ........................222Klaus Faserl, Andrew Chetwynd, Iseult Lynch, Herbert Lindner, Christopher Loessner, Marcin Moczulski, Jean Charles Berliet and Stephen Lock

[PP-29] Method optimization and evaluation for RNA purity analysis using CE-LIF technology ....................................................................................223Tingting Li, Christopher Loessner, Marcin Moczulski, Jean Charles Berliet and Stephen Lock

[PP-30] Simplify modification mapping at the intact protein level with on-line separation and analysis by CE-MS ...........................................................224Christopher Loessner, Marcin Moczulski, Jean Charles Berliet and Stephen Lock




Plenary Lectures

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[PL-1]

AN INSIDER’S VIEW OF THE EVOLUTION OF CLINICAL SCIENCE THROUGH 25 YEARS OF ANNUAL LACE SYMPOSIUMS

Norberto A. Guzman

Princeton Biochemicals, Inc., Princeton, New Jersey, USA. E-mail: [email protected]

Modern clinical chemistry began with the application of 20th century quantitative analysis using automated instrumentation to measure constituents of biological fluids, and further relating the resulting values to human health and disease. The significant scientific advances achieved in the last 30 years in immunochemistry and separation science have had a pro-found influence on the quantification of biomarkers to improve health, comfort and welfare of mankind. Today, the term “clinical science” is used in preference to clinical chemistry or clinical pathology since that a myriad of scientists, in addition to chemists and biologists contribute with their knowledge and expertise, to advance the transitional health care system of the 21th century. These scientists include physicists, engineers, and information technology professionals. The clinical science advancements include the delivery of healthcare that is safe, effective, sensitive, timely, patient-centered, efficient, and available in centralized and decentralized locations.

As a clinical biochemist, working in clinical and bioanalytical laboratories of medical centers and pharmaceutical industries for many years, I set my sights toward introducing bioanalytical applications using capillary electrophoresis (CE) to Latin American scientists with the help of international experts working in similar research fields. Although my first lectures on CE in Latin American countries dated back to the late 1980s, the first meeting of the Annual Sympo-sium Series of Latin American Capillary Electrophoresis (LACE) started in 1995.

This presentation will summarize the contribution of many scientists working in bioanalyt-ical applications using conventional capillary electrophoresis, microchip capillary electro-phoresis and coupling CE to multiple detection systems.

References:[1] Guzman NA, Hoebel BG, Hernandez L. BioPharm 2(1): 22-37 (1989).[2] Jellum E, Dollekamp H, Blessum C. J. Chromatogr. B 683(1): 55-65 (1996).[3] Guzman NA, Phillips TM. Electrophoresis 32(13): 1565-1578 (2011).[4] Guzman NA, Guzman DE. Journal of Chromatography B, 1021: 14-29 (2016).

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[PL-2]

HIGH THROUGHPUT PROTEIN ASSAYS USING MICROCHIP ELECTROPHORESIS

Robert T. Kennedy, Claire Ouimet, and Natalie Cleaveland

1 University of Michigan, Ann Arbor, MI, USA. Email: [email protected]

1.- ObjectivesMicrochip electrophoresis (MCE) enables fast separations due to the ability to apply high electric fields over short distances as well as good control over the injection to prevent extra column band broadening. A limit of MCE is that it is challenging to add new samples to a chip to enable truly high-throughput measurements and take full advantage of the speed of separation. In addition, few practical assays that can utilize the speed have been demonstrat-ed for proteins. In this work we present technology and application of rapid MCE protein separations for: 1) Western blots; 2) high-throughput screening (HTS) of enzyme activity; and 3) assay of protein-protein interactions (PPI). Included in this work is the use of droplet microfluidics to allow rapid sample introduction to the chips of nanoliter samples.

2.- MethodsWestern blotting is achieved by rapid separation on a chip, and then capturing the separated proteins on a membrane for immunoassay. Transfer to the membrane is achieved either by dragging the chip across a membrane or using a dispensing technology to deliver eluent to the membrane. For studying PPI, the simplest method is to mix interacting proteins and sep-arate; however, this requires that separation conditions and binding conditions are compat-ible. In fact, proteins often stick to capillaries and yield unusable separations. To overcome this problem, we use protein cross-linking CE (PXCE). In this approach, proteins are cross-linked by rapid reaction with glutaraldehyde prior to separation. Separation can take place under denaturing conditions, e.g. sieving electrophoresis, to provide reliable separations of complexes of different sizes. To achieve high-throughput, nanoliter samples are encapsulated in a fluorinated liquid as droplets within a tube. Droplets are pumped past an inlet that ex-tracts the samples and feeds them to an injection cross on a MCE chip.

3.- ResultsUsing PXCE we have found: reliable method development and separations, quantitative binding information from nM to uM Kd, and sensitivity to small molecule modulation en-abling screening. Assays have been developed for a variety of protein complexes including Hsp70: Chip, Hsp70 dimer, Antibody:antigen, and KIX with several targets. For the MCE Western blot, we have again found reliable method development. Several target proteins can be detected in cell lysate with little band broadening from deposition. Multiplexing is also possible. In this approach, several injections are made from a single sample and separate tracks laid down on the membrane. Each track is treated with a different primary antibody so that multiple proteins may be detected from a single sample. Up to 11 proteins have been detected using this approach. Using droplet interfaces, both PPI and MCE enzyme assays can

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be performed at up to 1 sample/s for over 1000 samples. This throughput allows HTS for PPI and enzyme modulators.

4.- ConclusionsMCE offers interesting new approaches for protein analysis. The Western blot is the most common protein assay; however, it has changed little from its earliest inception 30 years ago. The use of MCE allows faster Westerns on nanoliter samples. Furthermore, the multiplexing potential brings new capability to this established method. Protein-protein interactions play a fundamental role in governing cell function. PPIs are increasingly viewed as drug targets as well. Better understanding of PPI requires methods that can determine binding affinity, stoichiometry, and interaction sites. Drug development requires screens that can detect PPI and modulation by small molecules. Several techniques are used for such studies including isothermal calorimetry, fluorescence polarization, and various bead binding assays. All have limitations in terms of material required, protein modifications needed, and information ob-tained. The MCE method offers a new method that fills gaps in current technology for PPI. Droplet technology is an approach for manipulating small samples and automating sample injection at high-throughput.

Keywords: Protein, Western blot, High-throughput screening, Protein-Protein interaction

Acknowledgements: This work has been sponsored by NIH.

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[PL-3]

NANOPARTICLE-BASED SEPARATION MEDIA FOR ELECTRO- AND LIQUID PHASE-SEPARATION TECHNIQUES

Ziad El Rassi, Sarah Alharthi, Nisansala Ganewatta and Cemil Aydogan

Oklahoma State University, Department of Chemistry, Stillwater, OK, USA. Email: [email protected]

1.- ObjectivesDemonstrate the potentials of inorganic and organic-based nanoparticles (INP and ONP, respectively), namely modified and unmodified fumed silica nanoparticles (FSNPs) and multiwalled carbon nanotubes (MWCNTs) in liquid phase separation techniques such as HPLC, CE and CEC, which also entailed devising ideal supports for NP sorbents in HPLC and CEC and NP pseudo stationary phases in CE for NP electrokinetic chromatog-raphy (NPEKC).

2.- MethodsFor INP sorbents, the support consisted of glyceryl monomethacrylate (GMM) and ethylene dimethacrylate (EDMA) monolith [poly(GMM-co-EDMA)]. In one case, FSNPs were in-corporated into this monolith in a 4.6 mm i.d. stainless steel tubing forming a new monolithic column for HILIC. When compared to a monolithic column without FSNPs, the FSNPs incorporated column yielded important effects on HILIC separations. In a second instance, C18 modified FSNPs were incorporated into poly(GMM-co-EDMA) monolith for use in reversed phase chromatography. The FSNPs modified with 3-(trimethoxysilyl)propylmeth-acrylate yielded the “hybrid” methacryloyl FNSP monomer, which was subsequently mixed with GMM and EDMA in a porogen and the insitu copolymerization was performed in a stainless-steel column. The silanol groups of this hybrid monolith were grafted with C18 ligands by perfusing the column with a dimethyloctadecylchlorosilane solution in toluene while heating the column at 110 ˚C. In other studies, the preparation and characterization of monolithic capillary columns of poly(GMM-co-EDMA) with incorporated bare FSNPs and surface coated gluconamide FSNPs and their subsequent use in hydrophilic interac-tion capillary electrochromatography of small relatively polar solutes were performed. The poly(GMM-co-EDMA) monolith functioned as a true “support” for both types of polar FSNPs “stationary phases”. Regarding ONP sorbents, various studies were performed in-cluding two types of monolithic capillaries with incorporated hydroxyl functionalized MW-CNTs (MWCNT-OH), e.g., poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolith [poly(GMA-co-EDMA)], and its diol derivative. In another series of studies with ONP, MWCNTs in either unmodified, MWCNT-OH, MWCNT-COOH or MWCNT-SO3H forms were added to the CE electrolytes to perform NPEKC.

3.- ResultsThe optimized INP polar sorbents yielded excellent HILIC separations for polar acidic, weakly basic and neutral analytes including hydroxy benzoic acids, nucleotides, nucleosides,

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dimethylformamide, formamide and thiourea. Besides alkylbenzenes, the C18 INP allowed the analyses of aniline derivatives and phenolic compounds with high separation efficiencies while six standard proteins were baseline separated using a 10 min linear gradient elution at increasing ACN concentration. For the first time NPEKC permitted the separation of stand-ard proteins and high resolution of their tryptic peptide maps.

4.- ConclusionsThe various studies yielded important data with many small and large molecules, which will be detailed in the plenary lecture.

Keywords: Fumed silica nanoparticles; Multiwalled carbon nanotubes, HILIC, RPC, NPEKC.




Keynote Lectures

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[KL-1]

FABRICATION OF STRUCTURED MAGNETIC METALLIC NANO-PLATELETS

FOR BIO-ANALYTICAL APPLICATIONS

Jakub Novotny1,2, Petra Juskova1,† , Rudolf Kupcik2 , Zuzana Bilkova2 and Frantisek Foret1,3

1 Institute of Analytical Chemistry of the Czech Academy of Sciences, Brno, Czech Republic. Email: [email protected], [email protected]

2 Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic. Email: [email protected], [email protected]

3 Central European Institute of Technology, Masaryk University, Brno, Czech Republic

† Current address: Department of Biosystems Science and Engineering, ETH Zürich, Switzerland. Email: [email protected]

1.- ObjectivesWe have developed a simple method of preparation of thin-metal nano-platelets with com-plete control over size, shape and properties of nano-platelets of sub-micrometer thickness. Platelets with a thickness of 50–200 nm and with defined arbitrary shapes and sizes in the range of 15–300 mm were prepared from single or multiple metal layers by magnetron sputtering.

2.- MethodsMetal sputtering and lift-off photolithography was used for platelets preparation. The steps included: (A) pattern exposition on a photosensitive layer; (B) dissolution of the exposed (positive tone) photoresist as well as the sacrificial layer underneath with a suitable develop-er; (C) metal sputtering on the resulting 3D structures; (D) stripping off the remaining resist

3.- ResultsDeposition of different metals in layers enabled fabrication of bi- or tri-metallic platelets with a magnetic core and differently composed surfaces. Highly reflective nano-platelets with a magnetic core allowed manipulation by magnetic fields, while different metallic sur-faces served for functionalization by selected molecules. Submicron thin nano-platelets are extremely light - can be attached to surfaces by only a few chemical bonds. At the same time their area is sufficiently large for simple optical recognition of their shape – Fig.1.

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Fig. 1 Examples of the prepared nanoplatelets

4.- ConclusionsThe lift-off approach eliminates the need for different corrosive etchants for each metal layer. Chemical modification of the surface of various metals, provides a basis for applications of the nano-platelets including immunoassays. Inclusion of ferromagnetic properties into the particles enables utilization in separation and liquid movement control using a magnetic field.

Keywords: photolithography, nanoplatelets, surface interaction, optical detection

Acknowledgements: This research was funded by the GACR (16-09283Y), with additional support from European Regional Development Fund-Project “SINGING PLANT” (grant number CZ.02.1.01/0.0/0.0/16_026/0008446) and the European Regional Development Fund-Project ”Strengthening interdisciplinary cooperation in research of nanomaterials and their effects on living organisms” (grant number CZ.02.1.01/0.0/0.0/17_048/0007421).

References: [1] Novotny J, Juskova P, Kupcik R, Bilkova Z, Foret F. Micromachines 10: 106; doi:10.3390/

mi10020106 (2019).

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[KL-2]

12 YEAR’S EXPERIENCE OF LOW-COST ANALYTICAL DEVICE BASED ON CAPILLARY ELECTROPHORESIS FOR FALSIFIED OR SUB-STANDARD DRUG QUALITY CONTROL

Serge Rudaz1, Julie Schappler1, J.-L. Veuthey1, P. Bonnabry2, C. Rohrbasser3, S.O. Sarr4, S. Roth5 and O. Vorlet5

1 School of Pharmaceutical Sciences, University of Geneva, 1 Michel Servet – Geneva – Switzerland. Email: [email protected]

2 Geneva University Hospitals (HUG), CH-1211 Geneva 14, Switzerland3 Pharmelp, (www.pharmelp.ch), CH-1704 Fribourg, Switzerland4 Faculty of medicine and Pharmacy, University Cheikh Anta Diop of Dakar, Senegal5 College of Engineering and Architecture of Fribourg, CH-1705 Fribourg, Switzerland

1.- ObjectivesThe proportion of falsified or sub-standard medicines in emerging countries has dra-matically increased in the last few years. According to official sources, the proportion could reach 50% in Africa. Simple, reliable, and cost-efficient drug control approaches are needed and the currently used methods entail numerous drawbacks such as (i) the availability of reference substances, (ii) the maintenance of analytical instruments, (iii) the availability and costs of consumables. In this context, the use of capillary electrophoresis (CE) appears of utmost interest since the separation is achieved in a capillary of reduced dimension filled with an aqueous buffered solution of electrolytes.

2.- MethodsAnalytical approaches and device established on CE are greener and home-made alterna-tives could provide comparable accomplishments than commercial instrumentation. In 2006, we designed a low-cost CE device to help transitional countries to fight against falsified or sub-standard medicines. Over these last 12 years, this prototype was successfully imple-mented in 8 emerging countries including Mali, Cambodia, Senegal, Republic of Congo and Rwanda, leading to various missions, conventions, scientific communications, dissemination (>50 press releases and interviews), as well as international recognitions. Simple and generic methods were validated according to regulatory guidelines and applied.

3.- ResultsThis lecture will present results and issues evidenced by the use of a low-cost CE in emerg-ing countries over the last 12 years. To analyze a relatively high number of analytes and benefit from the device with basic chemistry knowledge, simple and generic methods were developed and validated. A strategy based on multiple injections was proposed for the si-multaneous qualitative and quantitative analysis of various drugs from the list of the 200 essential medicines defined by the WHO. The situation of Sénégal, where a low-cost CE was first used since 2012, will be discussed as an example of successful collaboration and

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implementation. Even if the first device was robust, some normal wear parts remain difficult to obtain in emerging countries According to the feedback gathered through the missions regarding the instrument, the methods, and the field constraints, a new generation of device is currently under development. This new version of the prototype is intended to be evolution-ary and sustainable. It is therefore important to standardize and guarantee the interoperability of components. To do this, users must take ownership of the machine, understand it and be able to repair it. It was mandatory to transform the prototype so that each component can be replaced locally or easily available via the Internet. The further steps of the project will also include a new/renewable energy source to supply HV regardless of the local electric facili-ties, and an integrated software to simplify data treatment and reporting.

4.- ConclusionsFalsified and Substandard medical products are an increasing threat to public health and most prevalent in low- and middle-income countries. A first low-cost CE was successfully built and implemented. Due to its reduced operating costs, this device is perfectly adapted to rapidly evaluate the quality of drugs, establish the presence and quantify the amount of the active principle(s), and give evidence regarding the presence of degradation impurities. CE can thus be considered an appropriate tool for drug quality control. A new generation proto-type is expected to push forwards the use of this technique in emerging countries.

Keywords: Substandard and Falsified medicines; Quality control; Capillary Electrophoresis, Emerging Countries

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[KL-3]

DEVELOPMENT OF CAPILLARY ELECTROPHORESIS HARDWARE FOR IMPLEMENTATION ON

SPACEFLIGHT MISSIONS OF EXPLORATION

Maria Fernanda Mora1, F. Kehl, E. Tavares da Costa, N. Bramall and Peter Willis

1 Jet Propulsion Laboratory, California Institute of Technology. Pasadena, California, USA. E-mail: [email protected]

Capillary electrophoresis (CE) and its miniaturized version, microchip electrophoresis (ME) are ideal candidates for in situ planetary missions, especially to areas where aqueous analysis is required [1,2]. CE is a liquid-based technique capable of high efficiency separations of a wide variety of molecules and, when coupled to laser-induced fluorescence (LIF) allows the detection of extremely low concentrations (ppb to ppt) of organics.

These techniques overcome the limitations of gas-phase techniques and hold unique promise in the search for signatures of life on other worlds. A powerful approach in this search involves seeking evidence of life at the molecular level, as distributions of organic molecules [3]. Abiotic processes generally produce samples containing smooth distributions of molecular properties while biotic processes only use specific subsets of these distributions to create the ordered structures of life. For example, the distribution of amino acids, including type, relative abundance, and chirality, can be used to determine if life is, or was once, present in a sample [4]. Here we describe CE and ME instrumentation developed at JPL and the steps we are taking to someday enable the implementa-tion of this technology in spaceflight missions. Towards this end, we have developed the Chemical Laptop, a portable, battery-powered, and fully automated microchip electrophoresis instrument coupled to laser-induced fluorescence detection [5]. This system would provide the sample pro-cessing capabilities required for in situ amino acid analysis with sub parts-per-billion sensitivity in a compact, low-mass, and low-power package. This instrument concept could be adapted to a variety of astrobiologically interesting targets like Europa, Enceladus, or Titan. This instrument is the first “end-to-end” ME-LIF astrobiology instrument capable of receiving a liquid sample and performing all operations required for analysis in an automated fashion. This system also serves as a general prototype that could be reprogrammed for terrestrial-based analyses as well.

References:[1] Mora MF, Stockton AM, Willis, PA. Electrophoresis 33: 2624-2638 (2012).[2] Willis P, Creamer J, Mora M. Anal. Bioanal. Chem. 407: 6939-6963 (2015).[3] Lovelock JE. Nature 207, 568-570 (1965).[4] Creamer JS, Mora MF, Willis PA. Anal. Chem. 89, 1329-1337 (2017).[5] Mora MF, Kehl F, da Costa ET, Bramall N, Willis PA. Anal. Chem. 2019, in preparation.

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[KL-4]

ULTRA-HIGH-THROUGHPUT MULTI-PARAMETRIC IMAGING FLOW CYTOMETRY

Andrew J. deMello, Gregor Holzner and Stavros Stavrakis

Department of Chemistry & Applied Biosciences, ETH Zürich, Vladimir Prelog Weg 1, 8093 Zürich, Switzerland. E-mail: [email protected]

1.- IntroductionFlow cytometry, based on point-based detection schemes, is recognized to be the gold-stand-ard tool for high-throughput manipulation, processing and analysis of cells in flow, but is typically limited with regard to the number of cells that can be interrogated per unit time and the information yield per experiment. To address such limitations, much recent activity has focused on the development of imaging flow cytometry platforms [1]. Imaging flow cytometry combines the high-throughput capabilities of conventional flow cytometry with single-cell imaging and enables high information content analysis of large cellular popula-tions. We have developed approaches for sheathless imaging flow cytometry that incorporate stroboscopic illumination for blur-free cellular analysis at throughputs exceeding 100,000 cells per second.[2] By combining inertial (or viscoelastic) focusing of cells in parallel (or single) microchannels and stroboscopic illumination, such chip-based cytometers are able to extract multi-color fluorescence, bright-field, and dark-field images. This enables the ac-curate sizing of individual cells and the analysis of heterogeneous cell suspensions while maintaining operational simplicity.

2.- MethodsA schematic of one of microfluidic imaging flow cytometers is shown in Figure 1. The cy-tometer consists of three sub-components: an input module (R1) incorporating a series of posts to remove cellular aggregates prior to analysis, a cell-focusing manifold (R2) con-taining six parallel and winding channels (each with a width and depth of 40 and 22 mm, respectively), and an imaging region (R3) where dual-color fluorescence, bright-field, and dark-field images of the cell flows can be extracted. In addition to lateral focusing, fluid inertia is used to control the axial position of cells within each microfluidic channel and the inter-cellular spacings. To eliminate motion blur associated with CMOS cameras when im-aging rapidly moving cells, we implement a stroboscopic illumination scheme and dual-view detection system, to extract blur-free images. Details of this approach have been reported elsewhere [3].

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Fig. 1. Schematic of an inertial microfluidic flow cytometer highlighting the inlet port (blue), inertial focusing channels (orange), imaging area for the detection (green), and the outlet port

for the collection (gray). (R1) Filter for removing the cell debris, cell aggregates, and the clumps. (R2) Schematic of the cells flowing at the beginning and at the end of the inertial focusing channel.

(R3) Representative images from all the possible imaging modalities of the detection. All scale bars represent 30 mm. Image adapted and reproduced from [2].

3.- ResultsMy talk will describe how we have applied the above method to the rapid enumeration of apoptotic cells and the high-throughput discrimination of cell cycle phases [2]. In addition, I will describe an entirely new approach for high-resolution, image-based detection of flowing cells within microfluidic formats.[4] Such a capability leverages and combines elasto-iner-tial microfluidics, stroboscopic illumination and machine learning. Moreover, the adoption of a wide channel microfluidic format, facilitates system automation and simplifies the op-tical detection scheme. The system allows for blur-free detection at throughputs in excess of 60,000 cells per second (fluorescence) and 400,000 cells per second (bright field). The imaging platform is capable of multi-parametric fluorescence quantification and subcellular localization of cellular structures down to 500 nm with microscopy image quality.

4.- ConclusionsThe described developments in imaging flow cytometry have significant potential for the large-scale screening of cells, quantitative analyses of heterogeneous cell populations and the systematic mapping of molecular interactions within real-world cell populations.

Keywords: Microfluidics; Imaging Flow Cytometry, Inertial flows, Viscoelastic fluids.

Acknowledgements: The authors acknowledge the Swiss National Foundation and ETH Zürich for partial support

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References:[1] Stavrakis S, Holzner G, Choo J, deMello AJ. Curr. Op. Biotechnol. 55: 36 (2018).[2] Rane AS, Rutkauskaite J, deMello AJ, Stavrakis S. Chem 3: 588 (2017).[3] Hess D, Rane AS, deMello AJ, Stavrakis S. Anal. Chem. 87: 4965 (2015).[4] Holzner G, Mateescu B, van Leeuwen D, Cereghetti G, Dechant R, A.J. deMello AJ,Stavrakis S. Submitted for publication (2019).

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[KL-5]

PURPOSE MADE CE INSTRUMENTS

Peter C. Hauser1 and Jasmine S. Furter2

1 Department of Chemistry, University of Basel, Basel, Switzerland. Email: [email protected] Department of Chemistry, University of Basel, Basel, Switzerland. Email: [email protected]

Capillary electrophoresis is highly versatile in that it is possible to determine small inorganic anions and cations, heavy metal ions, organic anions and cations and as well as biochemical species such as DNA fragments, peptides and proteins. Applications encompass a wide vari-ety including clinical and environmental analysis and the life sciences.

Bench-top CE instruments for laboratory use are available from different commercial sup-pliers. On the other hand, in contrast to HPLC a CE instrument does not require advanced technical components such as ultrahigh pressure pumps and sophisticated separation col-umns. It is thus possible to construct CE instruments relatively easily. The building of low cost instruments is of interest for laboratories with limited financial means, such as those in developing countries. However, there are also applications of CE which cannot be well ad-dressed with commercial benchtop instruments. This includes field portable and battery pow-ered instruments, automated on-site monitoring systems (process analysis), or instruments for the laboratory designed for special tasks such as fast analysis, preparative separations, or hyphenation with other operations such as automated sample extractions.

Simple instruments can be very basic and employ manual siphoning injection. However, this is not always easy to carry out, especially when done in the field, and it is preferable to automate this step in order not to be reliant on the skill of the operator. The most demanding part of these instruments is the detector, but capacitively coupled contactless conductivity de-tectors (C4D) may be constructed in the lab if electronic expertise is available or be bought at relatively low cost. It is also possible to build in-house LED or laser diode-based absorbance or fluorescence detectors.

Instruments with automated injection and flushing can be constructed from readily available parts. Pneumatic pressurization allows pulse free propulsion of liquids. The different steps in the operation can be controlled with electrically driven valves and flow rates may be set with flow restrictors consisting of defined lengths of narrow tubing. Sequencing of operations is achieved by programming a microcontroller system such an Arduino. By using miniature components compact instruments can be assembled in a highly flexible and adaptable micro-fluidic breadboard approach.

Keywords: Portable CE, process analysis, microfluidic breadboard.

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References:[1] Furter JS, Hauser PC. Anal. Chim. Acta 1058: 18-28 (2019).[2] Furter JS, Hauser PC. Electrophoresis 40: 410-413 (2019).[3] Fuiko R, Saracevic E, Koenka IJ, Hauser PC, Krampe J. Talanta, 195: 366-371 (2019).

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[KL-6]

CAPILLARY ELECTROPHORESIS AND ISOTACHOPHORESIS APPLIED FOR ANALYSIS AND

CHARACTERIZATION OF ANTIMICROBIAL PEPTIDES

Vaclav Kasicka1, Tereza Tumova1,2, Lenka Monincova1 and Vaclav Cerovsky1

1 Institute of Organic Chemistry and Biochemistry, The Czech Academy of Sciences, Flemingovo n. 2, 166 10 Prague 6, Czechia. Email: [email protected]

2 University of Chemistry and Technology, Faculty of Food and Biochemical Technology, Technicka 3, 166 16 Prague 6, Czechia

1.- ObjectivesThe aim of this work was to apply high performance capillary electromigration methods, zone electrophoresis (CZE) and isotachophoresis (CITP) [1], for qualitative and quantitative analysis and physicochemical characterization of antimicrobial peptides (AMPs) originated from venom of two species of Hymenoptera insects [2]. AMPs represent a promising new group of anti-infective agents strongly interacting with bacterial membranes. Prior to testing their biological activity, their purity and acid-base properties should be determined.

2.- MethodsCZE and CITP were performed on Agilent 7100 CE instrument (Waldbronn, Germany) equipped with UV detector set at 210 nm and contactless conductivity detector. For CZE at constant separation voltage 25 kV, bare or hydroxypropylcellulose (HPC) coated fused silica capillary of 48.5 cm total length (40 cm effective length to the UV detector) and 50/375 µm id/od, was used. For CITP at constant electric current 5 µA, total length of the 100/375 µm id/od HPC coated fused silica capillary was 58.5 cm. Conductivity and UV detectors were 44.5 and 50.0 cm distant from the capillary inlet end. Both CZE and CITP capillaries were thermostated at 25°C.

3.- ResultsPurity degree, counterion content, effective and ionic mobilities, acidity constants, and ef-fective charges of twelve synthetic AMPs (deca- to hexadecapeptides) containing 3–7 basic amino acid residues (His, Lys, Arg) at variable positions of peptide chain were determined from their combined CITP and CZE analyses.

The ionic mobilities and the apparent mixed acidity constants, pKa,mix, of the AMPs were de-termined by nonlinear regression analysis of the pH dependence of their effective mobilities measured in series of the background electrolytes (BGEs) within a wide pH range (1.80-12.10), at constant ionic strength (25 mM) and temperature (25°C) [3]. Then, the pKa,mix values were recalculated to the thermodynamic pKas using the Debye-Hückel theory. Ther-modynamic pKa values of His were in the range 3.72-4.98, pKa of α-amino group was in the range 6.14-6.93, and pKa of ε-amino group of Lys spanned the interval 7.26-9.84.

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Eff ective charge of the AMPs was determined by combination of CITP and CZE [4]. This approach is based on the linear dependence of the CITP zone length of the analyte on its eff ective charge and injected substance amount. Cationic CITP systems with leading elec-trolyte (LE) composed of 10 mM ammonium leading ion, 40 mM acetate counterion, pH 4.1, and terminating electrolyte containing 40 mM acetic acid, pH 3.2, provided sharp zones with good linearity and repeatability, see Fig. 1. Ionic mobilities of AMPs were determined by CZE in the BGE of the same composition as the LE. Using this procedure, the eff ective charge numbers of the AMPs were found to be in the range 1.65-5.04, i.e. signifi cantly re-duced as compared to the theoretical charge numbers (2.86-6.99) calculated from the pKa values. This reduction of eff ective charge due to tightly bound acetate counterions (coun-terion condensation) was in the range 17–47 % depending on the number and type of the basic amino acid residues in the AMPs molecules. Ionic mobilities of AMPs achieved values (26.5-38.6) × 10-9 m2V-1s-1 and in most cases were in a good agreement with the ratio of their eff ective charges and relative molecular masses.

Fig. 1: CITP-grams of AMPs HYL 1 (A) and HYL 2 (B) and reference compound (ammediol) (trace a), its fi rst derivative (trace b) and a blank (sample free) CITP run (trace c). x, non-identifi ed admixture of the ITP electrolyte system. Experimental conditions: LE, 10 mM NH4OH, 40 mM AcOH, pH 4.1;

TE, 40 mM AcOH, pH 3.2; hydroxypropylcellulose coated fused silica capillary, 100/375 µm id/od, 58.5/44.5 cm total/eff ective length; constant current 5 or 8 µA at 25 °C;

hydrodynamic injection 20 mbar for 5–20 s.

4.- ConclusionsCZE and CITP proved to be suitable and eff ective methods for analysis and physicochemical characterization of polycationic insect AMPs in a microscale. Reduction of eff ective charges of AMPs due to counterion (acetate) condensation was found to be signifi cant and will be taken into account in the studies of mechanism of action of these AMPs toward variable pathogens.

Keywords: Antimicrobial peptides; capillary electrophoresis; capillary isotachophoresis; eff ective charge; acidity constant; ionic mobilities

Acknowledgements: This work has been supported by the Czech Science Foundation (grant no. 17-10832S) and by the Czech Academy of Sciences (project RVO 61388963).

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References:[1] Kasicka V. Electrophoresis 39: 209-234 (2018).[2] Monincova L, Budesinsky M, Slaninova J, Hovorka O, Cvacka J, Voburka Z, Fucik V, Borovickova

L, Bednarova L, Straka J, Cerovsky V. Amino Acids 39: 763-775 (2010).[3] Tumova T, Monincova L, Cerovsky V, Kasicka V. Electrophoresis 37: 3186-3195 (2016).[4] Tumova T, Monincova L, Nesuta O, Cerovsky V, Kasicka V. Electrophoresis 38: 2018-2024

(2017).

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[KL-7]

IDEAL-FILTER CAPILLARY ELECTROPHORESIS

Sergey N. Krylov, Svetlana M. Krylova, An T. H. Le, Stanislav S. Beloborodov and Tong Ye Wang

York University, Toronto, Canada. Email: [email protected]

Our major goal is to make CE a frontline screening technology in early stage drug develop-ment using combinatorial oligonucleotide libraries [1]. The current state of the art screening technology is surface-based separation on magnetic beads. The target is immobilized on magnetic beads and the beads are mixed with the library. Target bound oligonucleotides are separated from the unbound oligonucleotides by a simple pulldown procedure. A major lim-itation of the surface-based approach is non-specific binding of 1 to 10% of the library to the surface of the beads which leads to very low efficiency of screening. We intend to replace the surface-based screening with homogeneous screening by CE.

We recently introduced ideal-filter capillary electrophoresis (IFCE) – a new version of CE-based screening [2–4]. IFCE which increases the efficiency of screening by 107 times with respect to that of the surface-based screening and by 104 times with respect to that achieved in earlier works on CE-based screening [5,6]. The increase was achieved by setting pH and ionic strength of the CE running buffer in the physiological range. The best efficiency was achieved when the oligonucleotide library and the target-oligonucleotide complexes moved in the opposite directions. IFCE can uniquely facilitate one-round selection of highly-pure pools of target binders [2,3]. Our initial work in this direction opened a unique window of practical opportunities and also posed a number of fundamental questions; the major question is, argua-bly, what causes such a drastic increase in efficiency of screening. Our current efforts focus on answering these fundamental questions and addressing remaining technical issues.

In our lecture, we will briefly explain the basic principles of IFCE-based screening of oligo-nucleotide libraries and present the most recent data on advancing this screening approach to its practical application in early-stage drug discovery.

Keywords: Ideal-filter capillary electrophoresis, Oligonucleotide libraries, Screening

Acknowledgements: This work has been sponsored by the Natural Sciences and Engineering Coun-cil of Canada

References:[1] Derek Lowe, “The Good Stuff Goes One Way. . .”, In the Pipeline, https://blogs.sciencemag.org/pipeline/archives/2019/02/06/the-good-stuff-goes-one-way.[2] Le ATH, Krylova SM, Kanoatov M, Desai S, Krylov SN. Ang. Chem. Int. Ed. 58(9) 2739-2743

(2019).

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[3] Le ATH, Krylova SM, Sergey N. Krylov SN. Electrophoresis, doi: 10.1002/elps.201900028.[4] Le ATH, Krylova SM, Krylov SN. Anal. Chem. 91(13): 8532-8539 (2019).[5] Berezovski M, Drabovich A, Krylova SM, Musheev M, Okhonin V, Petrov A, Krylov SN. J. Am.

Chem. Soc. 127(9): 3165-3171 (2005).[6] Musheev MU, Kanoatov M, Krylov SN. J. Am. Chem. Soc. 135(21) 8041-8046 (2013).




Oral Presentations

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[OP-1]

APTAMERS AND EVOLUTION

Linda B. McGown

Rensselaer Polytechnic Institute, Troy, New York, USA. Email: [email protected]

Combinatorial chemistry has a decades-long history in drug discovery and therapeutics, including its relatively recent application to the selection of oligonucleotide affinity reagents, or aptamers, to particular targets for use in biology, medicine and analysis. The basic tenet of the combinatorial approach is that a sufficiently large library of randomly synthesized molecules will include one or more members with the desired functionality. Over the years, however, it has become apparent that this tenet is flawed. This talk will discuss reasons for this in the context of aptamer selection to protein targets, contrasting the established, combinatorial selection route through chemical “evolution” to new approaches that take advantage of naturally occurring oligonucleotides resulting from earth’s long history of biological evolution.

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[OP-2]

NEW APPROACHES BASED ON ELECTROCHEMICAL IMMUNOSENSING MICROFLUIDIC PLATFORMS FOR NEONATAL SEPSIS BIOMARKERS DETERMINATION

Águeda Molinero-Fernández1, María Moreno-Guzmán2, Miguel Ángel López†1 and Alberto Escarpa†1,

1 Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, University of Alcalá, Alcalá de Henares, Madrid, Spain. E-mail: [email protected]

2 Department of Chemistry in Pharmaceutical Sciences, Analytical Chemistry, Faculty of Pharmacy, Universidad Complutense de Madrid, Avenida Complutense, s/n, 28040 Madrid, Spain

† Chemical Engineering and Chemical Research Institute “Andrés M. Del Río”, Universidad de Alcalá, Madrid, Spain

AbstractNeonatal sepsis remains as one of the leading causes of morbidity and mortality among both term and preterm infants, being especially problematic in very low birth weight (VLBW). While treatment delay is associated with increased mortality, the early diagnosis of neonatal sepsis is difficult due to the variable and nonspecific signs and symptoms, and the low sen-sitivity and delayed availability of the blood cultures (gold standard). Hence, among other clinical symptoms, the diagnosis and treatment initiation are based on the analysis of bio-markers as C-reactive protein (CRP), cytokines and procalcitonin (PCT). Due to the limited sample volume available in these patients and the need of serial analysis for prognosis and therapy monitoring, an accurate, sensitive, fast, and specially using very low sample volumes analytical procedure, could meet the requirements of clinicians and patients.

The combination of electrochemical immunoassays and microfluidic platforms provides ex-ceptional benefits such as high selectivity and sensitivity, together with faster analysis times, extremely low sample and reagent volumes, multiplexed analysis, an accurate fluid control and handling capabilities. New approaches for CRP and PCT determination will be presented as a promising tool for on-site/bed side clinical analysis, especially in neonatal sepsis diagno-sis, where fast response is needed and limited sample volume is available.

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[OP-3]

PRECONCENTRATION STRATEGIES FOR THE DETERMINATION OF CATHINONES

IN BIOLOGICAL SAMPLES

Alberto Pérez-Alcaraz*, Francesc Borrull, Marta Calull and Carme Aguilar

Department of Analytical Chemistry and Organic Chemistry, Faculty of Chemistry, Rovira i Virgili University, Tarragona, Spain. Email: [email protected]

Cathinones derivatives are a group of new psychoactive drugs appearing on the recreational drug market. They are readily available from “smart shops” and can be easily purchased through many websites, which sell them as “legal” alternatives of controlled amphetamines. Serious effects, including death can be produced by intake of these drugs. Consequently, analytical procedures for their efficient determination for clinical and forensic toxicology purposes are needed [1].’khat sessions’ have played a key role in the social and cultural traditions among several communities around Saudi Arabia and most East African coun-tries. The identification of cathinone as the main psychoactive compound of khat leaves, exhibiting amphetamine-like pharmacological properties, resulted in the synthesis of several derivatives structurally similar to so-called natural amphetamine. Synthetic cathinones were primarily developed for therapeutic purposes, but promptly started being misused and exten-sively abused for their euphoric effects. In the mid-2000’s, synthetic cathinones emerged in the recreational drug markets as legal alternatives (‘legal highs’).

All cathinone derivatives possess a stereogenic center allowing the existence of one pair of enantiomers that can differ in their pharmacodynamic and pharmacokinetic properties [2]. So, it is important to develop methods that allow the determination of each stereoisomer in biological samples. Among the analytical techniques currently used for this purpose, capil-lary electrophoresis (CE) represents an attractive alternative to chromatographic approaches due to its versatility, high separation efficiency, and low consumption of reagents and sam-ples. Moreover, the enantioseparations by CE are achieved by simply adding a chiral selector into the background electrolyte [3]. However, one important drawback of CE-based method-ologies is inherent poor sensitivity achieved.

To overcome this problem, different strategies have been developed. With this in mind, the main aim of this research was to compare different CE based methodologies for the chiral separation of a group of cathinones in urine samples that involve different preconcentration strategies. These strategies were based on chromatographic or electrophoretic principles. Within the chromatographic based strategies we applied the in-line coupling of SPE with CE where a microcartridge (or preconcentrator) is inserted near the inlet end of the sepa-ration capillary, and it becomes integral part of the separation capillary [4]. For techniques based on electrophoretic principles we used stacking strategies in which the sample was electrokinetically injected. These techniques were field-amplified sample injection (FASI)

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or electrokinetic supercharging (EKS) [5]. In FASI, the sample is prepared in a low conduc-tivity medium and the stacking is due to the difference of conductivities between the sample zone and the BGE zone, and in the case of EKS the samples were injected between a high conductivity leading electrolyte (LE) and a low conductivity terminating electrolyte (TE). All these strategies have been satisfactorily tested and validated to achieve the sensitive en-antiodetermination of cathinones in urine samples.

References:[1] Valente MJ, Guedes De Pinho P, de Lourdes Bastos M, Carvalho F, Carvalho M. Arch. Toxicol.

88: 15-45 (2014).[2] Tai S, Morrison C. Trends Anal. Chem. 2017, 86: 251-252 (2017).[3] Stavrou IJ, Agathokleous E, Kapnissi-Christodoulou CP. Electrophoresis 38: 786-819 (2017).[4] Ramautar R, Somsen GW, de Jong GJ. Electrophoresis 37: 128-137 (2016).[5] Šlampova A, Mala Z, Gebauer P. Electrophoresis 40: 40-54 (2019).

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[OP-4]

DEVELOPMENT OF AN APTAMER FUNCTIONALIZED POLYMER MONOLITH FOR THE SELECTIVE DETECTION

OF CONCANAVALIN A BY ON-LINE SOLID-PHASE EXTRACTION CAPILLARY ELECTROPHORESIS

María Vergara-Barberán1, Ancuta Moga1, Ernesto Francisco Simó-Alfonso1, Fernando Benavente2 and José Manuel Herrero-Martínez1

1 Department of Analytical Chemistry, University of Valencia, C/Dr. Moliner, 50, 46100-Burjassot, Valencia, Spain. Email: [email protected]

2 Department of Chemical Engineering and Analytical Chemistry, Institute for Research on Nutrition and Food Safety (INSA·UB), University of Barcelona, Barcelona, Spain

1.- ObjectivesIn this study, aptamer affinity monolithic microcartridges were developed for on-line sol-id-phase extraction capillary electrophoresis (SPE-CE). These devices were used for the iso-lation, preconcentration, separation and ultraviolet (UV) detection of concanavalin A (ConA) as a test solute in different matrices.

2.- MethodsA 1.5-cm preconcentration unit was prepared in-situ as a fritless monolithic bed integrated in-line in the inlet of a 100-µm fused-silica separation capillary (33.5-cm total length). For this purpose, a glycidyl-based monolith was synthesized and subsequently modified with streptavidin. Then, a biotinylated DNA aptamer targeting ConA was immobilized onto the streptavidin-modified polymer monolith. The analysis of human serum albumin, cytochrome c and the target protein ConA by nano-LC or CE demonstrated the aptamer binding on the monolith and the selectivity against ConA [1, 2].

3.- ResultsThe different steps of the SPE-CE method were optimized to analyze dilute solutions of ConA The developed aptamer-affinity system gave satisfactory repeatability and acceptable reusability. Linearity and limits of detection were also investigated. The applicability was also demonstrated by analyzing food samples.

4.- ConclusionsWe have demonstrated that ConA can be analyzed by SPE-CE due to the high affinity and selectivity afforded by the developed aptamer functionalized monolithic microdevice. The novel ConA-aptabody sorbent provides a promising substitute to ConA-antibody to develop miniaturized sample pretreatment units for food analysis and related applications.

Keywords: Aptamer; capillary electrophoresis; concanavalin A, on-line solid-phase extraction.

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Acknowledgements: This work has been sponsored by projects PROMETEO/2016/145 (Valencian Government, Spain), RTI2018-095536-B-I00 and RTI2018-097411-B-I00 (Ministry of Science, In-novation and Universities, Spain).

References:[1] Zhao Q, Li X-F, Le X-C. Anal. Chem. 80: 3915-3920 (2008).[2] Marechal A, Jarrosson F, Randon J, Dugas V, Demesmay C. J. Chrom. A 1406: 109-117 (2015).

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[OP-5]

MULTIPLEXED CE-MS FOR BIOMARKER DISCOVERY IN METABOLOMICS: IMPROVED SCREENING OF CYSTIC FIBROSIS IN NEWBORNS WITHOUT GENETIC TESTING?

Philip Britz-McKibbin1, Alicia DiBattista1, Osama Aldirbashi2 and Pranesh Chakraborty2

1 Department of Chemistry and Chemical Biology, McMaster University, Hamilton, ON, Canada

2 Newborn Screening Ontario, Children’s Hospital for Eastern Ontario, Ottawa, ON, Canada

1.- ObjectivesCystic fibrosis (CF) is a complex multi-organ disease that is among the most common life-shortening genetic disorders. Newborn screening (NBS) programs for early detection of CF rely on a two-stage immunoreactive trypsinogen and cystic fibrosis transmembrane conductance regulator (CFTR) mutation panel (IRT/DNA) algorithm that is sensitive, but not specific for early detection of CF neonates in the population. There is urgent need for new biomarkers for improving the screening algorithm of CF that addresses unaffected carrier identification when relying on genetic testing in NBS programs.

2.- MethodsFor the first time, we report the discovery of a panel of CF-specific metabolites from dried blood spot (DBS) specimens when using multisegment injection-capillary electrophore-sis-mass spectrometry (MSI-CE-MS) with stringent quality control. Optimization of a mi-croscaled methanol extraction procedure was first performed prior to ultrafiltration as re-quired for analysis of DBS extracts using MSI-CE-MS with full-scan and positive ion mode. Complementary multivariate and univariate statistical methods were used to identify metab-olites of significance after using a false-discovery adjustment, whereas structural elucidation of unknown metabolites was performed by high resolution MS/MS in conjunction with mo-bility matching and/or chemical reactivity.

3.- ResultsThis retrospective case-control study design identified 32 metabolites that were differentially expressed in asymptomatic and normal birthweight CF neonates without meconium ileus (n=36) as compared to age/sex-matched screen-negative controls (n=44) after a false discov-ery rate (FDR) adjustment (q < 0.05). Importantly, 16 metabolites from DBS extracts (q < 0.05, FDR) also allowed for discrimination of true CF neonates from screen-positive carriers and transient hypertrysinogenemic cases (n=60) who are presumptive CF until confirmed as unaffected with low sweat chloride (< 29 mM). Notably, CF-specific biomarker candi-dates satisfying a Bonferroni adjustment (p < 7.25 E-5) included several amino acids (Thr, Ser, Pro, Tyr) likely reflecting protein maldigestion or malabsorption, ophthalmic acid as an indicator of severe glutathione depletion, as well as an unknown trivalent peptide that was correlated with IRT (ρ = 0.332, p = 4.55E-4).

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4.- ConclusionsThis work identifi ed a panel of metabolites that improve the specifi city of CF screening al-gorithms using existing MS/MS infrastructure, which is needed to reduce unaff ected carrier identifi cation due to detection of CFTR mutations of unknown consequence.

Keywords: Metabolomics, Biomarkers, Newborn screening, Dried blood spots, Cystic fi brosis

Acknowledgements: This work has been sponsored by Cystic Fibrosis Canada, Genome Canada and the Natural Sciences and Engineering Research Council of Canada.

References:[1] DiBattista A, McIntosh N, Lamoureux M, Al-Dirbashi OY, Chakraborty P, Britz-McKibbin P.J. Proteome Res. 18: 841-854 (2019).

Fig. 1. An overview of the multiplexed separation method based on MSI-CE-MS for rapid and reliable metabolite screening from a single dried blood spot punch extract as required for diff erentiation

of aff ected neonates with CF from unaff ected carriers.

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[OP-6]

SHEATHLESS CE-MS FOR THE IN-DEPTH CHARACTERIZATION OF IgGs AND IgG SUBDOMAINS

Elena Domínguez-Vega1, C. Gstöttner1, T. Senard1, D. Falck and M. Wuhrer1

1 Center for Proteomics and Metabolomics, Leiden University Medical Center, The Netherlands. Email: [email protected]

1.- ObjectivesMany diseases are antibody-mediated or induce an immune response in our organism. Fur-thermore, most of the products exploited nowadays by the pharmaceutical industry are based on antibodies. Therefore, there is a high demand for analytical tools to allow in-depth char-acterization of immunoglobulins in pharmaceutical and clinical labs. The large heterogeneity of these molecules comprising several posttranslational modifi cations (PTMs), isotypes, al-lotypes and complex structural features makes their analysis challenging. Common methods to study immunoglobulins involve protein digestion or glycan release resulting in a con-siderable loss of information. Analysis of the immunoglobulins or their subdomains at the intact level off ers complementary information, providing isotype and allotype specifi city and permitting the study of the interplay of diff erent PTMs. In this work, we have demonstrated the capabilities of sheathless CE-MS for the characterization of a variety of monoclonal and polyclonal IgGs at the intact level. Technical developments such as hyphenation with high resolution FTICR MS as well as biopharmaceutical and clinical applications will be shown.

2.- MethodsSheathless integrated capillary electrophoresis electrospray ionization was carried out on a CESI 8000 instrument (Sciex, USA) coupled to either an Impact qTOF mass spectrometer or a 12T FTICR from Bruker Daltonics equipped with a nano-electrospray source. Protein sep-arations were performed using neutral pre-coated or in-house polyethylenimine (PEI) coated capillaries with a porous tip. Solutions of 1-20% acetic acid containing 0-20% methanol were used as background electrolytes.

Pharmaceutical formulations were analyzed directly after buff er exchange. Human IgGs were fi rst captured using Fc specifi c beads. For the analysis of antibody subunits the antibodies were digested in the hinge-region using the endoproteinase IdeS or SpeB followed by a reduc-tion of the disulphide bonds. Sample buff ers were exchanged for water using Vivaspin 10kDa MWCO fi lter tubes prior injection.

3.- ResultsVarious sheathless CE-MS approaches have been developed and applied to study the diversity of IgG proteoforms. To prevent protein adsorption, we employed dedicated positively-coat-ed capillaries providing effi cient protein separations. The fl exibility of sheathless CE to be hyphenated with various MS analyzers has been exploited during our research and adapted to the specifi c needs. First results of the hyphenation of sheathless CE with FTICR will be

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shown. Various antibody-based biopharmaceutical products including bispecific monoclonal antibodies have been characterized in detail. Analysis of Fc portions prepared from human plasma IgG will be shown which permits the allotype-specific study of glycosylation and co-occurring modifications. The proposed approaches provided reliable assignment of a va-riety of PTMs including deamidated forms.

4.- ConclusionsThis study demonstrates the power of sheathless CE-MS for the separation and character-ization of monoclonal and polyclonal IgGs relevant to the biomedical and pharmaceutical sciences. These developed methods exhibit the selectivity and sensitivity needed to reliably characterize a variety of biopharmaceutical and plasma samples. In addition to glycosylation, many other co-occurring protein modifications were revealed.

Keywords: Biopharmaceuticals, IgG, capillary electrophoresis, mass spectrometry, intact proteins.

Acknowledgements: This project has received funding from the European Union’s Horizon 2020 research and innovation programme (Analytics for biologics, Grant No. 765502 and Glysign, Grant No. 722095).

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[OP-7]

LC-MS FOR THE DISCOVERY OF POTENTIAL DIAGNOSTIC AND PROGNOSTIC BIOMARKERS

OF NON-HODGKIN LYMPHOMA

Ana Valéria Colnaghi-Simionato1,2, Anna Maria Alves de Piloto-Fernandes3, Patricia de Oliveira-Carvalho3,Victor Piana-de Andrade4,

Jayr Schmidt-Filho4 and Gustavo Henrique Bueno-Duarte1

1 Institute of Chemistry - Unicamp, Campinas, Brazil. 55 19 35213145, 55 19 35213145 [email protected]

2 Institute of Science and Technology in Bionalytics - Unicamp, Campinas, Brazil3 Laboratory of Multidisciplinary Research, São Francisco University, Bragança

Paulista, Brazil. 55 11 24548298, [email protected] AC Camargo Cancer Center, São Paulo, Brazil. 55 11 21895000, victor.andrade@

accamargo.org.br

1.- ObjectivesLymphomas are solid tumor, which normally surround the lymphatic nodules, the spleen and the bone marrow. Abnormal lymphocytes grow fast due to immune system failure, since reg-ular lymphocytes do not recognize cancerous cells as threatening. Lymphomas are classified as Hodgkin (more unusual and with a predictable dissemination) and non-Hodgkin (NHL) (higher incidence and more heterogeneous according to clinical and morphological pres-entations) [1]. Statistics shows that in the USA 74200 new cases of NHL will be diagnosed in 2019 (4.2% of all cases), and 3.3% of all cancer deaths will be related to NHL [2]. The prognosis of NHL is currently determined by the International Prognostic Index, which is derived from the physical characteristics, pathological observations and symptoms reported by the patient. Therefore, the IPI presents low discriminatory potential, making the search for prognosis biomarkers essential, in order to predict the evolution of the disease and to determine the appropriate treatment for each individual. The ‘‘omics’’ era has corroborated in such investigations since the development of new technologies has arisen along with it. One of the techniques applied to the investigation of tumor biomarkers is CE, and increasing applications of CE-MS in this field have been observed [3]. This talk will present untarget-ed metabolomics analyses by liquid chromatography coupled to quadrupole time of flight mass spectrometry (LC-QToF) of urine and serum samples from healthy subjects and NHL patients before and after the first chemotherapy cycle, in order to identify putative NHL di-agnostic and prognostic biomarkers.

2.- MethodsSingle-spot urine (n=38) and fasting serum (n=39) samples were obtained from NLH pa-tients before and after chemotherapy, as well as from healthy blood donor volunteers (51 serum samples and 42 urine samples). Samples were stored at -80°C until processing, which consisted of a simple dilution step in deionized water followed by ultrafiltration for

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deproteinization. Samples were analyzed by LC-QToF. For this purpose, a ACQUITY FTN liquid chromatograph coupled to XEVO-G2XSQTOF mass spectrometer (Waters) using MassLynx 4.1 software was used with a Poroshell 120 EC-C18 (Agilent Technologies) col-umn (100 × 2.1 mm; 2.7 μm). Mass spectra were acquired in both positive and negative elec-trospray ionization modes (ESI) to detect acidic and cationic metabolites, respectively. The sample injection volume was 3 μL. The mobile phase was composed by 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.4 mL/min for both ESI+ and ESI-. The B mobile phase gradient was 0-1.5 min: 20%; 1.5-2 min: 30%; 2-4 min: 50%; 6-8 min: 55%; 8-10 min: 60%; 10-12 min: 65%; 12-14 min: 70%; 14-16 min: 75%; 16-18 min: 80%. Some of the mass spectrometry parameters were: nebulizer gas temperature of 260 °C, drying gas pressure of 11 psi, sheath gas temperature of 150 °C, sheath gas flow of 12 L/min, nebulizer gas pressure of 60 psi for ESI+ and 40 psi for ESI-. Mass spectra were acquired in centroid mode, and the monitored mass range was 100 - 1700 Da.

3.- ResultsDue to high amount of collected data and extensive data treatment required for LC-QToF, obtained results are still under evaluation.

4.- ConclusionsLC-QToF is part of a multi-platform study of untargeted metabolomics of biofluids from NHL patients. Other techniques, such as CE-MS and GC-MS are also part of this study, in order to assess a broader panel of metabolites, pointing to those highlighting to groups differentiation. Thus, potential diagnostic and prognosis NHL biomarkers will be proposed.

Keywords: LC-MS; tumor biomarkers; biofluids

Acknowledgements: The authors would like to acknowledge the Coordination for the Improve-ment of Higher Education Personnel (CAPES), São Paulo Research Foundation (FAPESP), and the Brazilian National Council for Scientific and Technological Development (CNPq).

References:[1] Armitage JO, Gascoyne RD, Lunning MA, Cavalli F. Lancet 390: 298-310 (2017).[2] National Cancer Institute - Cancer Stat Facts: Non-Hodgkin Lymphoma, https://seer.cancer.gov/

statfacts/html/nhl.html.[3] Buzatto AZ, de Sousa AC, Guedes SF, Cieslarová Z, Simionato AVC, Electrophoresis 35: 1285–

1307 (2014).

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[OP-8]

CE-MS FOR METABOLOMICS EVALUATION OF THE MITOCHONDRIAL RESPONSE TO

KETAMINE-RELATED COMPOUNDS

Francisco J. Rupérez1, Andrea Faccio1,2, Nagendra S. Singh3, Santiago Angulo1, Marina F. Tavares2, Michel Bernier3, Coral Barbas1 and Irving W. Wainer3

1 Centre for Metabolomics and Bioanalysis (CEMBIO), Faculty of Pharmacy, University CEU San Pablo, Madrid, Spain. Email: [email protected]

2 Institute of Chemistry, University of Sao Paulo. Sao Paulo, Brazil3 Translational Gerontology Branch, National Institute on Aging, National Institutes of

Health, Baltimore, MA, USA

1.- BackgroundThe single largest contributor to non-fatal health loss is major depressive disorder and bipo-lar depression. Although therapies already exist, they must be improved in terms of speed, effectiveness and duration, together with the inter-individual heterogeneity in drug response. The discovery of the antidepressant effects of ketamine has opened the doors for the treat-ment of bipolar disorder (BD). These antidepressant effects are linked to metabolic trans-formation of the drug, a racemic mixture in its common form, into different metabolites with different activity. Furthermore, different enantiomers show different effects. Previous studies have shown that mitochondrial biogenesis and metabolism are altered by stress and depression, and part of the benefits of the therapies could be related to improvements in the mitochondrial function.

2.- MethodsCells from established PC12 pheochromocytoma cell line were non treated (Control) or treated with R-ketamine, S-ketamine, R-norketamine, S-norketamine, (2R,6R)-hydroxynorketamine, (2S,6S)-hydroxynorketamine, (2R,4R)-dehydronorketamine, (2S,4S)-dehydronorketamine. Mitochondria were isolated and their extracts were analysed (nontargeted metabolomics) by CE-MS (TOF) in order to unveil the metabolites that were different among conditions.

Signals were processed, and all related ions were grouped (molecular feature extraction). All features were multialigned, and the resulting matrix analyzed with univariate and multivar-iate methods (supervised and non-supervised). Those features that became significant were putatively annotated thanks to CEU mass mediator tool.

3.- ResultsMultivariate statistical analysis on the resultant data pointed at metabolites that contributed to the discrimination between the groups. CE-MS helped to discover differences in amino acids and their derivatives, as well as pyrimidines and purines.

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We found that different ketamine metabolites affected different metabolic routes such as bi-osynthesis of valine, leucine and isoleucine, and purine and pyrimidine metabolism. Moreo-ver, there were significant enantiospecific differences between ketamine-derived compounds possessing an ‘R’ versus ‘S’ configuration

4.- ConclusionsCE-MS has proven a valuable tool for suggesting that alteration in basal mitochondrial func-tion, due to genetic variation or underlying disease, is a key aspect of the antidepressant response produced by (R,S)-ketamine and related compounds

Keywords: Ketamine; CE-MS, Bipolar Depression; Mitochondria; Metabolomics; Biomarker.

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[OP-9]

LAB-ON-PAPER ELECTROANALYTICAL DEVICES: TOWARDS THE FULLY INTEGRATION OF STEPS

E. Costa-Rama1,2, E. Núñez-Bajo1, A. González-López1, O. Amor-Gutiérrez1, L. Blanco-Covián1, M.C. Blanco-López1, H.P.A. Nouws2, C. Delerue-Matos2,

P.I. Nanni3, R.E. Madrid3 and M.T. Fernández-Abedul1

1 Department of Physico-Chemistry and Analytical Chemistry, University of Oviedo, Oviedo, Spain. Email: [email protected]

2 REQUIMTE/LAQV, Instituto Superior de Engenharia do Porto, Politecnico do Porto, Portugal. Email: [email protected]

3 Departament of Bioengineering, FACET, National University of Tucuman, San Miguel de Tucuman, Argentina. Email: [email protected]

1.- ObjectivesThere is a marked trend towards decentralization of analysis. It is an urgent requirement in environmental, food, clinical and other fields of analysis. This is the moment to include a new milestone in the timeline of Analytical Chemistry. Our discipline is becoming bifid: complicated and expensive high-tech equipment is living together with low-cost and simple devices for doing the same thing, that is performing analytical processes. On the one hand, current difficult problems require exquisite sophistication in the instruments and methodol-ogies but, at the same time, the use of drones in environmental monitoring, the urge for fruit analyses after post-harvest treatments or the occurrence of episodes such as stroke are some of the examples that will require point-of-use devices.

Paper can undoubtedly play a main role in this scenario. Properties and benefits are well known. Remarkable are the possibility of fluid flowing by capillary forces, the ability to store functional materials or the integration with several detection schemes. Among them, the electrochemical detection fits perfectly with decentralization purposes. All the advantages make this material perfect for the integration of several steps of the analytical process to gen-erate simple but sample-to-result lab-on-paper analytical devices.

2.- MethodsIn this context, several devices that integrate different steps of the analytical process are here presented. Sampling or diluting is performed with simple microfluidic tongues; sepa-ration by electrophoresis is done with a paper microchip; enzymatic reactions, exemplified by glucose determination, are carried out on multiplexed biosensors; immunoassays can be performed in vertical or lateral flow formats and detection can adopt different approaches. Carbon or metal can be the basis of the electrodes, used as films directly on paper or using unconventional elements such as pins or staples. Using nanomaterials as well as taking ad-vantage of preconcentration strategies, provides with interesting and promising approaches for decentralized analysis.

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3.- ResultsIn Fig. 1, there are some examples of paper-based platforms including different steps of the sample-result process. A microfluidic sampler allows avoiding the use of micropipettes for adding the solutions onto a carbon-based paper platform (Fig. 1A). Several working areas can be measured simultaneously, as depicted in Fig. 1C, if a multi-potentiostat is employed. Selectivity is an important issue that can be approached in two different ways: i) using bi-oreagents or ii) integrating separation techniques. Then, paper was wax-printed and diffused to generate channels for electrophoretic separation. Folding the paper allowed the creation of functional layers for: i) injection, ii) separation and iii) detection (Fig. 1B). Alternatively, biocatalysts (enzymes) or affinity reagents (antibodies) can be also incorporated for selective methodologies. Regarding the detection, sputtered or wire metals can be employed for detec-tion but carbon ink is the most common. This advantageous material can be added directly on paper but off-paper electrodes are also possible. Miniaturised electrochemical thick or thin layers can be combined for detection but also unconventional elements as pins or staples (Fig. 1D). Sensitivity can be increased through the electrode nanostructuration and also com-bination with preconcentration strategies.

Fig. 1: Examples of: A) microfluidic tongue acting as sampler for electroanalytical detection [1], B) multi-layered electrophoresis microchip [2], C) top and bottom views of a multiplexed electrochemical

glucose sensor [1] and D) staple-based electrochemical platform for paper substrates [3].

4.- ConclusionsPaper-based electroanalytical devices have been demonstrated to be very useful for decen-tralized analysis in very different areas: environmental, food or clinical fields. Several ex-amples with integration of different steps and approaches are presented. The combination is required for obtaining a sample-to-result platform that could be further coupled with the oth-er elements: e.g., energy source or instrumentation and control systems, to have a real-world useful device.

Keywords: Paper-based devices, Electroanalysis, Lab-on-paper platforms, Low-cost analysis.

Acknowledgements: The authors would like to thank the Spanish Ministry of Economy and Com-petitiveness (MINECO, CTQ2014-58826-R) and also University of Oviedo (PAPI-19-Puente), the

BC

D

A

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EU and the Fundação para a Ciência e a Tecnologia (FCT) / UEFISCDI / FORMAS for funding (consortium REWATER financed under the ERA-NET Cofund WaterWorks2015 Call. This ERA-NET is an integral part of the 2016 Joint Activities developed by the Water Challenges for a Chang-ing World Joint Programme Initiative (Water JPI). E. Costa-Rama also thanks the Government of Principado de Asturias and Marie Curie-Cofund Actions for the post-doctoral grant “Clarín-Cofund” ACA17-20. A. González-López thanks the Governement of Principado de Asturias for a pre-doctoral “Severo Ochoa” grant and O. Amor-Gutiérrez the University of Oviedo for her pre-doctoral grant.

References:[1] Amor-Gutierrez O, Costa Rama E, Fernandez-Abedul MT. Biosen. Bioelectr. 135: 64-70 (2019).[2] Gonzalez-Lopez A, Garcia-Manrique P, Blanco-Lopez MC, Fernandez-Abedul MT. Procedia Technol.

27: 21-22 (2017).[3] Nanni PI, Gonzalez-Lopez A, Nunez-Bajo E, Madrid RE, Fernandez-Abedul MT. ChemElectroChem,

5: 4036-4045 (2018).

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[OP-10]

DIRECT PREPARATION OF MICROFLUIDIC DEVICES USING 3D PRINTING FOR APPLICATIONS ON NANOPARTICLES

SYNTHESIS AND MICROCHIP ELECTROPHORESIS

José Alberto Fracassi da Silva, Lucas P. Bressan1, Brenda Maria de Castro Costa1, Taíssa Moreira da Silva Lima1, Aline Guadalupe Coelho1,

Michael Beauchamp2 and Adam T. Woolley2

1 Institute of Chemistry, University of Campinas, Campinas-SP, Brazil. Email: [email protected]

2 Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah, USA. E-mail: [email protected]

1. ObjectivesThe main objectives of the proposed research are to develop [1] low-cost microfluidic de-vices based on fused deposition modelling (FDM) to synthesize core-shell Au@Ag nanopar-ticles and DLP-based 3D-printed microfluidic devices with narrow channels to be used in microchip electrophoresis with contactless conductivity detection (C4D).

2. MethodsWe used a FDM-based 3D printer (Sethi3D) with a nozzle of 0.2 µm and poly(lactic acid) (PLA) to direct print microfluidic device for synthesis of nanoparticles. Gold nanoparticles (AuNPs, 2.4 mmol L-1) seeds were inserted in one inlet of the device, subsequently mixing with NaBH4 (1 mmol L-1 in 3 mmol L-1 NaOH) and then with AgNO3 (0.75 mmol L-1). The synthesized core-shell nanoparticles were collected at the outlet and then characterized by UV-Vis spectroscopy and transmission electron microscopy (TEM).

A labmade C4D system described in [1] was used and operated with a sinusoidal excitation signal with a frequency of 252 kHz and 7.0 Vpp (peak-to-peak). The voltages for injection and CE separation were applied by a four-channel high voltage power supply ER430 (eDaq, Sydney, Australia). Microchips for electrophoresis were made in PEGDA resin using a DLP-based 3D printer [2]. The microchip layout contained two 40-mm width channels arranged in a cross geometry. Close to the outlet reservoir two spiral channels were placed around the separation channel. These spiral channels were filled with melted Gallium in order to form metallic electrodes (Figure 1). Data acquisition and electrokinetic control were performed using Labview and QuadSequencer (eDAQ), respectively.

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Fig. 1 - (A) 3D serpentine flow channels surrounding the separation channel in microfluidic device. (B) Top view of DLP-based 3D-printed microfluidic device containing Gallium electrodes.

3. ResultsThe device was printed using PLA, since it is a widespread material used in FDM that pos-sesses low-cost and requires simple printing configurations, such as low extrusion and bed temperature. The device was printed in less than 2 h, and no post processing was required after the printing was done. We show the CAD rendering of the device as well as an actual image of the device used in the core-shell nanoparticles synthesis in Figure 2a.

The synthesis inside the microfluidic chip allowed us to effectively create a silver shell around the gold nanoparticles seeds (AuNPs), as seen in the UV-Vis spectra (Figure 2b), where a shift in the surface plasmon resonance occurs from 529 nm (AuNPs) to 390 nm. TEM images of the synthesized nanoparticles also display a characteristic shell around the nucleus (Figure 2c).

Fig. 2 – a) CAD representation of the 3D-printed microfluidic device; b) Image of the 3D-printed microfluidic device; c) UV-Vis spectra of the AuNPs and of the core-shell nanoparticles and d) TEM

image of the core-shell nanoparticles where the core and the shell are highlighted.

Regarding the DLP-made microchips, the best results were obtained by using a BGE com-posed of 20 mmol L-1 MES/histidine (pH = 6.0) at 25 °C. The separation voltage of 800 V, injection voltage of 500 V, and injection time of 3 s were selected for the determination of K+, Na+ and Li+. In addition, the linearity of the method was evaluated using standard solution of K+, Na+ and Li+ (25-200 µmol L-1). Adequate linearity (r ˃ 0.996) was achieved for all calibration plots (Figure 3), which shows a suitable response of the detector to concentration of the analytes.

A B

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Fig. 3 - Electropherograms obtained from the introduction of standard solutions containing K+, Na+ and Li+ (25-200 µmol L-1). Respective calibration curves are also shown.

4. ConclusionsA low-cost 3D-printing device was developed and printed using FDM to synthesize core-shell Au@Ag nanoparticles in less than 2 hours, without further processing.

DLP is fully capable of 3D printing truly microfluidic flow channels. In addition, the pro-posed device was successfully applied for separation of K+, Na+ and Li+. This way, DLP-based 3D-printed microfluidic devices have great potential for determination of other ana-lytes, such as, drugs, adulterants and metabolites by MCE-C4D.

Keywords: core-shell; nanoparticles; FDM; DLP, contactless conductivity detection.

Acknowledgements: The authors would like to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2018/06478-3) and INCTBio. This study was in part funded by the Coordenação de Aperfeiçoamen-to de Pessoal de Nível Superior-Brasil (CAPES)-Finance Code 001.

References:[1] da Silva JAF, Guzman N, do Lago CL. J. Chrom. A 942: 249-258 (2002).[2] Gong H, Bickham, BP, Wooley AT, Nordin GP. Lab Chip 17(17): 2899-2909 (2017).

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[OP-11]

E PLURIBUS UNUM: UNRAVELING THE MOLECULAR HETEROGENEITY OF PRO-B-TYPE

NATRIURETIC PEPTIDES AND ITS CONSEQUENCES AS BIOMARKER FOR HEART FAILURE

Herbert H. Lindner, Benno Amplatz and Klaus Faserl

Division of Clinical Biochemistry, Biocenter, Medical University of Innsbruck, Austria. Email: [email protected]

1.- ObjectivesCurrently, N-terminal pro–B-type natriuretic peptide (NT-proBNP) and its physiologically active counterpart, BNP, are most frequently used as biomarkers for diagnosis, prognosis, and disease monitoring of heart failure (HF) and were implemented into the guidelines for HF-management more than 10 years ago.

Commercial NT-proBNP and BNP immunoassays cross-react to varying degrees with unpro-cessed proBNP, which is also found in the circulation. ProBNP processing and immunoassay response are related to O-linked glycosylation of NT-proBNP and proBNP. Therefore, there is a clear and urgent need to identify and quantify the glycosylation sites in the endogenously circulating peptides to gain further insights into the different naturally occurring forms and to establish a method that enables to determine the actual levels of N-terminal pro–B-type natriuretic peptides.

2.- MethodsNT-proBNP and proBNP peptides were characterized in plasma samples of severe HF pa-tients (NT-proBNP: >10000 ng/L) by developing and using a variety of different methods including antibody cleavage and fixation, immunoaffinity purification, sequential exoglyco-sidase treatment for glycan trimming, β-elimination and Michael addition chemistry, as well as high-resolution nano-LC and CE-MS methods.

3.- ResultsWe were able to identify not only 9 distinct glycosylation sites on circulating (NT-) proBNP in HF patients, but also a large number of N- and C-terminally truncated forms. For each of the identified proteolytic glycopeptides, a nonglycosylated form also was detectable and fur-thermore, remarkable differences in the glycosylation pattern were found within the patient collective.

4.- ConclusionsUnderstanding the structural attributes and molecular heterogeneity of the different natriu-retic peptide B forms is a prerequisite for improved design of assays as well as their clinical application in the diverse settings of HF. An important step in this direction is to identify the

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various peptides to get further insights into the different circulating forms. Based on these results we developed an immuno-affinity-MS based strategy for the quantitative analysis of the actual endogenous levels of various pro-B-type natriuretic peptides.

Keywords: B-type natriuretic peptides, glycosylation, LC-MS, CE-MS

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[OP-12]

CAPILLARY ELECTROPHORESIS FOR THERAPEUTIC RNAS

Robert Weinberger1 and James Timmins2

1 CE Technologies, Inc., Chappaqua , New York, USA. E-mail: [email protected] 2 Helix Diagnostics, Inc., Madison, Wisconsin, USA

After decades of fits and starts, therapeutic and diagnostic RNAs may finally gain some Trac-tion [1]. The first RNA drug approval was granted to Alnylam in August 2018 to treat a rare genetic disorder that causes the buildup of amyloid protein deposits in human organs. A De-cember 2018 IPO launched Moderna into the public domain with a market capitalization of $9 billion. Dozens of companies have entered the competition, some partnering with big pharma.

Before discussing CE, the various types of RNA employed in therapy and diagnosis will be reviewed along the technologies required for drug delivery, drug stability and reduction of immunogenicity. Briefly, messenger RNA can be used to synthesize, in-vivo, proteins that are under-expressed due to illness, genetic or otherwise. mRNA is large, consisting of a thousand to several thousand ribonucleotides. Unlike DNA, which is stable, smaller RNA agents are by natural design unstable and thereby must be modified to be effective for drug use. While intact mRNA agents are larger and can be separated by CE, degradation techniques are required to fully characterize the molecule [2]. Other new RNA drugs not discussed here include aptam-ers, noncoding RNAs, and guide RNAs for emerging CRISPR gene editing therapeutics.

MicroRNAs are small (~18-25 nt) non-coding molecules that may regulate gene expression by either enhancement or repression. Circulating miRNA are promising biomarkers for tum-ors and liver toxicity (e.g. miR-122). Thus far, RT-PCR, miRNA arrays, and RNA sequenc-ing have been employed for detection, but other methods – hybridization combined with CE-LIF, HPLC, and MS – are used more frequently in clinical bioanalysis. Other schemes may yet evolve. Circulating concentrations are often quite low giving PCR an advantage, however smaller modified RNAs can be difficult for polymerase activity.

Small interfering RNA (~20-25 nt) is utilized to suppress expression of a protein by binding to mRNA, causing degradation. Hybridization and CE has been extensively employed in clinical development for determinations of blood plasma/serum concentrations, drug me-tabolism, stability studies and to confirm full-length synthesis of dosage form manufacture.

Fortunately, CE separation science developed for the human genome project is a versatile platform and can be directly employed for various RNA separations.

References: [1] Jarvis LM, Cross R. Is this the decade of RNA?, C&E News, January 15, 2019.[2] Chen B, Yuan B.-F, Feng Y-Q. Analytical Methods for Deciphering RNA Modifications, Anal.

Chem. 91: 743−756 (2019).

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[OP-13]

ELECTROKINETIC SEPARATIONS APPLIED TO SIMPLE AND COMPLEX BIOLOGICAL SAMPLES

Virginia A. Robinson-Fuentes1, Xiomara Zavala-Sánchez1, Maria Soledad Vázquez-Garcidueñas1 and Gerardo Vázquez-Marrufo2

1 Faculty of Medicine, Universidad Michoacana de San Nicolas de Hidalgo. Morelia, Michoacán, Mexico. Email: [email protected]

2 Faculty of Veterinary Medicine, Universidad Michoacana de San Nicolas de Hidalgo. Morelia, Michoacán, Mexico

MEKC has been applied for the analysis of analytes with all sorts of complexities and sizes, from small molecules to cells. This presentation deals with examples of work that has been carried out in our research group, using electrokinetic separations.

Mycotoxins are secondary metabolites of filamentous fungi that represent a serious health problem since they can target different systems and organs. In general, food samples can be contaminated with more than one mycotoxin, so it is necessary to develop a method for the simultaneous determination of Ochratoxin A (OTA), Aflatoxin B1 (AFB1) and Zearale-none (Zea). These three mycotoxins were chosen since they are the most toxic to humans and animals, can persist after food processing and are found in the meat of cattle fed with contaminated feed [1, 2]. This method consisted of a sodium tetraborate buffer (25 mM, pH 9.1) with 50 mM SDS, a 50 mm id x 40 cm capillary; applied voltage of 17 KV, T = 23°C and detection at 201, 214 and 280 nm. The analysis time is less than 15 min and validation parameters showed that this method met appropriate requirements. Samples of corn known to be contaminated with AFB1, were analyzed aand the presence of AFB1 was confirmed, along with that of Zea.

Mushroom production is a multimillion dollar industry so the contamination of these crops by filamentous fungi can cause the Green mold disease leading to great economical losses [3]. Capillary electrophoresis has been used as an alternative technique for fungal identifi-cation. Several electrophoretic conditions were tested and the following, provided efficient signals: HEPES buffer (pH 7, 15 mM) and Tricine buffer (pH 7.8, 10 mM), both with 25 mM CTAB; a 100 mm i.d x 50 cm capillary, T = 25°C and detection at 208 nm. These conditions were applied to the analysis of conidia of four wild species of Trichoderma and spores of Agaricus bisporus var. Portobello. However, in this context, capillary electrophoresis has been used to obtain information of the cell wall composition of such cells and their behavior under different circumstances.

Keywords: Aflatoxin B1, Agaricus bisporus, MEKC, Ochratoxin A, Trichoderma spp, Zearalenone.

Acknowledgements: CIC (UMSNH) and CONACYT (CB 2012-182508)

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References:[1] Alshannaq A, Yu J-H. Occurrence, Toxicity, and Analysis of Major Mycotoxins in Food. Int. J

Environ Res Pub H, 14, 6, 632, 2017.[2] Castellanos-Onorio, O., Gonzalez-Rios, O., Guyot, B., Fontana, T. A., Guiraud, J. P., Schorr-

Galindo, S., Suárez-Quiroz, M. (2011). Effect of two different roasting techniques on the Ochratoxin A (OTA) reduction in coffee beans (Coffea arabica). Food Contl, 22, 8, 1184-1188, 2011.

[3] Hatvani L, Antal Z, Manczinger L, Szekeres A, Druzhinina IS, Kubicek CP, Nagy A, Nagy E, Vágvölgyi C and Kredics L. Green Mold Diseases of Agaricus and Pleurotus spp. Are Caused by Related but Phylogenetically Different Trichoderma Species. Phytopathol. 97, 4, 532-537, 2007.

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[OP-14]

IMMUNOAFFINITY CAPILLARY ELECTROPHORESIS FOR THE ANALYSIS OF BIOMARKERS IN BLOOD

Nora M. Vizioli

Department of Analytical Chemistry and Physicochemistry, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, and the Nacional Council of Scientific and Technical Research (CONICET), Buenos Aires, Argentina. E-mail: [email protected]

Improving the detection capabilities of low abundance proteins and correlating translational and post-translational modifications in proteins with their function are main goals in bio-analytical chemistry [1]. In that sense, immunoaffinity capillary electrophoresis comes to provide the advantages of the high affinity of antibodies fixed on a solid support and the high resolution of capillary electrophoresis [2]. Such devices cannot only be used for concentra-tion enrichment, isolation, or extraction of proteins but also to achieve microreactions, al-lowing the on-line analysis of reaction products. The antibodies can be immobilized directly on a portion of the inner surface of the capillary wall, a membrane, beads, monolithic struc-tures, or even a multibore capillary. In comparison with off-line solid phase extraction tech-niques, immunoaffinity capillary electrophoresis requires lower sample volume, and only a few microliters of organic solvent or buffers are consumed. The entire process is carried out within the CE instrument and only small quantity of sorbent material is needed, which makes on-line preconcentraction cheaper than the off-line method. In addition, sensitivity in CE is increased at least 100-fold, allowing the detection of analytes from 1 ng/mL with UV absorbance detector to 1 pg/mL with a LIF detector, and even as low as fg/mL. The potential of immunoaffinity capillary electrophoresis will be demonstrated through its application to quantify specific protein levels in blood.

Acknowledgements:Financial support for this work has been provided by CONICET, and Univer-sidad de Buenos Aires.

References: [1] Štěpánová S, Kašička V. J. Sep. Sci. 42: 398-414 (2019).[2] Guzman NA, Guzman DE. J. Chromatogr. B 1021: 14-29 (2016).

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[OP-15]

SCREENING GUT MICROBIOTA FOR THE IDENTIFICATION OF BACTERIA WITH HARMFUL METABOLIC

TRAITS FOR CARDIOVASCULAR HEALTH

Carolina Simó and Virginia García-Cañas

Laboratory of Molecular Nutrition and Metabolism, Institute of Food Science and Research, Spanish National Research Council (CSIC), Madrid, Spain. Email: [email protected]

1.- ObjectivesRecent studies on gut microbiota have highlighted the connection between the microbial metabolite trimethylamine (TMA) produced by various taxa of the gut microbiota from di-etary quaternary amines, mainly choline and L-carnitine [1]. When TMA is absorbed in the intestine and reaches the liver, it is rapidly oxidized to trimethylamine N-oxide, which has been recently implicated as a risk factor for cardiovascular disease [2]. Target TMA analysis applied to in vitro bacterial systems may help to determine the microbial capacity to produce or metabolize TMA under specific conditions and, therefore, to enable functional studies uncovering the potential contribution of specific bacterial strains to TMA metabolism in the gut. The main goal of the present work was the development of a new simple CE method with UV detection for a rapid, high-throughput, and cost-effective analysis of TMA to study the capacity of isolated gut bacteria from volunteers to produce TMA from L-carnitine.

2.- MethodsTo selectively isolate gut bacteria able to produce TMA from L-carnitine from volunteers’ fecal samples, a novel microbiological culture medium was formulated. The analyses were carried out on a P/ACE 5010 CE system equipped with a diode array UV-Vis detector. Sam-ple derivatization was performed using 2,4′-dibromoacetophenone (DBA)

3.- ResultsThe method development stage was focused on the study of variables affecting the separa-tion and sample derivatization. The optimal CE separation conditions were established to be 0.75 M formic acid at pH 2.05 with an applied separation voltage of + 14 kV, whereas the optimal derivatization reaction conditions were achieved using 80 mM DBA and 60 min of reaction time at 70 °C. The bacterial isolates from fecal samples donated by volunteers were tested for their potential to produce TMA in selective culture medium using the optimized procedure.

4.- ConclusionsThe developed CE-UV method was demonstrated to be rapid, simple, and reproducible for the analysis of TMA in bacterial culture. The method was successfully applied to assess the capacity of isolated gut bacteria to metabolize L-carnitine to TMA.

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Keywords: Trimethylamine; gut microbiota, metabolic function, capillary electrophoresis, cardio-vascular disease.

Acknowledgements: This work was funded by the Spanish Ministry of Science, Innovation and Universities (project AGL2017-89055-R)

References:[1] Wang Z, Klipfell E, Bennett BJ, Koeth R, Levison BS, Dugar B, Feldstein AE, Britt EB, Fu X,

Chung YM, Wu Y, Schauer P, Smith JD, Allayee H, Tang WH, DiDonato JA, Lusis AJ, Hazen SL. Nature 472: 57-65 (2011).

[2] Tang WH, Wang Z, Levison BS, Koeth RA, Britt EB, Fu X, Wu Y, Hazen SL. N. England J. Med. 368: 1575-1584 (2013).

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[OP-16]

BIPOLAR ELECTROCHEMISTRY/FLUORESCENCE DETECTOR FOR MICROCHIP ELECTROPHORESIS

Susan M. Lunte, Manjula Wijesinghe, Dulan Gunasekara and Indika Warnakula

University of Kansas, Lawrence, , Kansas, USA. E-mail: [email protected]

1.- ObjectivesAmperometric detection coupled to microchip electrophoresis has been used extensive-ly to detect electrochemically active species in biological samples. However, the detec-tion limits are usually in the micromolar range and, therefore, not adequate to quantify many biologically important analytes including reactive oxygen and nitrogen species at the single cell level. Optical detection techniques, such as fluorescence and chemi-luminescence, offer nanomolar-to-picomolar detection limits, but frequently require derivatization of an analyte for detection, which limits selectivity. To take advantage of the strengths of both approaches, a bipolar electrochemistry-based fluorescence detec-tion method has been developed by our group [1]. This work has been further modified to obtain the electrochemically generated fluorescence signal without the need for a potentiostat.

2.- MethodsThe experimental setup consists of a PDMS chip containing a simple-t (5 cm × 15 µm × 40 µm with side arms) for the microchip separations and a separate reporter channel (straight channel 5 cm × 15 µm × 40 µm without side arms) to flow the fluorescent probe. A 15-µm pyrolyzed photoresist film electrode on glass was aligned in the in-channel configuration with the simple-t chip at a distance of 15 µm from the channel end. For the flow channel, a 30 µm gold electrode was aligned to in-channel configuration at a distance of 500 µm from the channel end (Figure 1A). The two electrodes were connected by a Cu wire to create a single bipolar electrode (Figure 1B). The potentials at the ends of the bipolar electrode were determined by the field strengths applied to the separation and flow channels, obviating the need for a potentiostat. Fluorescence generated at the reporter channel was measured using an inverted microscope equipped with a PMT. A 488 nm cyan diode laser was used as the excitation source.

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Fig. 1: Bipolar fluorescence-based detection for microchip electrophoresis. (A) Schematic of the exper-imental system, (B) actual experimental setup, (C) bipolar electrogenerated fluorescence electrophero-

gram using a separation buffer consisting of pH 6.2, 15 mM phosphate buffer with 2 mM SDS.

3.- ResultsTwo model analytes, benzoquinone and resazurin, were used to demonstrate the success of the new method to separate and detect the electrochemically active analytes separated by ME. Figure 1C shows the bipolar fluorescence response generated for the two model analytes. The potential drop required to drive the electrochemical reduction reaction at the separation channel and the oxidation reaction at the reporting channel was provided by the separation voltages at the sample-t microchip and the voltages applied across the reporter channel; therefore, a potentiostat was not necessary.

4.- ConclusionsA method has been developed based on using a bipolar electrode to convert a reduction cur-rent response to fluorescence. The bipolar electrode potentials are determined by the combi-nation of the voltages applied across the separation channel and reporter channel, obviating the need for a potentiostat. Initial results indicate that at mid-nanomolar range the detection limits are achievable for both analytes using this approach.

Keywords: Electrochemical detection; Microchip electrophoresis; Bipolar electrochemistry; Reactive oxygen and nitrogen species.

Acknowledgements: This work has been sponsored by the National Science Foundation and NIH NIGMS P20 1036 38

References:[1] Wijesinghe MB, Gunasekara DB, Lunte SM. microTAS Proceedings 1292-1294 (2014).

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[OP-17]

CARBON BLACK BASED ELECTROCHEMICAL TRANSDUCERS FOR MICROFLUIDIC DETECTION

Flavio Della Pelle1, Daniel Rojas1,2, Michele Del Carlo1, Luis Vázquez3, Dario Compagnone1 and Alberto Escarpa2,4

1 Faculty of Bioscience and Technology for Food, Agriculture and Environment University of Teramo 64100, Teramo (Italy). Email: [email protected]

2 Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, Faculty of Sciences, University of Alcalá, Alcalá de Henares, Madrid, Spain

3 Institute of Materials Science of Madrid (CSIC), Madrid, Spain4 Chemical Research Institute “Andrés M. del Río”, University of Alcalá, Alcalá de

Henares, Madrid, Spain

1.- ObjectivesThis work aims to demonstrate that carbon black nanoparticles (CBNPs) realize effective and cheap transducers for the detection at the microscale level. In order to allow direct de-tection (without additional transducers) onto the CBNPs surface, as well as integration in a microchip electrophoresis platform, the transducers have been introduced onto poly(methyl methacrylate) (PMMA) supports using a press-transfer technology. Thus, the CBNPs-based transducer fabrication has been optimized and the resulting devices have been deeply char-acterized, in terms of both surface features and electrochemical performance (off-chip and on-chip). Finally, the fabricated devices have been implemented on board of microfluidic chips for amperometric determination of neurotransmitters and environmental organic con-taminants (pesticides), in order to prove the ability to solve real world analytical issues.

2.- MethodsPress-transferred CBNPs transducers fabrication main steps are [1]: a) CBNPs dispersion was filtered with an analytical stainless steel 13 mm vacuum filter holder, using a Teflon filter with a pore size of 0.1 mm; b) Teflon filters were cut out in 13x1 mm2 pieces to obtain a CB wire; c) the CB wire was placed on the PMMA support; d) the CBNPs were transferred onto the sheet by applying a pressure of 5.8±0.1 tons for 60 s. The Teflon filter was removed using tweezers; e) electrical contacts were made using conductive silver that was later electrically isolated using insulating paint. The glass chip was fabricated by Micronit Microfluidics (BV, NL) and consisted of a glass plate (88x16 mm2) with a four-way injection cross, a 74 mm long separation channel, and side arms measuring 5 mm long. The amperometric detector (end-channel detection) consisted of an Ag/AgCl wire as reference electrode, a platinum wire as counter electrode, and a CBNP transducer as working electrode.

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Fig. 1: Press-transferred carbon black nanoparticles film on board of microfluidic chip.

Amperometric detection, electrochemical impedance spectroscopy (EIS) and cyclic voltam-metry (CV) were performed by using a Potentiostat Autolab PGSTAT12 from Eco Chemie. A LabSmith HVS448 high-voltage sequencer with eight independent high-voltage channels and programmable sequencing for an entire level of voltage manipulation (Lab-Smith, Liv-ermore, CA) was used as the voltage source. The CBNPs-based press-transferred transducers were characterized by field-emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), current-sensing atomic force microscopy (CS-AFM), and Raman spec-troscopy. While transmission electron microscopy (TEM) and X-ray photoelectron spectros-copy (XPS) were used to investigate the CBNPs features.

3.- ResultsCarbon black nanoparticles (CBNPs) press-transferred film-based transducers revealed that the CBNPs films retained good nano CB conductivity, and a significant correlation between the morphology and the resistance were observed. Indeed, the amount of press-transferred CBNPs is the key parameter to obtain films with improved conductivity, which is in good agreement with the electrochemical response. In addition, the conductivity of such optimum films was not only Ohmic; in fact, tunneling/hopping contributions were observed [1]. The CBNPs film act as an exclusive electrochemical transducer, resulting to be an elective de-vice to be coupled with microchip-electrophoresis. Thus, the CBNPs film was employed as detector coupled to microfluidic chip confirming remarkable properties, offering low de-tection potentials, and negligible surface fouling, allowing to achieve enhanced analytical performance compared to commercial graphite electrodes. Finally, these conductive CBNP press-transferred films have been successfully applied for the transduction of target mole-cules, and excellent electrochemical performances have been obtained, despite the complex chemical media investigated [1-3]. In particular, neurotransmitters and pesticides, have been successfully analyzed in model solutions and real samples [1,2]. The proposed devices cou-pled to microchip-electrophoresis have proved to be an excellent approach to carry out a multiresidual screening analysis characterized by rapidity, low sample and reagent consump-tion, and low waste generation.

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4.- ConclusionsThe affordable press-transfer technology reported here allowed the design and control of the electrical properties of conductive CBNPs films. The obtained results demonstrated both the analytical potential of CBNPs as a novel alternative to other carbon nanomaterials, the solid establishment, and the suitability of the press-transfer technology and open novel ave-nues in microfluidics and others related micro- and nanochemistry applications. Moreover, in our perception, these results prove that the proposed strategies represent a viable tool for the analysis of complex real samples, for on-site environmental monitoring, and for rapid diagnosis. Moreover, preliminary results obtained by our group, have proved how the CB nano-hybridized films with transition metal dichalcogenides (2D-graphene like materials) show unique and useful properties. From this promising results, one of our research goals aim to exploit these features to fabricate new transducers for microchip-electrophoresis.

Keywords: Microchip; Electrophoresis; Nanocarbon; Pesticides.

References:[1] Della Pelle F, Vazquez L, Del Carlo M, Sergi M, Compagnone D, Escarpa A. Chemistry-A Eur. J.

22(36): 12761-12766 (2016).[2] Della Pelle F, Del Carlo M, Sergi M, Compagnone D, Escarpa A. Microchimica Acta, 183(12):

3143-3149 (2016).[3] Della Pelle F, Di Battista R, Vazquez L, Del Carlo M, Sergi M, Compagnone D, Escarpa A. Appl.

Materials Today, 9: 29-36 (2017).

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[OP-18]

FLUORESCENCE DETECTION OF PROTEINS IN CAPILLARY AND MICROCHIP ELECTROPHORESIS

Angel Puerta, Mercedes de Frutos and José Carlos Díez-Masa

Institute of Organic Chemistry (IQOG-CSIC), Madrid, Spain. Email: [email protected]

1.- Introduction and objectivesDeployment of Personal Medicine requires development of new biomarkers. These bio-markers, either image or molecular, should be sensitive, selective, and cost-effective. Protein analysis using miniaturized electrophoresis, both in capillary and in microchip formats, has potential to analyze complex mixtures of proteins in liquid biopsies, such as plasma, urine, saliva or cerebrospinal fluid.

However, one of the major limitations of miniaturized electrophoresis to this aim is sensi-tivity. Using UV detection, the limit of detection for proteins present in biological fluids is approximately10-6 M.

Induced fluorescence detection, either using lasers (LIF) or light emission diodes (LEDIF) as excitation light sources, is an attractive alternative. For natural samples, native fluorescence detection of proteins works well only in a limited number of cases. Therefore, derivatization of proteins with a fluorescent probe is the most common approach. Nevertheless, protein derivatization is not a trivial task due to the instability of most fluorescent reagents and to formation of multiple derivatization products of proteins. These drawbacks are particularly critical in biological fluids where many proteins are present in low concentration (< 10-6 M).

In our laboratory, we have used various high sensitivity methods for protein detection in capillary and microchip electrophoresis, both of which are useful in fields such as food and health. This communication presents a comparative study of these approaches.

2.- Results and discussionComparison of native detection of proteins using thermo-optical lenses, UV detection, and fluorescence detection for analysis of whey proteins are considered. Non-covalent derivat-ization of proteins could be very helpful for the study of hydrophobic proteins. However, most of the proteins of interest in food and health are rather hydrophilic, therefore covalent derivatization is the sole valid approach when high sensitivity is required. Using in-capillary covalent derivatization detection of subnanomolar concentration of proteins in milk samples can be achieved.

A particularly challenging case is the sensitive detection of glycoproteins when proteoform separation is required. For these proteins in-capillary fluorescent derivatization does not allow separation of proteoforms. For some of these glycoproteins in-vial covalent derivatization

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through thiol moiety can allow separation of isoforms of some glycoproteins, although high sensitivity is compromised.

3.- ConclusionsFrom these case-studies for the detection of proteins at low concentrations in capillary and chip electrophoresis some general guidelines can be concluded.

Keywords: Capillary Electrophoresis, CE, Microchip Capillary Electrophoresis, MCE, Light-Emitting Diode (LED), Fluorescence derivatization. Protein. Glycoprotein.

Acknowledgements: Authors thank the Comunidad of Madrid and European funding from FSE and FEDER programs for financial support (project S2018/BAA-4393, AVANSECAL-II-CM).

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[OP-19]

DETERMINATION OF GLYCOPROTEINS BY MICROCHIP ELECTROPHORESIS WITH ELECTROCHEMICAL DETECTION

Agustín G. Crevillen1, Tania Sierra2 and Alberto Escarpa2,3

1 Department of Analytical Sciences, Faculty of Sciences, Universidad Nacional de Educacion a Distancia (UNED), Madrid, Spain. E-mail: [email protected]

2 Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, University of Alcalá, E-28871 Alcalá de Henares, Madrid, Spain

3 Chemical Research Institute “Andrés M. del Río” (IQAR), University of Alcalá, E-28805 Alcalá de Henares, Madrid, Spain

1.- ObjectiveWe propose a methodology for the determination of glycoproteins by microchip electropho-resis with electrochemical detection (ME-ED). As proof of concept, Transferrin (Tf) and α-1-acid glycoprotein (AGP), both glycoproteins, were detected and quantified in serum samples.

Typically, the main detection modes applied in ME for protein determination are fluores-cence and MS [1]. Electrochemical detection (ED) has not been explored in spite of its inter-esting features such as easy miniaturization without losing analytical performance and lower cost, making this detection mode more adequate for developing point-of-care testing (POCT) systems [2]. ED has some disadvantages since as it is not as sensitive as fluorescence or MS detection and not all proteins are electroactive. However, these handicaps could be overcome by labeling the protein with an electroactive tag (in the same way as fluorescence detection) [3]. In this sense, Palecek´s group reported the electrochemical detection of glycoproteins by using an osmium (VI) complex as electrochemical probe [4].

2.- MethodsTransferrin (Tf) and α-1-acid glycoprotein (AGP) were labeled with an Os(VI) complex (electrochemical tag). Then both glycoproteins were separated and detected by ME-ED (see figure 1). The end-channel amperometric detector consisted of an Ag/AgCl electrode as a ref-erence electrode, a platinum wire as counter electrode, and a screen-printed carbon electrode as working electrode.

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Fig. 1: A) Reaction of glycoproteins with the electrochemical tag (Os(VI) complex). B) Microchip layout used for glycoprotein separation (SR: sample reservoir, RB: running buffer reservoir, SW: sample waste

reservoir, ED: electrochemical detection cell).

3.- ResultsHydrodynamic voltammograms of each glycoprotein (tagged and intact) revealed that oxi-dation potential of intact glycoproteins started at +0.60 V while tagged glycoproteins started at +0.50 V (vs Ag/AgCl). With the detection potential at +0.5 V, only tagged glycoproteins were detected (see figure 2), avoiding the interference of the rest of proteins. The ME-ED method was evaluated by analyzing Tf and AGP glycoproteins in certified human serum reference material. Both glycoproteins were determined with excellent accuracy (Er≤4%) in less than 400 s.

Fig. 2: Tagged glycoprotein (black line), intact proteins (red line). Conditions: 25 mM sodium tetraborate pH 9.2; separation voltage +3.00 kV; injection voltage = +3.00 kV for 20.0 s. Peaks: (1) Tf and (2) AGP

4.- ConclusionsFor the first time, glycoproteins were separated and detected by ME, using a Os(VI) complex tag-based electrochemistry. This tag binds selectively to glycans present in glycoproteins and the corresponding synthesized adduct yields an anodic signal at +0.50 V. Thus far, at this completely unexplored detection potential, there is not interference from other proteins.

This work opens the door to ME-ED for glycoprotein analysis which seemed to be reserved only for fluorescence and MS detection thereby exploiting the specific advantages of ED for POCT and bed side devices such as easy miniaturization and low cost.

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Keywords: glycoproteins, human serum, osmium (VI), microchip electrophoresis, amperometric detection, electrochemical tag

Acknowledgements: This work has been financially supported by the TRANSNANOAVANSENS program from the Community of Madrid (P2018/NMT-4349), Spanish Ministry of Economy, In-dustry and Competitiveness (CTQ2017-86441-C2-1-R) and MINECO (MAT2016-80394-R).T.S. acknowledges the FPI fellowship from the University of Alcalá.

References:[1] Štěpánová S, Kašička V. J. Sep. Sci., 42: 398-414 (2019).[2] Gencoglu A, Minerick AR. Microfluid. Nanofluidics. 17: 781-807 (2014).[3] Sierra T, Crevillen AG, and Escarpa A. Electrophoresis, 38: 2695-2703 (2017).[4] Trefulka M, Paleček E. Electrochem. Commun. 48: 52-55 (2014).

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[OP-20]

ANALYTICAL METROLOGY FOR NANOMATERIALS: THE ROLE OF CAPILLARY ELECTROPHORESIS

AND RELATED TECHNIQUES

Ángel Ríos

Department of Analytical Chemistry and Food Technology. University of Castilla – La Mancha, Ciudad Real, Spain. Email: [email protected]

1.- ObjectivesUnderline the current challenge presented by analytical metrology applied to nanomaterials and the role that Capillary Electrophoresis and related techniques can play.

2.- AbstractNanoscience and Nanotechnology (N&N) have had a deep impact in Analytical Chemistry [1]. On the one hand, analytical chemists welcome the challenge and opportunities that N&N offers in this area There are powerful nanotools to improve analytical performances of results of analytical processes and analysis of the nanoworld. On the other hand, the basic (Nano-science) and applied (Nanotechnology) developments and achievements need information from the nanoworld to to fulfill their respective objectives and to make founded and timely decisions. In this last way, the determination of nanomaterials (nanoparticles in many cases), in specific types of samples is a recognized challenge in today’s analytical science.

Analytical metrology for nanomaterials (AMNM) merges as the metrology applied to na-nomaterials for analytical purposes. It has two different facets, not mutually exclusive [2]:

(i) The characterization of NMs by themselves. This is the situation for companies/labora-tories producing NMs for uses in different fields (industrial, cosmetics, foods, etc.). This characterization is involved in the quality control of NMs production, and from this point of view, NMs are considered as target samples. Here, the objective is to obtain reliable in-formation about the characteristics of NMs, which is a key aspect to establish reproducible preparation schemes for instance, and hence to produce reproducible nanomaterials between different batches.

(ii) The determination of NMs in particular types of samples. This aspect can be seen follow-ing two different approaches. The first one is by applying a full analytical process in order to obtain a detailed and complete information. The second one is by applying a screening method to give a rapid information about the presence (or not) of the specific NMs in particu-lar types of samples. In fact, this is a qualitative method for the classification of the samples (presence/absence of NMs at a present threshold).

For these objectives hydrodynamic instrumental separation techniques can be very useful. Thus, capillary electrophoresis offers a wide variety of analytical alternatives [2,3], some of

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which are summarized in this communication. Other related techniques, such as Hydrody-namic Chromatography and Field Flow Fractionation [4], offer complementary alternatives for the analysis and determination of nanoparticles. In any case, some bottlenecks appear when these methodologies are used to solve true analytical problem in control laboratories. All of these aspects will be pointed out in this presentation.

Keywords: Nanomaterials; Analytical metrology; Capillary Electrophoresis, Separation instrumen-tal techniques.

Acknowledgements: Support for this work was provided by the projects MINECO CTQ-2016-78793-P (The Spanish Ministry of Economy and Competitiveness) and SBPLY/17/180501/000262 (Junta de Comunidades de Castilla-La Mancha, Spain).

References:[1] Soriano ML, Zougagh M, Valcarcel M, Rios A. Talanta 177: 104-121 (2018).[2] Lopez-Sanz S, Guzman FJ, Rodriguez Martin-Doimeadios RC, Rios A. Anal. Chim. Acta, 1059:

1-15 (2019).[3] Adelantado C, Rodriguez N, Rodriguez RC, Zougagh M, Rios A. Anal. Chim. Acta 923: 82-88

(2016).[4] Lopez-Sanz S, Rodriguez-Farias N, Rodriguez Martin-Doimeadios RC, Rios A. Anal. Chim. Acta

1053: 178-185 (2019).

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[OP-21]

CHIRAL IONIC LIQUIDS AS SYNERGISTIC SELECTORS FOR ENANTIOMERIC SEPARATIONS

BY CAPILLARY ELECTROPHORESIS

María Castro-Puyana1,2, Maider Greño1, Sandra Salido-Fortuna1 and María Luisa Marina1,2

1 Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, University of Alcalá, Alcalá de Henares, Madrid, Spain. Email: [email protected]

2 Instituto de Investigacion Quimica Andrés M. del Río (IQAR), University of Alcalá, Alcalá de Henares, Madrid, Spain

1.- Objectives Nowadays, ionic liquids are successfully applied in all branches of chemistry, including an-alytical separation techniques. Chiral ionic liquids (CILs) are a subtype of ionic liquids that have a chiral moiety. Despite the properties of CILs that make them particularly attractive to achieve enantiomeric separations, their use in CE is still in its infancy. In this presentation, their potential to be used in Capillary Electrophoresis (CE) as the sole chiral selectors or combined with neutral cyclodextrins (CDs) in dual chiral systems will be shown.

2.- MethodsVarious chiral methodologies were developed by CE to evaluate the discrimination power of different CILs and CDs as sole chiral selectors or combined in dual systems to achieve the enantiomeric separation of multiple compounds including non-protein amino acids and drugs.

3.- ResultsA synergistic effect was observed between CILs and CDs. In addition, the combination of both selectors caused in some cases, the reversal of the enantiomer migration order with re-spect to that observed when either selector was used in single a chiral systems.

4.- ConclusionsCILs possess great potential to be employed as synergistic selectors in CE to achieve the enantiomeric separation of chiral compounds. However, further investigations are needed not only to exploit their full potential of CILs as chiral selectors in CE but also to elucidate their precise role during separation.

Keywords: Chiral ionic liquids, cyclodextrins, dual systems, enantioseparation.

Acknowledgements: Authors thank the Spanish Ministry of Economy and Competitive-ness (MINECO) for project CTQ2016-76368-P and the Comunidad of Madrid and Eu-ropean funding from FSE and FEDER programs for project S2018/BAA-4393 (AVAN-SECAL-II-CM). M.C.P. thanks MINECO for her “Ramón y Cajal” research contract

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(RYC-2013-12688). M. G. thanks the Spanish Ministry of Science, Innovation and Uni-versities for her pre-doctoral contract FPU17/01635. Authors thank the Center for Applied Chemistry and Biotechnology (CQAB) (University of Alcalá) for the synthesis of CILs.

References:[1] Greno M, Marina ML, Castro-Puyana M. J. Chromatogr. A 1568: 222-228 (2018).[2] Greno M, Salgado A, Castro-Puyana M, Marina ML. Electrophoresis, DOI: 10.1002/

elps.201800483.

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[OP-22]

ENANTIOSELECTIVE STUDY OF DRUG BIODEGRADATION BY CHIRAL CAPILLARY ELECTROPHORESIS

Yolanda Martín-Biosca1, L. Escuder-Gilabert1, S. Sagrado1,2 and M.J. Medina-Hernández1

1 Departamento de Química Analítica, Universitat de València, Burjassot, Spain. Email: Yolanda.martin @uv.es

2 Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM). Universitat Politècnica de València, Universitat de València, Valencia, Spain

1.- ObjectivesThe enantioselectivity in ecotoxicity, biodegradation processes and environmental fate have to be considered to provide realistic risk assessment of chiral compounds [1]. The main objective of this communication is to study the enantioselectivity for the biodegradation process of two common chiral drugs, citalopram and verapamil, using highly sulphated cy-clodextrins as chiral selectors in capillary electrophoresis (CE) [2,3].

2.- MethodsBiodegradation experiments were performed in batch mode using a minimal salts medi-um inoculated with an activated sludge (collected from a Valencian Waste Water Treat-ment Plant) and supplemented with the corresponding enantiomeric mixture. The cultures were incubated at 20ºC for 28 days. Abiotic degradation of verapamil and citalopram enantiomers exposed to light and in the dark was also assessed. The concentration of the enantiomers of verapamil and citalopram were monitored using 0.7 % (w/V) HS-β-CD and 0.1 % (w/V) S-γ-CD solutions as chiral selectors, respectively. Separations were car-ried out using the complete filling technique.

3.- ResultsFor verapamil, the degradation reached values between 63-69%, both in biotic and abiotic assays. Biodegradation predominated over the physicochemical degradation during the first five days of the assay. From day 5, the physicochemical degradation became more important. No differences were observed in the behavior of either enantiomers. In the case of citalo-pram, neither biodegradation nor physical-chemical degradation were significant, with the values during the whole assay being below 10%. Likewise for verapamil, no degradation differences were observed for either enantiomers.

4.- Conclusions

The use of highly sulphated cyclodextrins as chiral selectors in CE employing the complete filling technique, enables the adequate separation and determination of the enantiomers of verapamil and citalopram. The results of the biodegradation studies indicate that verapa-mil could be considered as a non-persistent compound, whereas citalopram is considered

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as potentially persistent in the environment. In either case, no evidences of enantioselective degradation was observed.

Keywords: Biodegradation; Enantioselectivity; Chiral capillary electrophoresis

Acknowledgements: This work has been supported by the Project CTQ2015-70904-R (MINECO/FEDER, UE).

References:[1] Kasprzyk-Hordern B. Chem. Soc. Rev. 39: 4466-4503 (2010).[2] Escuder-Gilabert L, Martin-Biosca Y, Medina-Hernandez MJ, Sagrado S. J. Chromatogr. A 1363:

331-337 (2014).[3] Escuder-Gilabert L, Martin-Biosca Y, Medina-Hernandez MJ, Sagrado S. J. Chromatogr. A 1467:

391-399 (2016).

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[OP-23]

USE OF WHOLE ELECTROPHORETIC PROFILES AND CHEMOMETRIC TOOLS FOR THE DIFFERENTIATION

OF THREE OLIVE OIL QUALITIES USING THE COMPOUNDS EXTRACTED BY LIQUID-LIQUID EXTRACTION

AND SUPERCRITICAL FLUID EXTRACTION

Natividad Jurado-Campos1, Laura Valbuena1, Rocío Rodríguez-Gómez1, Natalia Arroyo-Manzanares2 and Lourdes Arce1

1 Department of Analytical Chemistry. Institute of Fine Chemistry and Nanochemistry, University of Córdoba, Campus de Rabanales, Marie Curie Annex Building, E-14071 Córdoba, Spain. Email: [email protected]

2 Department of Analytical Chemistry, Faculty of Chemistry, Regional Campus of International Excellence “Campus Mare-Nostrum”, University of Murcia, Murcia, Spain. Email: [email protected]

1.- Objectives

The differentiation of virgin olive oils of grades extra (EVOO), virgin (VOO) and lampante (LOO) is currently carried out by expert panel tests through an organoleptic evaluation of fruity aroma and defects, such as fustiness, rancidity, winey, and musty [1]. Authentication of olive oil is a matter of great importance due to due to the price differences between grades, what might result in a higher profit if the oils are classified having a higher grade or vice versa.

In this work, an analytical methodology, to support the panel test activity, based on analysis of virgin olive oils extracts using capillary electrophoresis coupled to ultraviolet detector (CE-UV) is proposed. In addition, two different sample treatments were explored and compared: liquid-liquid extraction (LLE) and supercritical fluid extraction (SFE). In order to obtain the greatest success in the classification of the oil, data were processed using chemometric tools.

2.- MethodsThe LLE procedure was based on the previous work of Arroyo-Manzanares et al. [2]. It con-sisted of weighing 1 g of the tempered oil in a 5 mL Eppendorf tube. Then, 1 mL of hexane was added to remove fats and 950 µL of a mixture of methanol/water (1:1, v/v) was added as extraction solvent. In addition, 50 µL of two internal standards (naphthol and benzoic acid in methanol/water (1:1 v/v) at 400 mg·L-1) were added. After mixing by hand and vortexing for 30s, the two phases were separated after 15 min. The upper fatty phase was removed, and the lower aqueous phase was transferred (300 µL) to a vial with insert for CE analysis.

The SFE procedure was previously optimized using an experimental design (a Box-Behnken design, involving 15 runs). Three factors were considered, and they were studied in the fol-lowing ranges: percentage of methanol (5-20 %) used as modifier of polarity of supercritical

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CO2, time of extraction (15-45 min) and pressure (10-20 MPa). As response variables, the peak areas of ferulic and vanillic acid were considered. Both compounds have previously been described in olive oils samples. For this purpose, a volume of 300 µL of oil was fortified for both standards at 400 mg L-1 and placed over a filter paper. The filter paper was placed into the vessel of sample to carry out the extraction procedure. The optimum conditions for the maximum efficiency of the extraction were as follows: 5% of methanol, 30 min as time of extraction and 20 MPa as working pressure.

The CE-UV analysis were carried out using also the method described by Arroyo-Man-zanares et al. [2]. The buffer was 45 mM sodium tetraborate at pH 9.0 and the sample was injected using hydrodynamic mode at 0.5 psi for 8 s. The electrophoretic separation was carried out at 20 kV and 25ºC being the average current value 50 µA. Electropherograms were recorded at 200 nm, using normal polarity. Before each extract analysis, the capillary was flushed with 0.1 M NaOH (1 min), milli-Q H2O (1 min) and separation buffer (2 min) as conditioning procedure.

To process the data, two alignments and a baseline correction were carried out. The corrected electropherograms were used to obtain chemometric models based on Principal Component Analysis (PCA), Linear Discriminant Analysis (LDA) and K-Nearest Neighbors (KNN) [2] for olive oils classification. For this data processing, Matlab (The MathWorks, Natick, MA, USA 2002) and PLS Toolbox 5.5 (Eigenvector Research, Inc., Manson, WA, USA) software were used.

3.- ResultsA total of 60 virgin olive oils samples (20 EVOO, 20 VOO and 20 LOO) were analyzed in duplicate using SFE-CE-UV and LLE-CE-UV.Extra-non Extra binary models (to differentiate olive oil with and without defects) and Lampante-non Lampante binary models (to differentiate inedible and edible olive oil), as well as, ternary models (to differentiate between the three categories) were created. These results expressed as classification success rate are included in the Table 1.

Table 1: Success rate for the classification of oil samples.

Sample treatment

Extra-non Extra model

Lampante-non Lampante model

Ternary model

SFE 98.2 99.1 96.4LLE 99.1 99.1 99.1

4.- ConclusionsBoth sample treatments, SFE and LLE, have shown successful results and could be em-ployed to support the panel test. However, LLE is a simpler and faster technique that allows to work with several samples in parallel. For this reason, the procedure LLE-CE-UV would be more appropriate to carry out the authentication of olive oil.

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Keywords: capillary electrophoresis; supercritical fluid extraction; liquid-liquid extraction, olive oil authentication, chemometrics.

Acknowledgements: The authors acknowledge financial support from the Government of Spain (Ministerio de Economía y Competitividad) and from Interprofesional del aceite de oliva español under the framework of the Innolivar project. N.J.C. wishes to thank the Spanish Ministry of Educa-tion, Culture and Sport for award of a pre-doctoral grant (FPU15/00639).

References:[1] Commission Regulation (EEC) No 2568/91 of 11 July 1991 on the characteristics of olive oil and

olive-residue oil and on the relevant methods of analysis. Official Journal, L 248, 1-83.[2] Arroyo-Manzanares N, Gabriel F, Carpio A, Arce L. Talanta 197: 175-180 (2019).

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[OP-24]

ASSESSING CHIRALITY AND CONFORMATION BY GAS-PHASE ELECTROPHORESIS

Govert W. Somsen1, Raquel Pérez-Míguez2, Robert L.C. Voeten1,3, Maria Castro-Puyana2, Maria Luisa Marina2, Elena Domínguez-Vega1,4 and Rob Haselberg1

1 Division of Bioanalytical Chemistry, Amsterdam Institute for Molecules, Medicines and Systems, Vrije Universiteit, Amsterdam, The Netherlands. E-mail: [email protected]

2 Department of Analytical Chemistry, Faculty of Sciences, University of Alcalá, Alcalá de Henares, Spain

3 TI-COAST, Amsterdam, The Netherlands4 Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden,

The Netherlands

1.- ObjectivesStudy the feasibility of trapped ion mobility spectrometry coupled to quadrupole time-of-flight mass spectrometry (TIMS-QTOFMS) for revealing differences in three-dimensional arrangements of small molecules, polymers and proteins.

2.- MethodsTIMS is a recently developed mode of ‘gas-phase electrophoresis’. In TIMS a DC elec-tric field gradient is applied to hold ions stationary in a constant flow of drift gas. Ions are eluted from the TIMS analyzer by reducing the analytical field strength over time, yielding IM-based separations of relatively high resolution. IM is inversely proportional to the ion’s collision cross section (CCS), which depends on the size and shape of the gas-phase ion. TIMS is readily combined with (tandem) MS, allowing structural assignment of IM-sepa-rated species.

3.- ResultsIM-based resolution of enantiomers of amino acids was achieved by forming diastere-omers through derivatization with a chiral agent. Positive electrospray ionization (ESI) yielded sodiated diastereomers which were separated efficiently by TIMS [1]. TIMS-QTOFMS also demonstrated suitability for distinguishing and elucidating isomeric branched polyester oligomers. Recorded mobilograms revealed multiple peaks per oli-gomer mass indicating the presence of conformers, which could be assigned to branching variants. In addition, we studied the detection and resolution of protein conformational states by TIMS. Experimental settings maintaining native protein structures (i.e. prevent-ing voltage-induced unfolding) were assessed. TIMS data of individual charge states of model proteins showed coexistence of different protein conformations as function of pH, revealing protein unfolding in solution.

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4.- ConclusionsTIMS facilitates rapid separation of isobaric compounds (both small and large molecules) of different structure and conformation.

Keywords: Trapped ion mobility spectrometry; chiral separation; amino acids; conformation; polymers; proteins.

References:[1] Perez-Miguez R, Bruyneel B, Castro-Puyana M, Marina ML, Somsen GW, Dominguez-Vega E.

Anal. Chem. 91(5): 3277-3285 (2019).

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[OP-25]

INTEGRATING NUCLEIC ACID EXTRACTION WITH MICROSCALE ANALYSIS: ONE STEP CLOSER TO

DEMOCRATIZING MOLECULAR BIOLOGY

Jeff Chapman1, David Saul2 and Jo Stanton3

1 MicroGEM International, South Hampton, United Kingdom. Email: [email protected] MicroGEM International, Hamilton, New Zealand3 University of Otago, Dunedin, New Zealand

For more than 30 years we have been discussing the potential for microscale scale techniques like capillary electrophoresis, to democratize molecular analysis, allowing complex assays to be moved from the hands of a few into the hands of many. With cloud-based computing, the use of machine-based learning, and with our ability to encode data interpretation into algo-rithms, the scientific community has made significant strides towards this goal. Furthermore, with advanced microfluidics designs, and online enrichment and amplification techniques, devices have been developed that substantially reduce the footprint of analytical technology, making personal portable devices well within reach. Yet there continues to be a missing link with sample preparation, in that depending upon the complexity of your sample, a labora-tory and trained personnel are still required to prepare the sample for the analytical device. We all know too well the adage “Garbage In – Garbage Out” with regards to proper sample preparation. Molecular Biology has provided us with powerful assays for food authentica-tion, human identification, pathogen detection/quantitation, disease diagnosis and improved therapeutics through precision medicine. Yet, in most cases the data quality of these assays’ rests squarely on the quality of the input DNA, and the methods currently used to extract it, are time consuming and requiring of both a laboratory and technical expertise.

With this work, we present a radically different approach to DNA extraction compared with current laboratory methods. We have developed an extraction chemistry that uses a novel cocktail of thermophilic proteinases and mesophilic cell wall degrading enzymes that sys-tematically lyse cells, destroy nucleases, digest proteins and release nucleic acids. We have coupled this with an extractor cartridge, made from thermo-responsive polymers, to facilitate both extraction and removal of enzymatic inhibitors. The temperature-controlled extraction is performed in a chamber at the top of the cartridge. The temperature used to activate the proteinase also leads to a pressure increase in the closed tube and shrinkage of the chamber, forcing the extract through a heat-burstable valve and through a purification media located at the bottom of the cartridge. The purification segment removes debris, as well as inhibitory polyphenols and polysaccharides. This is accomplished with only a change in temperature, allowing for a device with no moving parts. This single-step closed system approach, allows for a rapid, hands-free preparation of DNA without the danger of cross-contamination. To test the portability and transferability of this approach, our collaborators deployed this ex-traction cartridge with farmers in Uganda, Tanzania and Kenyan for the detection of Cassava

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Mosaic Virus in Cassava root, leaves and from a single white fly shown to be carrying this vector. The virus was identified using nanopore sequencing, with read lengths as long as 276,793 bp. Further, I will provide an update on our progress with integrating this cartridge with electrophoresis for rapid human identification and with QPCR with the objective to create a fully integrated hand-held molecular diagnostic device.

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[OP-26]

CE-MS AS A USEFUL TOOL IN THE CONTROL OF CHEMICAL HAZARDS IN FOODS: SOME SELECTED EXAMPLES

Ana M. García-Campaña, Francisco J. Lara, David Moreno-González, Carmen Tejada-Casado, Maykel Hernández-Mesa,

Laura Gámiz-Gracia and Monsalud del Olmo-Iruela

Department of Analytical Chemistry, Faculty of Sciences, Campus Fuentenueva, University of Granada, 18071 Granada, Spain. Email: [email protected]

1.- ObjectivesThe global concern about food safety and environmental quality has led to the development of a legal framework to control residues of organic compounds (e.g. pesticides and veterinary drugs). In this context, the establishment of analytical methods achieving satisfactory per-formance in terms of sensitivity, identification, sample throughput and applicability is man-datory. Multiresidue methods based on chromatographic techniques have been extensively applied, while sample preparation has evolved through the development of miniaturized and environmentally friendly procedures according to Green Chemistry principles. In this way, capillary electrophoresis (CE), mainly coupled with mass spectrometry (MS), emerges as an efficient alternative, due to the development of off-line, on-line and in-line preconcentration strategies to overcome its main limitation, i.e., the lack of sensitivity inherent to the use of small sample volume and narrow bore capillaries[1,2].

2.- MethodsIn this communication, we show the potential of CE for the monitoring of residues of im-portant compounds widely used in agriculture and animal husbandry, such as antibiotics, anthelmintics and pesticides. Different techniques have been applied. The first involves the construction of in-line analyte concentrators using solid-phase extraction sorbents to increase sensitivity with minimum sample pretreatment [3]. The second employs a volatile surfactant, ammonium perfluorooctanoate, that is compatible with MS in order to separate neutral spe-cies by micellar electrokinetic chromatography (MEKC). This surfactant prevents analyte signal suppression and contamination of the mass spectrometer4. In addition, CE has shown to be an excellent choice for the analysis of highly polar compounds, avoiding some of the drawbacks often found in their determination by liquid chromatography (LC), such as low retention factors or poor peak shapes.

3.- ResultsThe combination of CE with miniaturized sample pretreatments such as dispersive liquid-liq-uid microextraction5, or the use of selective methodologies based on molecularly imprinted polymers6 make the proposed methods useful for routine analysis. These analytical meth-odologies have been applied to monitor some relevant chemical hazards (aminoglycosides, fluoroquinolones, benzimidazoles, etc.), in different foods such as meat, milk, honey, etc. The developed methods were validated in terms of limits of detection and quantification,

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linear range, matrix effect, precision and trueness, according to the EU regulation, demon-strating their satisfactory sensitivity and accuracy for the analysis of real samples.

4.- ConclusionsThe different examples presented in this communication show that CE coupled with MS can be an efficient alternative to LC in food safety. This is of special interest in those cases where chromatography has found some limitations such as the determination of highly polar or ionic residues and contaminants. In this respect, further developments in food safety based on CE could be expected in the near future.

Keywords: Capillary electrophoresis, mass spectrometry, chemical residues, sample treatments

Acknowledgements: This work has been sponsored by the financial support of the Spanish Ministry of Economy and Competitiveness (Projects ref: AGL2015-70708-R and RTI2018-097043-B-I00, co-financed with FEDER funds). FJL is grateful for personal funding through the Special Research Program of the University of Granada.

References:[1] Lara FJ, Moreno-Gonzalez D, Hernandez-Mesa M, Garcia-Campana AM. In: Food Safety

Applications of Capillary Electromigration Methods. Colin F. Poole (Ed). Elsevier, (Amsterdam), pp. 511-545 (2018).

[2] Hernandez-Mesa M, Moreno-Gonzalez D, Lara FJ, Garcia-Campana AM. In: Capillary Electrophoresis. Food Chemistry Applications. Chemistry, Molecular Sciences and Chemical Engineering. Jan Reedijk (Ed). Elsevier, (Amsterdam), pp. 358-366 (2019).

[3] Moreno-Gonzalez D, Lara FJ, Gamiz-Gracia L, Garcia-Campana AM. J. Chromatogr. A 1360: 1-8 (2014).

[4] Moreno-Gonzalez D, Gamiz-Gracia L, Bosque-Sendra JM, Garcia-Campana AM. J. Chromatogr. A 1247: 26-34 (2012).

[5] Tejada-Casado C, Moreno-Gonzalez D, Lara FJ, Garcia-Campana AM, del Olmo-Iruela M. J. Chromatogr. A 1490: 212-219, (2017).

[6] Moreno-Gonzalez D, Lara FJ, Jurgovska N, Gamiz-Gracia L, Garcia-Campana AM. Anal. Chim. Acta 891: 321-328 (2015).

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[OP-27]

APTAMER AFFINITY SORBENTS FOR THE ANALYSIS OF PROTEIN BIOMARKERS IN BIOLOGICAL FLUIDS

BY ON-LINE SOLID-PHASE EXTRACTION CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY

Fernando Benavente1, Roger Pero-Gascon1, Maxim V. Berezovski2 and Victoria Sanz-Nebot1

1 Department of Chemical Engineering and Analytical Chemistry, Institute for Research on Nutrition and Food Safety (INSA·UB), University of Barcelona, Barcelona, Spain. Email: [email protected]

2 Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario, Canada

1.- ObjectivesIn this study, on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS) is described for the purification, preconcentration, separa-tion, and characterization of protein biomarkers in biological fluids. It is presented as a case study the analysis of α-synuclein (α-syn) in blood. α-Syn is a major component of Lewy bodies (LB), the typical aggregated protein deposits found in Parkinson’s disease (PD).

2.- MethodsA single-stranded DNA aptamer sorbent is used to bind with high affinity and selectivi-ty α-syn. A fritless particle-packed microcartridge is integrated in-line near the entrance of the separation capillary and no valves are necessary for the operation in AA-SPE-CE-MS. After loading a large volume of sample (∼50-100 μL), the capillary is rinsed to eliminate non-retained molecules and filled with background electrolyte (BGE). Then, the analyte is preconcentrated eluting in a small volume of an appropriate solution (~25-50 nL) before the separation and the detection by mass spectrometry [1].

3.- ResultsUnder the optimized conditions with standards, repeatability (2.1 and 5.4% percent relative standard deviation (%RSD) for migration times and peak areas) and microcartridge lifetime (around 20 analyses/microcartridge) were good. The method was linear between 0.5 and 10 µg·mL-1 and a limit of detection (LOD) of 0.2 µg·mL-1 (100 times lower than by CE-MS, 20 µg·mL-1) was achieved. The method was applied to the analysis of endogenous α-syn from red blood cells (RBCs) lysates of healthy controls and PD patients.

4.- ConclusionsIn view of these results, their high affinity and selectivity and improved tolerance to acidic and basic conditions, aptamers can be regarded as a powerful alternative to antibodies for targeted analysis of biomarkers by SPE-CE-MS.

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Keywords: aptamer; capillary electrophoresis; mass spectrometry; in-line solid-phase extraction; on-line solid-phase extraction; α-synuclein

Acknowledgements: This work was supported by projects CTQ2014-56777-R and RTI2018-097411-B-I00 (Ministry of Economy and Competitiveness, Spain) and the Cathedra UB Rector Francisco Buscarons Úbeda (Forensic Chemistry and Chemical Engineering).

References: [1] Pont L, Pero-Gascon R, Gimenez E, Sanz-Nebot V, Benavente F. Anal. Chim. Acta 10.1016/j.

aca.2019.05.022 (2019-in press).

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[OP-28]

DEEP N-GLYCOMIC ANALYSIS OF HUMAN SERUM BY CAPILLARY ELECTROPHORESIS AND CESI-MS:

THE CHALLENGE OF SAMPLE PREPARATION

Andras Guttman1 and Marton Szigeti2

1 Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Hungary. Email: [email protected]

2 Horvath Csaba Memorial Laboratory of Bioseparation Sciences, University of Debrecen, Hungary

1.- ObjectivesScale up of the sample preparation of human serum to increase the dynamic range of N-gly-comics analysis is hindered by precipitation during the denaturation step, that otherwise is necessary for the endoglycosidase enzymes to reach their consensus sequence and release the glycans. In addition, the large amount of small human serum sugars, e.g., glucose, may inhibit the enzymatic carbohydrate release reaction.

2.- Methods Considering these two issues, in this paper we introduce an efficient sample preparation workflow to facilitate deep N-glycomics analysis of the human serum by capillary electro-phoresis with laser induced fluorescence (CE-LIF) detection and to accommodate the higher sample concentration requirement of electrospray ionization mass spectrometry connected to capillary electrophoresis (CE-ESI-MS).

3.- ResultsA novel, temperature gradient denaturing protocol was applied on amine functionalized mag-netic bead partitioned glycoproteins. During this process, the high amount of the low degree of polymerization sugar content of the human serum was significantly decreased, accommo-dating the PNGase F mediated release of the N-linked carbohydrates. The liberated oligosac-charides were tagged with aminopyrene-trisulfonate, utilizing a modified evaporative labeling protocol.

4.- ConclusionsProcessing the samples with this new workflow enabled deep CE-LIF analysis of the human serum N-glycome and provided the appropriate amount of material for CE-ESI-MS analysis.

Keywords: capillary electrophoresis, endoglycosidase digestion, fluorophore labeling

Acknowledgements: The authors gratefully acknowledge the support of BIONANO_GI-NOP-2.3.2-15-2016-00017 project.[

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[OP-29]

FOODOMICS: TEN YEARS ON THE ROAD

Gerardo Álvarez-Rivera, Diego Ballesteros-Vivas, Alberto Valdés, Zully Suárez-Montenegro, J. David Sánchez-Martínez, José A. Mendiola,

Miguel Herrero, Elena Ibáñez and Alejandro Cifuentes

Foodomics Laboratory, CIAL, CSIC, Madrid, Spain. Email: [email protected]

In the last ten years, the main line of research in our lab has focused on obtaining bioac-tive compounds using green pressurized extraction. These were subjected to a Foodomics evaluation of their activities against different high incidence diseases as colon cancer and Alzheimer’s disease. In this work, we will present some selected results on these topics in-cluding results from:

a) the development of new green extraction processes to obtain bioactive compounds from different natural sources (algae, microalgae, food by-products, plants, etc.);

b) the determination of the bioactivity of the new extracts against different colon cancer in-vitro and in-vivo models and Alzheimer in-vitro models;

c) the development of advanced analytical approaches including metabolomics profiling based on LC-MS, CE-MS, GC-MS and comprehensive LCxLC-MS/MS for the chemical characterization of the bioactive extracts;

d) the identification of genes, proteins and metabolites differentially expressed in cancer cells using whole-transcriptome microarrays, nano-LC-MS for proteomics and/or non-targeted whole-metabolome approaches based on LC-MS and CE-MS and;

e) the development of algorithms for the analysis of these MS-based datasets.

This work is an excellent example of the important challenges that still have to be addressed by Foodomics to solve the binomial Food & Health dependency and it will allow us to dis-cuss some of the current and future challenges in this area of research.

Keywords: Colon cancer, Alzheimer, Foodomics, Bioactive compounds, green extraction.

Acknowledgements: This work has been sponsored by AGL2017-89417-R and ABACUS-H2020 projects.




Flash Communications

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[FC-1]

DEVELOPMENT OF A SELF-PUMPING MICROFLUIDIC HERRINGBONE DEVICE FOR PESTICIDE ANALYSIS

Daniel B. Carrão1,2, Ruth F. Menger2, Wei Wang3, Rob B. Channon2, Anderson R. M. de Oliveira1, Arun K. Kota3 and Charles S. Henry2,3

1 Departamento de Química, Laboratório de Metabolismo In Vitro e Técnicas de Separação, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil. Email: [email protected]

2 Department of Chemistry, Colorado State University, Fort Collins, Colorado, USA3 Department of Mechanical Engineering, Colorado State University, Fort Collins,

Colorado, USA

1. ObjectivesThe increasing world population in the last decades has increased the demand for food but global food production loss due to pests can be up to 80% [1]. In order to improve food qual-ity and productivity, pesticides are commonly applied [2]. Unfortunately estimates indicate that less than 1% of applied pesticides reach the target pests with the majority pesticides con-taminating the surrounding food, soil, water and air. Human exposure to pesticides through contaminated food and/or the environment is a safety concern, since even trace amounts of pesticides can cause human health issues such as infant mortality, Alzheimer’s disease, carcinogenicity, neurotoxicity, reproductive toxicity, metabolic toxicity, among others [3]. Multiple traditional analytical techniques are employed for pesticide analysis in real sam-ples. However, few of them allow on-site analysis. Microfluidic analytical devices are a new trend in analytical methods. Their advantages include rapid analysis, on-chip detection, low sample volume, low cost, and analysis on-site [4]. The present project aims to develop a new microfluidic analytical device for the analysis of pesticides on-site. The goal was to keep the fabrication as simple as possible to keep device costs low for future applications. In this work, we propose a self-pumping microfluidic herringbone device that automates reagent mixing using passive pumping to perform on-site pesticide detection. The fabrication, charac-terization and proof of concept of this microfluidic device is described.

2. MethodsThe self-pumping microfluidic herringbone device was fabricated using two glass slides, double-sided adhesive tape and a laser cutter. First, grooves in the shape of herringbones were etched into both glass slides. Then, double-sided adhesive tape was used to define the microfluidic channels and join both glass slides (Fig. 1). To evaluate the extent of mixing in the device, yellow and blue dye was used. The formation of green color was measured using ImageJ image processing software.

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Fig. 1: Fabrication schematic of self-pumping microfluidic herringbone device

3. ResultsGrooves in the shape of herringbones are etched into the substrate with a laser cutter, making fabrication of the device simple and fast (10 minutes) compared to conventional methods of photolithography [5]. The herringbone grooves act as a chaotic mixer, causing turbulent flow that accomplishes fast mixing of small volumes (30 µL) [5]. A microfluidic channel formed by two hydrophilic double-sided adhesive generates fast flow (1 cm/s). Complete mixing was shown using yellow and blue dye (Fig. 2a). As a proof of concept, two reactions were successfully performed using the device: i) nickel and dimethylglyoxime and ii) horseradish peroxidase reaction (Fig. 2b-c). To our knowledge, this is the first report of a herringbone mixer in a self-pumping microfluidic device.

Fig. 2: a) Complete mixing of yellow and blue dye. b) Nickel and dimethylglyoxime reaction. c) Horseradish peroxidase reaction.

4. ConclusionsThe fabrication and the characterization of the self-pumping microfluidic herringbone de-vice, as well as proof of concept reactions were reported. In the future, this device will be used to analyze organophosphorus pesticides by a colorimetric enzymatic inhibition assay. The assay is based on the catalytic conversion of indoxyl acetate (colorless) into blue-colored indigo dipolymer by acetylcholinesterase enzyme. The presence of an organophosphorus

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pesticide inhibits the enzyme, resulting in a decrease in color formation. This can be measured to determine pesticide concentration in the field.

Keywords: Microfluidic device, herringbone, self-pumping, pesticide

Acknowledgements: This work has been sponsored by Fundação de Amparo à Pesquisa do Estado de São Paulo (Processo FAPESP 2018/14668-7).

References:[1] Oerke EC. J. Agric. Sci. 144: 31-43 (2006).[2] Basheer AA. Chirality 30: 402-406 (2018).[3] de Albuquerque NCP, Carrao DB, Habenschus MD, de Oliveira ARM. J. Pharm. Biomed. Anal.

147: 89-109 (2018).[4] Tetala KKR, Vijayalakshmi MA. Anal. Chim. Acta. 906: 7-21 (2016).[5] Stroock AD, Dertinger SKW, Ajdari A, Mezic I, Stone HA, Whitesides GM. Science 295: 647-

651 (2002).

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[FC-2]

LOW-COST AND ACCESSIBLE RAPID-PROTOTYPING OF MICROFLUIDIC DEVICES

Juan F. Hernández-Rodríguez1, Daniel Rojas1,2 and Alberto Escarpa1,3

1 Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, Faculty of Sciences University of Alcalá, E-28871 Alcalá de Henares, Madrid, Spain. E-mail: [email protected]

2 Faculty of Bioscience and Technology for Food, Agriculture and Environment University of Teramo 64023, Teramo, Italy

3 Chemical Research Institute “Andrés M. del Río”, University of Alcalá, E-28871 Madrid, Spain

1.- ObjectivesTo develop a rapid prototyping cleanroom-free method that allows the fast and reproducible fabrication of PDMS microfluidic devices in-lab using just common labware.

To couple electrochemical detection to the developed microfluidic devices

2.- MethodsMicrofluidic circuit designs were drawn in a computer-aided design (CAD) software or by hand using a technical pen of definite stroke thickness. The printed layout was secured on top of the working table and a glass slide was put on top. The tape that was going to constitute the mold was attached onto the glass slide. The pattern of the design was transferred to the mold simply by cutting out the lines with a scalpel of the drawing below using a scapel. The excess of tape was carefully removed using tweezers. After that, the pattern was transferred to PDMS by soft lithography.

3.- ResultsThe reliability of the method was tested by producing channels of different widths (200 µm, 500 µm, 1000 µm) and heights (1, 2, 3 tape layers). Also, common microfluidic circuits such as flow-focusing configuration and Y-shaped channel were used to check the functionality of the device in comparison to the ones produced by conventional photolithographic meth-ods. PDMS-based microfluidic fabrication coupled with electrochemical detection has been designed and optimized. The microfluidic chip was successfully coupled to a miniaturized Pt-based electrochemical cell using a microfluidic flow-segmented and on-line detection approach.

4.- ConclusionsA low-cost, and rapid-prototyping method has been developed to fabricate PDMS microflu-idic devices. The suitability of this approach has been demonstrated towards the fabrication of a T-junction microfluidic device for on-line electrochemical detection using a miniaturized Pt-based electrochemical flow cell and adopting a segmented flow scheme. The exceptional

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user-friendliness of this microfluidic rapid-prototyping method and the suitability of its cou-pling with electrochemical detection became overall the key-advantage, thereby accelerating the inclusion of microfluidics in the everyday workflow of any laboratory.

Keywords: Unconventional microfabrication, microfluidics, rapid prototyping, electrochemical detection.

Acknowledgements: This work has been sponsored by S2018/NMT-4349 (TRANSNANOAVAN-SENS-CM) project. European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie (grant agreement Nº 713714) and co-funding of University of Teramo and Abruzzo region.

References:[1] Nath P, Fung D, Kunde YA, Zeytun A, Branch B, Goddard G. Lab on a Chip 10: 2286-2291

(2010).[2] Hoang-Tuan N, Thach H, Roy E, Huynh K, Perrault C. Micromachines 9: 461 (2018).

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[FC-3]

DETERMINATION OF AMMONIUM IN THE VITREOUS HUMOUR BASED ON CAPILLARY ELECTROPHORESIS: PRELIMINARY APPLICATION IN THANATOCHEMISTRY

Covadonga Palacio, Rossella Gottardo and Franco Tagliaro

Department of Diagnostics and Public Health, Section of Forensic Medicine, University of Verona, Policlinico G.B. Rossi, Piazzale L.A. Scuro 10, 37134 Verona, Italy

1.- ObjectivesThe aims of the research were the development of a new reliable tool for the determination of the post-mortem interval using NH4

+ in vitreous humour. Moreover, the present work carried out a preliminary investigation on the behaviour of ammonium concentrations in the vitreous humour after death to verify in practice the feasibility of this new approach for the determination of the time since death. Finally, the last aim was to study the variability of the ammonium concentration in both eyes.

2.- Methods Electrophoresis conditions: The electrophoretic separation was carried out in a running buffer composed of 5 mM imidazole, 5 mM 18-crown-6 ether and 6 mM d,l-α-hydroxybutyric acid (HIBA) at pH 4.5. To overcome the lack of optical absorption of ammonium, indirect UV detection was applied, at a wavelength of 214 nm. [1]

Standard and sample preparation: A 100 mM stock solution of NH4+ was prepared by dissolv-

ing an adequate amount in water. Ammonium working standard solution was prepared daily to the appropriate concentration by the dilution with water. BaCl2 (IS) solution was used as internal standard at a concentration of 40 μg/mL. Vitreous humour samples were collected from 14 medico-legal autopsies (age ranging from 19 to 95 years) or external examination of corpses of violent or sudden deaths, in which the time of death was exactly known. The vitreous humour samples were prepared by diluting 1:20 with the IS. Real samples were analysed in triplicate [1].

Optimization and validation: The method was validated in terms of linearity, precision, bias and sensitivity, and its applicability was evaluated on real post-mortem cases [1]. The in-tra-day precision was verified at three concentration levels (low, middle, and high). To cal-culate the inter-day precision, determinations were performed on three different non- con-secutive days.

Moreover, a specific study on the variability of ammonium concentration in the two eyes belonging to the same subject was carried out in 20 medico-legal cases [2].

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3.- ResultsThe stability and reproducibility in a real vitreous humour matrices was studied. Three real samples were selected and analysed over a period of 5 days with five injections per sample. In all of the studies, the concentration of the NH4

+, as well as the migration time of this ion were recorded and statistically analysed in terms of relative standard deviation (%RSD). The analytical precision was acceptable in all cases, being the RSDs well below 20% [1].

The analytical accuracy was calculated as bias, using water solutions spiked with ammonium at three different concentrations (0.312, 1.25 and 5.0 mM). However, to study the analytical bias in real matrix, a standard addition experiment was performed spiking known amounts of NH4+ standard (15 and 10 mM) into real vitreous humour samples. The bias was calculated according to the formulas reported by the Scientific Working Group for Forensic Toxicology (SWGTOX) [3]. Overall, the bias was fairly acceptable in all cases [1].

For the linearity studies, five solutions with increasing NH4+ concentration were measured at

the following concentrations: 0.16, 0.31, 0.63, 1.25, 2.5, 5.0 mM. The results were linearly correlated according to the equation y = (0.0357 ± 0.0008 SD) x + (0.033 ± 0.002 SD); r2= 0.998 (y = relative peak area; x = concentration of NH4

+ in mM). LOD and LOQ were deter-mined to be 0.039 and 0.31 mM, respectively[1].

A preliminary study to investigate an expected correlation between the ammonium concen-tration in the vitreous humour and the post-mortem interval (PMI) was conducted on 14 sub-jects whose time since death was well known on the basis of circumstantial information. The correlation between PMI (hours) and vitreous NH4

+ concentration (mM) determined in 14 forensic deaths with known time since death. Equation y = (0.0132) x + (−0.012); r2= 0.970, in which y = NH4

+ concentration (mM); x = PMI (hours) [1].

The statistical difference between the ammonium concentrations in the two eyes belong-ing to the same subject was evaluated in 20 medico-legal cases. In all of the cases, the mean concentrations of NH4+ range from 0.07 to 6.45 mM with PMI values varying from 31 to 89 h. A good correlation (Pearson correlation= 0.94) is found between those two values. The different concentration of each eye was examined using a paired sample t-test (α=0.01). The findings suggest that no statistically significant difference was found (p value for two tails= 0.87) [2].

4.- ConclusionsA CE method for the determination of ammonium in the vitreous humour has been developed and validated with the aim of providing a new and reliable tool for thanatochemistry studies. In addition, the variability of the ammonium concentration in both eyes was studied, and no statistically significant difference was found.

Keywords: ammonium; capillary electrophoresis; post mortem interval; vitreous humour.

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References:[1] Gottardo R. et al. Clin. Chem. Lab. Med. 57, 504-509 (2019).[2] Gottardo R, Palacio C, Tagliaro F. To be submitted (2019).[3] Scientific Working Group for Forensic Toxicology, S. Scientific working group for forensic

toxicology (SWGTOX) standard practices for method validation in forensic toxicology. J. Anal. Toxicol. 37, 452-474 (2013).

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[FC-4]

IN SITU SYNTHESIS OF GOLD NANOPARTICLES ON A THREAD BASED MICROFLUIDIC DEVICE AND THEIR APPLICATION

IN SURFACE-ENHANCED RAMAN SCATTERING

Dosil P. de Jesus, Cristina B. Adamo, Ayandra S. Junger, Lucas P. Bressan, José Alberto F. da Silva and Ronei J. Poppi

Institute of Chemistry, University of Campinas, Campinas-SP, Brazil. Email: [email protected]

1.- ObjectivesThe main objectives of this work were: (1) demonstrate, for the first time, a simple, easy, and fast synthesis of gold nanoparticles (AuNPs) on a microfluidic device based on cotton thread; (2) Apply the synthesized AuNPs supported on the cotton thread for surface-enhanced Ra-man scattering (SERS) detection.

2.- MethodsCotton threads were initially treated by boiling them in a sodium hydroxide solution (10 mg/mL) at 100ºC for 5 min. After that, the threads were washed with deionized water and dried at room temperature.

The synthesis of the gold nanoparticles was based on the Turkevich method [1], and it was conducted on cotton threads, 2 cm in length with an average diameter of (12.9 ± 0.6) µm. A 3D printed platform in acrylonitrile butadiene styrene (ABS) containing a solution reser-voir was fabricated to assemble (stretch) the threads. This platform was kept on a heating plate up to reach the required temperature for the synthesis. Next, the solutions of the rea-gents (2% v/v HAuCl4 and 2% m/v sodium citrate) were added to the reservoir. The reagent solution wicked then through the thread (by capillary force), where the AuNPs were in situ synthesized.

The SERS measurements were performed in a dispersive spectrometer (Raman Sta-tion,PerkinElmer 400F, USA), with a cooled charged-coupled device (CCD) detector, at 785 nm near-infrared laser, operating at 125 mW, spot laser of 100 µm was used. The spectra were acquired with 6 exposures of 3 s each.

3.- ResultsThe synthesis of the AuNPs was evaluated using the temperatures of 25°C, 50°C, and 90 °C. The size of the AuNPs varied from 20 to 40 nm and decreased as the temperature increased. Figure 1 shows the scanning electron microscopy images (SEM) with two different magnification of a thread after the AuNP synthesis.

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A B

Fig. 1: SEM images of cotton threads after synthesis of AuNP. (A) Magnification of 4000 x and (B) 60,000 x.

The synthesized AuNPs coating the cotton thread were evaluated with SERS detection of crystal violet (CV) solutions. This cotton thread was immersed for 30 min in aqueous CV solution, and after drying, SERS spectra were obtained. Figure 2 shows the SERS spectra of a cotton thread without AuNP, after the AuNPs synthesis, and after immersion in CV solution.

Fig. 2: SERS spectra of only the thread, thread with AuNPs, and thread with AuNPs and crys-tal violet (CV) at a concentration of 1 × 10-5 mol L-1.

SERS spectrum of CV with good intensity, particularly for the peak at 1170 cm-1 (ring C-H bending) was achieved.

By using the same thread coated with AuNPs, nicotine in aqueous solutions was also detect-ed by SERS, and a calibration curve in the concentration range of 2 to 8 mg L-1 was attained.

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4.- ConclusionsFor the first time, in situ synthesis of AuNPs was conducted on a thread-based microfluidic device. The flow of the reagent solutions through the cotton thread by capillary force allowed a simple, easy, and fast synthesis of the AuNPs. The cotton thread was also used as support for the AuNP in successful SERS detection of crystal violet and nicotine.

Keywords: Textile Thread, Raman spectroscopy, Microfluidics, Nanoparticle synthesis

Acknowledgements: The authors would like to acknowledge Conselho Nacional de Desenvolvi-mento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and INCT Bioanalítica. This study was financed in part by the Coordenação de Aper-feiçoamento de Pessoal de Nível Superior-Brasil (CAPES)-Finance Code 001.

References:[1] Turkevich J, Stevenson PC, Hillier J. Discuss. Faraday Soc. 11: 55-75 (1951).

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[FC-5]

COMBINATION OF OFF-LINE SAMPLE TREATMENTS AND ON-LINE PRECONCENTRATION FOR THE ANALYSIS OF

5-NITROIMIDAZOLES RESIDUES IN FOOD, ENVIRONMENTAL AND BIOLOGICAL SAMPLES

BY CAPILLARY ELECTROPHORESIS

Maykel Hernández-Mesa, Diego Airado-Rodríguez, Carmen Cruces-Blanco and Ana M. García-Campaña

Department of Analytical Chemistry, Faculty of Sciences, Campus Fuentenueva, University of Granada, Granada, Spain. Email: [email protected]

1.- ObjectivesThis work proposes a new micellar electrokinetic chromatography (MEKC) method based on an ultra-sensitive on-line preconcentration approach, specifically cation-selective exhaus-tive injection (CSEI)-sweeping [1], for the analysis of 5-nitroimidazoles (5-NDZs) in differ-ent food, biological and environmental matrices.

2.- MethodsPrior to injection, the capillary was rinsed with a low conductivity buffer (50 mM phosphate buffer pH 2.5), followed by a plug of a higher conductivity buffer (100 mM phosphate pH 2.5, 50 mbar, ≈ 31.5 % total capillary volume) and a plug of water (50 mbar, 2 s). Analytes, dissolved in a solvent of lower conductivity than the separation medium (i.e., 5 mM phos-phoric acid solution containing 5 % of methanol), were electrokinetically injected at 9.8 kV for 632 s in a bare fused-silica capillary (57.2 cm, 50 µm I.D.). As a result, the desired field-enhanced sample injection (FESI) effect was achieved. Finally, separation was carried out applying -30 kV at 20°C in 44 mM phosphate buffer (pH 2.5), containing 8 % tetrahy-drofuran and 123 mM SDS. UV-detection was used and signals were monitored at 320 nm. A scheme of CSEI-sweeping-MEKC method is shown in Fig. 1.

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Fig. 1: Procedure stages: A, capillary is rinsed with low conductivity buffer (LCB), followed by a plug of a high conductivity buffer (HCB) and a water plug; B, electrokinetic injection at positive polarity is

performed, being cationic analytes stacked at the interface between water and HCB zones; C, cationic analytes are stacked at HCB zone because of the long injection, but not at water or matrix zones; D, background solution (BGS) is placed in both ends of the capillary and a negative voltage is applied,

removing matrix sample from the capillary; E, ordinary MEKC separation takes place.

In order to achieve a successful sample injection, different sample treatments were evaluated. Salting-out assisted liquid-liquid extraction (SALLE) combined with solid phase extraction (SPE) using Oasis® HLB cartridges (60 mg, 30 µm) was proposed for egg samples (2 g). SALLE was carried out employing acetonitrile (4 mL) in presence of 0.8 g of NaCl [2]. Dis-persive liquid-liquid microextraction (DLLME) was applied to water samples (5 mL). Dibro-momethane (1156 µL) and 2-butanol (1363 µL) were employed as extraction and dispersive solvents, respectively. Moreover, a salting out effect was achieved during the extraction by the addition of 16% (w/v) NaCl to samples [3]. Finally, the simple analysis of diluted urine and serum samples (143 and 22-fold, respectively) was also investigated [4].

3.- ResultsSensitivity enhancement factors (SEFs) between 73 and 262-fold were achieved for the pro-posed CSEI-sweeping-MEKC approach in comparison with a traditional MEKC method involving an injection of 5 s. DLLME-CSEI-sweeping-MEKC was successfully applied to the determination of 5-NDZ residues in river and well water samples. Limits of detection (LODs) lower than 2.44 µg/L were achieved. Relative standard deviations (RSDs) lower than 10% were obtained for repeatability and intermediate precision studies in most cases. Furthermore, the selectivity and sensitivity of this automated preconcentration technique al-lowed the determination of 5-NDZs at therapeutic levels (0.8-20.0 µg/mL) in diluted urine and serum samples, avoiding tedious off-line sample treatments. High precision, in terms of RSD of peak areas, was obtained for repeatability and intermediate precision (9.3 and 14.5%, respectively). Finally, SALLE-SPE-CSEI-sweeping-MEKC was applied to the analysis of

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5-NDZ residues in egg samples and egg-derived products, reaching LODs lower than 5.0 µg/kg. Repeatability and intermediate precision ranged between 5.0 and 17.9%.

4.- ConclusionsAlthough low sensitivity has been traditionally attributed to CE-UV methods, high sensi-tivity was achieved by the combination of on-line and off-line preconcentration strategies. CSEI-sweeping was evaluated as on-line preconcentration approach, whereas SPE and DLLME procedures were assessed as sample treatments. Consequently, LODs at low µg/L and µg/kg levels were achieved for the analysis of 5-NDZ residues in food and environ-mental samples by MEKC-UV. Furthermore, CSEI-sweeping demonstrated to be a powerful strategy for the direct analysis of 5-NDZs in urine and serum samples at therapeutic levels (µg/mL) without involving tedious sample treatments.

Keywords: 5-nitroimidazoles, cation selective exhaustive injection, sweeping, micellar electroki-netic chromatography

References:[1] Quirino JP, Terabe S. Anal. Chem. 72(5): 1023-1030 (2000).[2] Airado-Rodriguez D, Hernandez-Mesa M, Garcia-Campana AM, Cruces-Blanco C. Food Chem.

213: 215-222, (2016).[3] Hernandez-Mesa M, Airado-Rodriguez D, Cruces-Blanco C, Garcia-Campana AM. J. Chromatogr.

A 1341: 65-72 (2014).[4] Hernandez-Mesa M, Airado-Rodriguez D, Garcia-Campana AM, Cruces-Blanco C.

Electrophoresis 36: 2538-2541 (2015).

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[FC-6]

DETERMINATION OF OFLOXACIN BASED ON FLUORIMETRIC DETECTION COUPLED TO

CAPILLARY ELECTROPHORESIS USING GRAPHENE QUANTUM DOTS AS FLUORESCENT ENHANCER

Samah Lahouidak1,2,3, M. Laura Soriano2,4, R. Salghi3, Mohammed Zougagh1,2,4 and Ángel Ríos1,2*

1 Department of Analytical Chemistry and Food Technology, Faculty of Chemical Science and Technology, University of Castilla-La Mancha, Ciudad Real 13071, Spain

2 Regional Institute for Applied Chemistry Research (IRICA), Ciudad Real 13071, Spain

3 Laboratoire d’Ingénieries des Procédés de l’Energie et de l’Environnement, ENSA, B.P. 1136, Agadir, Morocco

4 Department of Analytical Chemistry and Food Technology, Faculty of Pharmacy, University of Castilla- La Mancha, Albacete 02071, Spain. Email: [email protected]

1.- ObjectivesThe use of green nanomaterials for developing analytical methods for the detection of an-tibiotic residues in milk samples is a challenge to be overcome for reducing human risk. The administration of certain antibacterial fluoroquinolones to dairy cows is restricted by the authorities who stablished maximum residue limits in foodstuff. To control and reliably predict the residues in milk, we report an EC method based on the use of graphene quantum dots (GQDs) as fluorescent enhancer of fluoroquinolones for lowering the detection and quantification limits. In this communication, we provide relevant information to enhance the fluorescent signal of fluoroquinolones in capillary electrophoresis for separating and increas-ing the sensitivity of the CE method. Results regarding the novel CE fluorescence method for the determination of ofloxacin in milk samples using GQDs to boost the native fluorescence of ofloxacin are discussed herein.

2.- MethodsThe proposed CE method uses an instrument equipped with dual UV diode array and fluo-rescence detection to select the inherent fluorescent of the diverse fluoroquinolones. Good separation of seven fluoroquinolones is obtained after optimizing diverse variables, mainly the buffer type, pH, and voltage. The selected buffer, pH and voltage were respectively. The sensitivity of the CE method is significantly increased when adding GQDs, as shown in Figure 1. It is a crucial to inject GQDs prior the standards/sample in order to increase the antibiotic fluorescence response. Milk samples were clean‐up for deproteination using an acid treament and the antibiotic was afterwards preconcentrated onto a home-made silica-C18 packed sorb-ent (MEPS) material before injection to the CE

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Fig. 1: Stacked electropherograms of ofl oxacin (at 1 mg·L-1) when exciting at 280 nm, in presence (a) and absence (b) of GQDs in the proposed CE method.

3.- ResultsFluorescent water-soluble GQDs were used as additive in the CE method, being injected in the electrophoretic capillary prior the standards/sample. Sample preparation of milk samples were easily carried out with a clean‐up and preconcentration step, allowing a good line-ar correlation within a concentration range of 50 - 1000 ng/mL for ofl oxacin. The detec-tion and quantifi cation limits achieved were of 10.7 and 35.5 ng/mL, respectively, being suitable for routine milk analysis following the regulation of the European Commission[1]. The interaction of GQDs is specifi c to certain hydrophobic antibiotics, namely the family of fl uoroquinolone, showing an enhancement of the native fl uorescent. Electrostatic interaction between the anionic GQDs and cationic piperazine moiety of fl uoroquinolones is expect-ed (Figure 2); thus, such interaction of the GQD with fl uoroquinolone may diminish the non-radiative energy losses, resulting in an increase of the fl uorescence quantum yield. The applicability of the proposed CE method based on GQDs for the analysis of fl uoroquinolones in milk enables their detection at lower concentrations when coupling a photoluminescence detector. Evaluation of the variables for the separation of seven fl uoroquinolones with CE is demonstrated. However, due to the diverse maximum emission wavelengths of the target analytes and the limitation of the fi xed single wavelenth photoluminescence detector coupled to CE, we only based our results on the analysis of ofl oxacin to demonstrate improvement in detectability.

Fig. 2: Enhancement of the inherent fl uorescent of ofl oxacin when interacting with GQDs.

4.- ConclusionsA simple CE method based on non-toxic fl uorescent GQDs acting as emission enhancer of a fl uoroquinolone is proposed. The synthesis of GQDs is simple and easy, being a non-toxic material for its use in routine analysis. The method is characterized by the use of relative-ly low solvent consumption using a CE instrument with photoluminescence detectors that

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allow the determination of fluoroquinolones. We developed a sensitive method with the use of GQDs to enhance sensitivity for quantifying ofloxacin in milk samples. This study can be extended to the determination of other photoluminescence analytes which requires low detection and quantification limits accordingly to the desired application.

Keywords: graphene quantum dots; fluoroquinolones; ofloxacin, fluorescence, milk.

Acknowledgements: We thanks to the Spanish Ministry of Economy and Competitiveness (MINE-CO) and JJCC Castilla-La Mancha and European Commission for funding this work with Grants CTQ2016-78793-P and JCCM PEIC-2014-001-P, SBPLY/17/180501/ 000333, respectively.

References:[1] Huanga X-G, Zhanga H-S, Lia Y-X, Lia M-F. J. Chil.Chem. 54(3): 204-207 (2009).

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[FC-7]

TUNABLE AND pH-INDEPENDENT ELECTROOSMOTIC FLOW FOR CAPILLARY ELECTROPHORESIS SEPARATIONS

Dušan Koval, Veronika Šolínová and Renáta Konášová

Institute of Organic Chemistry and Biochemistry, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic

1.- ObjectivesModification of the capillary surface has been a pivotal theme since the advent of cap-illary electrophoresis (CE). Need for efficient separation of vast variety of analytes fueled development of a plethora of strategies for capillary modification in order to suppress analyte adsorption and control analysis time by modulation of electroosmotic flow. However, for use in CE-MS some adjustments are required in order to address specificity of the hyphenated technique.

While a wide variety of buffer additives have been in use for CE with UV or fluorescence detection, CE-MS is quite restrictive for possible contaminants entering the MS. Thus, ei-ther untreated or permanent neutral coatings are typically used in CE-MS. Modifications by charged polymers such as successive multiple ionic-polymers (SMIL) are possible but less frequently used. On the other hand, small molecules, detergents and polymers as additives to the background electrolyte (BGE) are ruled out because of their deleterious effect on the MS signal.

2.- MethodsRecently, we turned our attention to capillary modifications providing tunable electroosmotic flow. To be MS-compatible, we rely on covalently bound copolymers prepared by in-capil-lary polymerization. Anionic surface coatings were formed by PAMAMPS, i.e. copolymer of acrylamide and anionic 2-acrylamido-2-methyl-1-propanesulfonate.

Then, cationic surface modification is achieved with PAMAPTAC, i.e. copolymer of acryla-mide and (3-acrylamidopropyl)trimethylammonium chloride.

3.- ResultsA series of PAMAMPS and PAMAPTAC coatings ranging from 0 to 60% of charged monomer revealed electroosmotic flow gradually rising from zero to about +/- 40 x 10-9 m2V-1s-1. EOF proved to be stable and independent of pH at least in the 2 – 7.5 interval. Effect of variable normal EOF has been demonstrated so far on chiral separation of heli-cal cationic helquats in the presence of sulfated cyclodextrins in acidic phosphate buffer. In turn, tunable reversed EOF has been used for separation of cationic drug mixtures. Extensions to different analytes such as peptides and proteins as well as applications in CE-MS are underway.

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Keywords: Capillary electrophoresis, covalent capillary coating, polymers, tunable electroosmotic flow

Acknowledgements: The authors acknowledge a support from GAČR (17-12648S, 17-10832S) and IOCB (RVO 61388963).

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[FC-8]

MICROCHIP CAPILLARY ELECTROPHORESIS (MCE) FOR ANALYSIS OF DIFFERENT SAMPLES INCLUDING

PHARMACEUTICALS IN BODY FLUIDS

Ángel Puerta, Mercedes de Frutos and José Carlos Díez-Masa

Institute of Organic Chemistry (IQOG-CSIC), Madrid, Spain. Email: [email protected]

1.- Introduction and ObjectiveThere is a constant trend in Separation Science towards miniaturization, and in this context the impact of plastic and glass microchips on electrophoretic separations is remarkable. Mi-crochips give rise to faster separations, with minimal consumption of samples and reagents even by comparison to capillary electrophoresis (CE) in silica capillaries. Among the differ-ent detection techniques that have been used in Microchip Capillary Electrophoresis (MCE), Fluorescence Detection (FD) is very popular due its inherent sensitivity. The features of the Light-Emitting Diodes (LEDs) [1] and their compact dimensions, make them suitable for the combination with MCE devices [2,3] to perform LED-Induced Fluorescence (LEDIF) detection.

The objective of this work is to show the application of MCE-LEDIF devices built in our laboratory for the analysis of different compounds in standard solutions and in biological fluids, specifically urine.

2.- MethodsElectrophoretic separations have been carried out in polymethylmethacrylate (PMMA) mi-crochips with gated injection and different electrophoretic modes (capillary zone electropho-resis and micellar electrokinetic chromatography). LEDIF detection was performed using a detector previously developed in our laboratory [4]. Collinear configuration for LEDIF detection has been selected as this configuration is simple, and provides high fluorescence collection efficiency [5].

3.- Results and conclusionsDifferent fluorescent dyes and their degradation products have been analysed by MCE-LE-DIF using different electrophoretic modes. A methodology has been developed for the anal-ysis of B2 vitamin (riboflavin) in the urine of a healthy individual along the time after the pharmaceutical drug intake.

Keywords: Microchip Capillary Electrophoresis, MCE, Light-Emitting Diode, LED, Light-Emitting Diode Induce Fluorescence, LEDIF, B2 vitamin, Fluorescent dyes.

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Acknowledgements: Authors thank the Comunidad of Madrid and European funding from FSE and FEDER programs for financial support (project S2018/BAA-4393, AVANSECAL-II-CM).

References:[1] Rodat-Boutonnet A, Naccache P, Morin A, Fabre J, Feurer B, Couderc F. Electrophoresis 33(12):

1709-1714 (2012).[2] Hall GH, Glerum DM, Backhouse CJ. Electrophoresis 37(3): 406-413 (2016).[3] Macka M, Piasecki T, Dasgupta PK. Annu. Rev. Anal. Chem. 7: 183-207 (2014).[4] Puerta A. Poster Communication P-01, XV Scientific Meeting of the Spanish Society of

Chromatography and Related Techniques, SECyTA 2015, Castellon, Spain.[5] Xiao D, Yan L, Yuan H, Zhao S, Yang X, Choi MM. Electrophoresis 30(1): 189-202 (2009).

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[FC-9]

ANALYSIS OF CIRCULATING microRNA BIOMARKERS IN SERUM BY ON-LINE SOLID-PHASE EXTRACTION

CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY

Roger Pero-Gascon1, Maxim V. Berezovski2, José Barbosa1, Victoria Sanz-Nebot1 and Fernando Benavente1

1 Department of Chemical Engineering and Analytical Chemistry, Institute for Research on Nutrition and Food Safety (INSA·UB), University of Barcelona, Barcelona, Spain. Email: [email protected]

2 Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario, Canada

1.- ObjectivesMicroRNAs (miRNAs) are single-stranded, 19-23 nucleotides long, nonprotein-coding RNAs. Deregulation of miRNAs has been associated with different diseases, especially can-cer. The methods currently applied for routine analysis of miRNAs are very sensitive (e.g., reverse transcriptase quantitative polymerase chain reaction, RT-qPCR), but do not allow the direct detection of the target miRNAs or their post-transcriptional modifications. In this study, an on-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) method is described for the purification, preconcentration, separation, and charac-terization of endogenous miRNAs and their post-transcriptional modifications in serum [1].

2.- MethodsA commercial silicon carbide resin is used as a sorbent. The microcartridge is integrated in-line near the entrance of the separation capillary and no valves are necessary for the operation in SPE-CE-MS. After loading a large volume of sample (∼50 μL), the capillary is rinsed to eliminate non-retained molecules and filled with background electrolyte. Then, the analyte is preconcentrated eluting in a small volume of an appropriate solution (50 nL of 60% (v/v) acetonitrile) before the separation and the detection by mass spectrometry.

3.- ResultsUnder optimized conditions with standards, the microcartridge lifetime (around 25 analy-ses) and repeatability (2.8 and <6.4% RSD for migration times and peak areas) were good, the method was linear between 25 and 100 nmol·L-1, and LOD was 10 nmol·L-1 (i.e., 50 times lower than by CE-MS, 500 nmol·L-1). The potential of the SPE-CE-MS method to screen for B-cell chronic lymphocytic leukemia (CLL) was demonstrated analyzing se-rum samples from healthy controls and patients. miRNAs, specifically miR-21-5p and a 5′-phosphorylated miRNA with 3′-uridylation (iso-miR-16-5p), were only detected in the CLL patients.

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4.- ConclusionsThe results achieved show that SPE-CE-MS can be regarded as a promising technique for the direct, high-throughput, sensitive and multiplex analysis of miRNA and their post-transcrip-tional modifications in complex samples. It could become a unique complement to current methods applied in routine analysis.

Keywords: capillary electrophoresis; in-line solid-phase extraction; mass spectrometry; microRNA; on-line solid phase extraction.

Acknowledgements: This work was supported by projects CTQ2014-56777-R and RTI2018-097411-B-I00 (Ministry of Economy and Competitiveness, Spain) and the Cathedra UB Rector Francisco Buscarons Úbeda (Forensic Chemistry and Chemical Engineering).

References:[1] Pero-Gascon R, Sanz-Nebot V, Berezovski MV, Benavente F. Anal. Chem. 90: 6618-6625 (2018).

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[FC-10]

DEVELOPMENT OF A CAPILLARY ELECTROPHORESIS METHODOLOGY FOR THE STEREOSELECTIVE

SEPARATION OF TETRAMETRIN

M. Greño1, M. A. García1,2, Maria Luisa Marina1,2 and Maria Castro-Puyana1,2


2 Instituto de Investigacion Quimica Andrés M. del Río, University of Alcalá, Alcalá de Henares Madrid, Spain

1.- ObjectivesThe aim of this work was to develop, for the first time, an analytical methodology by Cap-illary Electrophoresis (CE) for the chiral separation of tetramethrin, a pyrethroid that has two pairs of stereoisomers (trans and cis) in a 80:20 ratio. Until now, just two articles have reported the chiral separation of tetramethrin isomers by liquid chromatography [1,2].

2.- MethodsTo select the optimal separation conditions, screening of different neutral cyclodextrins (CDs) and bile salts in borate buffer was carried out. The influence of different parame-ters such as buffer nature and concentration, pH, chiral selectors concentration, voltage, and temperature on the stereoselective separation of tetramethrin was investigated.

3.- ResultsBoth hydroxypropyl-β-CD (HP-β-CD) or heptakis(2,3,6-tri-O-methyl)-β-CD (TM-β-CD) in combination with sodium deoxycholate (DSC) enabled the separation of the four isomers of tetramethrin. Similar resolution values were achieved in less than 13 min (1.9 and 0.9 for trans and cis isomers when HP-β-CD + DSC was employed and 1.6 and 1.3 for trans and cis isomers when using TM-β-CD + DSC). Opposite enantiomer migration orders were ob-served depending on the cyclodextrin used.

4.- ConclusionsThe developed methodology allowed the separation, for the first time, of the four stereoi-somers of tetramethrin by CE in short analysis times with high Rs values. Moreover, the optimized methodology was applied for the determination of tetramethrin in a commercial antiparasitic spray.

Keywords: Capillary Electrophoresis, pyrethroid, tetramethrin

Acknowledgements: Authors thank the Spanish Ministry of Economy and Competitiveness for re-search project CTQ2016-76368-P. M.C.P also thanks this Ministry for her “Ramón y Cajal” research

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contract (RYC-2013-12688). M.G thanks the Spanish Ministry of Science, Innovation and Universi-ties for her pre-doctoral contract FPU17/01635.

References:[1] Xhe X, Wenwei X, Hua H, Lirui P, Xu X. J. Chromatogr. Sci. 46(9): 783-786 (2008).[2] Corcellas C, Eljarrat E, Barcelo D. Anal. Bioanal. Chem. 407(3): 779-786 (2015).

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[FC-11]

ENANTIODETERMINATION OF CATHINONES IN URINE BY FIELD-AMPLIFIED SAMPLE INJECTION-CAPILLARY

ELECTROPHORESIS AND ELECTROKINETIC SUPERCHARGING-CAPILLARY ELECTROPHORESIS

Albert Pérez-Alcaraz, Francesc Borrull, Carme Aguilar and Marta Calull

University Rovira i Virgili, Tarragona, Spain. Email: [email protected]

1.- ObjectivesIn recent years, the consumption of synthetic cathinones has increased due to their stimulat-ing properties and low price compared with amphetamines. After their consumption, usually nasally insufflated or ingested, cathinones enter in the organism where they produce their stimulating effects [1]. Therefore, the determination of cathinones in different biological ma-trices, as urine, can provide a useful tool to control their abuse. Moreover, synthetic cathi-nones present chiral centres, which implies the presence of two enantiomers (R and S) and each enantiomer can present different pharmacokinetic and pharmacodynamic behaviour. For that reason, the development of methods allowing the enantioseparation of these compounds is important. CE is an effective technique for the achievement of chiral separations by simply dissolving a chiral selector in the BGE [2]. However, one of the main limitations of CE is its inherent poor sensitivity. To overcome this drawback, a number of sample preconcentration strategies have been developed. Among them stacking based methods are widely used [3].

In view of these facts, we propose two electrophoretic preconcentration methodologies, field-amplified sample injection (FASI) and electrokinetic supercharging (EKS), in combi-nation with CE for the enantiodetermination of cathinones in urine samples at low concen-tration levels.

2.- MethodsAs FASI and EKS involve an electrokinetic sample injection, the analysis of complex sam-ples as urine cans negatively affect their performance. So, a previous extraction and clean up step based on LLE was developed for both methodologies. To achieve the enantioseparation of the target compounds by CE a mixture of cyclodextrins was added to the BGE. In FASI a low conductivity solvent plug was introduced in the capillary prior to the electrokinetic sam-ple injection, and in the case of EKS the samples were electrokinetically injected between a high conductivity leading electrolyte (LE) and a low conductivity terminating electrolyte (TE).

3.- ResultsHigh sensitivity enhancement factors were achieved in both methodologies. The obtained values were between 744 and 1310 for EKS and between 444 and 472 for FASI. Both methods were satisfactory validated in terms of linearity, repeatability, reproducibility,

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LODs and LOQs. The obtained LODs in urine were in the range between 15-45 ng/mL and 3-9 ng/mL for FASI and EKS, respectively.

4.- ConclusionsLLE/FASI-CE and LLE/EKS-CE have shown their effectiveness for the enantiodetermina-tion of cathinones in urine samples at low concentration levels. Both methodologies provided several advantages as their simplicity, their quite high sensitivity, low sample and reagents consumption and that the enantioseparation of the cathinones was easy to achieve. EKS-CE presented higher sensitivity than FASI-CE since whit this strategy it is possible to inject a larger sample amount.

Keywords: Cathinones; Chiral separation; EKS; FASI; Urine analysis.

Acknowledgements: The authors would like to thank the Ministerio de Ciencia, Innovación y Universidades and the European Regional Development Fund (ERDF) (Project: CTQ2017-88548-P) for the financial support given.

References:[1] Prosser JM, Nelson LS. J. Med. Toxicol. 8: 33-42 (2012).[2] Stavrou IJ, Agathokleous EA, Kapnissi-Christodoulou CP. Electrophoresis 38: 786-819

(2017).[3] Šlampova A, Mala Z, Gebauer P. Electrophoresis 40: 40-54 (2019).

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[FC-12]

ION ANALYSIS STRATEGIES FOR HABITABILITY ASSESSMENT IN PLANETARY SCIENCE

Elizabeth Jaramillo, Aaron Noell, M. Fernanda Mora and Peter Willis

NASA, Jet Propulsion Laboratory, California Institute of Technology, Pasadena, California, USA

1. ObjectivesThe recent discovery of subsurface oceans of liquid water on the icy moons of the outer solar system has driven the need for in situ instrumentation and methods capable of investigating the habitability of these worlds. In situ methods and instruments inherently require a minimi-zation of protocol complexity without sacrificing the returned science. The characterization of soluble ionic species in a sample addresses the need for a high science return by providing chemical information such as the oxidation-reduction potential and the availability of chemi-cal energy that are needed to assess the habitability of an environment. We are developing in situ methods and instruments capable of meeting these requirements for use on ocean worlds.

2. MethodsWe are employing two complimentary techniques to fully determine the ionic composition of unknown samples. First, we are developing a 3-D printed microfluidic ion selective electrode (ISE) array that can measure multiple inorganic cations simultaneously, without the need for sample pre-treatment or dilution.

Fig. 1: 3-D printed microfluidic ion selective electrode array.

Second, we are developing flight-compatible methods using capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) that can simultaneously separate a wide range of organic and inorganic anions that are relevant to planetary science and astrobiology.

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3. ResultsUsing a solution of artificial sweater, we have successfully demonstrated the ability of our 3-D printed ISE array to quantify the primary cations in seawater without sample pre-treatment. The associated error in the measurement is ≤ 0.2 log(a) units for each ion. The measurement takes < 5 min and uses 0.5 mL of sample.

Ion log(a,M) of mock seawater Measured log(a,M) % error

Ca -2.61 ± 0.01 -2.45 ± 0.01 6%

Mg -2.11 ± 0.01 -1.92 ± 0.01 9%

Na -0.50 ± 0.01 -0.6 ± 0.2 23%

Cl -0.53 ± 0.01 -0.7 ± 0.2 33%

Table 1: Comparison of the known activity and the results of the analysis using the ISE array for a solution of artificial seawater

Regarding the CE-C4D methods, we have also demonstrated the ability to simultaneously separate 7 inorganic anions and 9 carboxylic acids that are relevant to astrobiology. We are currently validating both techniques using natural samples relevant to the study of ocean worlds.

Fig. 2: Simultaneous separation of 16 anions of interest in astrobiology.

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[FC-13]

MAGNETO-CATALYTIC JANUS MICROMOTOR IN LABS-ON-A-CHIP APPLICATIONS: MOBILE

SENSORS AND MICROREACTORS

Marta Pacheco1, Beatriz Jurado-Sánchez1,2 and Alberto Escarpa1,2

1 Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, University of Alcalá, Alcalá de Henares, Madrid, Spain. E-mail: [email protected]

2 Chemical Research Institute “Andrés M. del Río”, University of Alcalá, Alcalá de Henares, Madrid, Spain

1.- ObjetivesWe propose the synthesis of asymmetric Janus micromotors that encapsulate platinum nanopar-ticles for the decomposition of a fuel (hydrogen peroxide) towards efficient propulsion, ferrite nanoparticles for magnetic guidance and specific detection elements or molecules precursors for labs-on-a-chip applications. On a first approach, the use of magneto-catalytic Janus micromotors encapsulating phenylboronic acid and graphene quantum dots as mobile sensors for detection of bacteria endotoxins in lab-on-a-chip devices will be discussed. For a second approach we will il-lustrate the tailored synthesis of CdS quantum dots and Au nanoparticles with “mobile” catalytic Janus micromotors encapsulating suitable precursors (Cd2+ or citrate).

2.- Methods For the achievement of the first objective, Janus micromotors are synthesized by encapsu-lation of receptor functionalized graphene quantum dots modified PtNPs and Fe3O4 NPs, using a water-oil emulsion synthetic approach. The conditions for detection are optimized using a fluorescence optical microscope. Initially, the micromotor presents an intense fluo-rescent emission that disappears or attenuates (quenching) in the presence of the endotoxin of interest (Fig. 1). The lipopolysaccharide (LPS) of Escherichia coli 0111: B4 and the lipopol-ysaccharide of Salmonella enterica are used as model toxins [1,2].

Fig. 1: Magneto‐catalytic graphene quantum dots-based Janus micromotors for bacterial endotoxin detection. A) Bubble propulsion and optical microscopy images of the Janus micromotors before and

after the addition of the lipopolysaccharide (LPS). B) Optical microscopy images of the magnetotactic behavior of the Janus micromotor during its navigation in the different channels of a

polydimethylsiloxane (PDMS) chip. Scale bars: 20 μm; NP=nanoparticle.

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Following the second objective, Janus micromotors are designed and developed as reactors in the micrometric scale integrating Cd2+ for the synthesis of CdS quantum dots and Au3+ or citrate for the synthesis of gold nanoparticles (AuNPs) (Fig. 2). The CdS quantum dots syn-thesized on the motor are characterized and evaluated by high resolution transmission elec-tron microscopy (HRTEM), UV-Vis spectrometry, and fluorescence optical microscopy. The AuNPs are characterized by high resolution transmission electron microscopy (HRTEM) and UV-VIS spectrophotometry by evaluating their catalytic activity by the reduction reaction of 4-nitrophenol [3].

Fig. 2: Janus micromotors as mobile microreactors for nanoparticle synthesis. Schematic illustration of the synthesis of CdS quantum dots (top) and AuNPs (bottom)

using Cd2+ or citrate loaded Janus microreactors navigating in “reagent solutions” containing sulphur ions and ATP or Au3+, respectively. Scale bars, 20 mm.

3.- ResultsA self-propelled optical sensor with good analytical and selective characteristics has been designed for the detection of E. coli in samples of clinical relevance such as urine and blood serum and of S. enterica in samples of high food industry interest such as milk, mayonnaise and egg.

A self-propelled catalytic Janus micromotor has been used as a mobile microreactor for the synthesis of nanoparticles. We have designed micromotors loaded with Cd2+ to synthesize quantum dots of CdS using ATP as a stabilizing agent and micromotors loaded with Au3+ or citrate have been designed to synthesize gold nanoparticles.

4.- ConclusionsWe have developed a powerful ON-OFF fluorescence sensing approach based in a Janus mi-cromotor encapsulating PABA‐GQDs for the detection of bacteria endotoxins. Continuous mixing induced by the motion of multiple micromotors across a contaminated sample result-ed in greatly enhanced mass transport to increase the reaction rate between the endotoxins

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contaminated solution and micromotors as compared to the use of static micromotor counter-parts. The new micromotor-based fluorescent detection is particularly promising for clinical and food safety assurance and security biodefense applications against the use of bacteria toxins.

In addition, it is the first time that a catalytic Janus micromotors is used as an “all-in-one” mobile microreactors for nanoparticles synthesis(CdSQDs and AuNPs). The use of confined reagents along with the efficient microreactor navigation allows for a drastic reduction of waste volumes and hold a promise to address growing concerns about the impact of chemi-cals released in the environment and for a myriad of analytical, biomedical and material syn-thesis applications. Moreover, the moving nature and magnetic properties of the microreactor vessel allow controlled synthesis in ultra-small setting and controlled transport and release in pre-determined setting, with many possibilities that remain to be explored in the near future.

Keywords: Endotoxins, Janus, micromotor, microreactor, nanoparticles.

Acknowledgements: M. Pacheco acknowledges the FPU fellowship received from the Spanish Ministry of Education (FPU 16/02211). B. J-S acknowledges support from the Spanish Ministry of Science, Innovation and Universities (RYC-2015-17558, co-financed by EU) and from the Univer-sity of Alcalá (CCG2018/EXP-018). AE acknowledges financial support from the Spanish Ministry of Science, Innovation and Universities (CTQ2017-86441-C2-1-R) and the TRANSNANOAVANS-ENS program (S2018/NMT-4349) from the Community of Madrid.

References:[1] Jurado-Sanchez B, Pacheco M, Rojo J, Escarpa A. Angew. Chem. Int. Ed. 56: 6957 (2017).[2] Pacheco M, Jurado-Sanchez B, Escarpa A. Anal. Chem., 90, 2912, 2018.[3] Pacheco M, Jurado-Sanchez B, Escarpa A. Chem. Sci. 9: 8056 (2018).

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[FC-14]

METABOLOMIC STUDY OF THE EX VIVO EFFECT OF KYNURENIC ACID AFTER ISCHEMIA-REPERFUSION IN RATS

Douglas Oscar Ceolin Mariano1, Daniel Carvalho Pimenta2, Juliana Mozer Sciani3, Mario Angelo Claudino3, Aline Gonçalves Mora3, Sabrina Janussi3,

Coral Barbas1 and Francisco Javier Rupérez1

1 Center for Metabolomics and Bionalysis (CEMBIO), Fundación Universitaria San Pablo-CEU, Madrid, Spain. Email: [email protected]

2 Laboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo, Brazil3 Laboratório multidisciplinar de pesquisa, Universidade São Francisco, Bragança

Paulista, Brazil

1.- ObjectivesKynurenic acid (KYNA) is one important Tryptophan-derived compound formed through Kynurenine pathway. Some studies have showed that this molecule might exhibit neuropro-tective effect, with neuroprotective/scavenger/antioxidant role after occlusion of the middle cerebral artery in rats. Besides, KYNA may exhibit an angiogenic effect. Our preliminary re-sults suggest that KYNA is capable to promote or induce the formation of tube-like structure in an in vitro model of vasculogenesis. The molecular mechanisms involved in the beneficial effects of KYNA have not been elucidated yet, and a metabolomics approach with Capillary Electrophoresis–Mass Spectrometry (CE-MS) is a proven and valuable method to discover which compounds might be involved in angiogenic or cardioprotective effects.

2.- MethodsSprague-Dawley rats (n=5) were submitted to an ischemia reperfusion (I/R) injury model (30 min ischemia and 5 min reperfusion). After that, blood was extracted, and plasma separated and incubated in the absence or presence of KYNA (0.1 and 1 mM) for 30 min. Subsequent-ly, we extracted all metabolites from plasma samples and analyzed them by Capillary elec-trophoresis–Mass Spectrometry (CE-MS) system (CE - 7100 Agilent coupled to a TOF-MS 6224 Agilent).

3.- ResultsAround 300 features were detected after data treatment. After that, we performed a Princi-pal Components Analysis (PCA) and it was possible to observe a clear difference between KYNA and the control groups. One-way ANOVA followed by Benjamini-Hochberg multiple testing correction showed that 19 features were significantly different between the groups. The features were putatively annotated by performing a simultaneous query in publicly avail-able databases with CEU Mass mediator (http://ceumass.eps.uspceu.es). Spermidine and the prolyl-glycine dipeptide arose as two of the most prominent changing compounds.

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4.- ConclusionsCardiovascular diseases are one of majority causes of death or morbidity in the world and it is important to seek new possibilities to prevent or treat heart diseases. KYNA ex vivo treatment (1 mM), increased the concentration of spermidine in plasma. This polyamine is a known stimulator of cell division and proliferation, as well as angiogenesis, whereas pro-lyl-glycine could be associated to the activity of proteases involved in remodeling.

Keywords: Capillary Electrophoresis; Ischemia reperfusion injury model; Kynurenic acid; Metabolomics.

Acknowledgements: DCM received funding from Airbus Defense and Space through the CLX-2 program developed with Comando da Aeronáutica (COMAER) and the Brazilian Government.

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[FC-15]

DEVELOPMENT OF A CE-MS METABOLOMICS APPROACH TO STUDY IN VITRO HIGH GLUCOSE-

INDUCED CHANGES IN HK-2 CELLS

Samuel Bernardo-Bermejo1, Elena Sánchez-López1, María Castro-Puyana1,2, Selma Benito3,4, Francisco Javier Lucio-Cazaña3 and María Luisa Marina1,2

1 Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering,University of Alcalá, Alcalá de Henares, Madrid, Spain. Email:[email protected]

2 Institute of Chemical Reseach Andrés M. del Río (IQAR), Universidad de Alcalá, Alcalá de Henares, Madrid, Spain

3 Department of Biology and Systems, University of Alcalá, E-28871 Alcalá de Henares, Madrid, Spain

4 Networking Biomedical Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain

1.- ObjectivesDiabetic nephropathy is a prevailing complication of diabetes mellitus and the most com-mon cause of end-stage kidney disease worldwide. The aim of this work was to develop, for the first time, an untargeted metabolomic approach based on capillary electrophoresis-mass spectrometry (CE-MS) for identifying potential biomarkers of diabetic nephropathy in HK-2 cells.

2.- MethodsTo study an in vitro model of high glucose (HG)-induced metabolic alterations in HK-2 cells, three different groups of cells cultivated in different media (physiological glucose (glucose control), high glucose and mannitol (osmotic control)) were analyzed using the developed CE-MS metabolomic method approach. In order to expand the metabolite coverage, both intracellular and extracellular fluids were investigated.

3.- ResultsOptimization of sample preparation and CE-MS analysis steps was carried out to extract as much information as possible from HK-2 cells. Principal components analysis and partial least square discriminant analysis revealed the differences existing among the three experi-mental groups. Variable importance in the projection values combined with Mann Whitney Univariate test helped discover the important molecular features in the given models. Dif-ferent metabolites, mainly from the amino acidic metabolism, were identified as statistically significant in both fluids.

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4.- ConclusionsA novel platform based on CE-MS was developed for the metabolomic analysis of HK-2 cells. This platform was demonstrated to be a good candidate to study HG-induced changes in this in vitro model to improve current knowledge on the diabetic nephropathy mechanism.

Keywords: capillary electrophoresis-mass spectrometry, diabetic nephropathy, HK-2 cells, metabolomics.

Acknowledgements: Authors thank the Ministry of Economy and Competitiveness (Spain) for re-search project CTQ2016-76368-P. S.B.B and M.C.P. thank the same Ministry for their predoctoral (BES-2017-082458) and “Ramón y Cajal” (RYC-2013-12688) research contracts, respectively. E.S.L. thanks the University of Alcalá for her postdoctoral contract.




PosterPresentations

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[PP-1]

INDIRECT CALIBRATION IN CAPILLARY ELECTROPHORESIS WITH CONDUCTIVITY DETECTION

Michele Alves Santana and Claudimir Lucio do Lago

Department of Fundamental Chemistry, Institute of Chemistry, University of Sao Paulo, Sao Paulo, Brazil. Email: [email protected]

1- ObjectivesFor monocharged species in BGE and analytes, the measured conductivity detected in elec-trophoresis condition is given by [1-3]:

where ∆κ is the conductivity variation, F is the Faraday constant, K is a cell constant, µa, µs, µo are the analyte, co-ion, and counter-ion respective mobilities, and Ca is the analyte concen-tration. This equation suggests that any species of same mobility as the analyte can be used for calibration purposes, which has been, previously demonstrated for monocharged species [4]. The purpose of this work is to explore the possibilities for the quantification of analytes with different charges using other species with similar mobilities for calibration purposes.

2 - MethodsFour series of standards were chosen: carboxylic acids, dicarboxylic acids, alkaline cat-ions and aliphatic diamines. For the negatively charged analytes, the background elec-trolytes were 10 mmol/L NaHCO3 (pH 8.3) and 30 mmol/L MES/Histidine 30 mmol/L (pH 6.0). For the positively charged analytes, the BGE was 0.5 mol/L acetic acid (pH 2.5). Calibration curves were made for each of the standards.Using the sensitivity values obtained from the curves, sensitivity versus mobility graphs were produced. For these analysis, capillary electrophoresis equipment with capacitively coupled contactless con-ductivity (C4D) was used.

3 – ResultsThe curves shown on Figures 1 and 2 were made by plotting the sensitivity versus mobility for the studied species. As the mobility range of the species is small, the equation can be ap-proximated to a straight line, which is confirmed by the correlation coefficient as calculated.

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Fig. 1: Sensitivity versus mobility. a) Curve with standards: p-aminobenzoate, butyrate, propionate and acetate. BGE: NaHCO3 10 mmol/L (pH 8.3). The charge of the standard species in pH 8.3 is -1. b) Curve

with standards: p-aminobenzoate, butyrate, propionate and acetate. BGE: MES/Histidine 30 mmol/L (pH 6.0). The charge of the standard species in pH 6.0 is -1.

Fig. 2: a) Curve with standards: adipate, phthalate, succinate and malate. BGE:NaHCO3 10 mmol/L (pH 8.3). The charge of the standard species in pH 8.3 is -2.

b) Curve with standards: 1,3-diaminopropane, 1,4- diaminobutane, 1,5-diaminopentane and 1,7-diami-noheptane. BGE: Acetic acid 0.5 mol/L (pH 2.5). In this case, a PVAcoated capillary was used.

The charge of the standard species in pH 2.5 is +2.

The results suggest that it is possible to fi nd the sensitivity for an analyte without the need to construct a calibration curve for said analyte, using only its mobility value. This strategy can be useful in instances where one doesn’t have the analyte standard, for example in cases of unstable species, new drugs, illicit drugs, and metabolites.

4 - ConclusionsThese results suggest that it is possible to determine the sensitivity of an analyte by only using its mobility value, without the need to construct a calibration curve. This strategy can be useful in instances where one does not have the analyte standard as is the case of unstable species, new drugs, illicit drugs, and metabolites. The method is valid for doubly charged analytes and partially charged BGE constituents.

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Keywords: Capillary Electrophoresis, Conductivity Detection, Indirect Calibration.

Acknowledgements: This work has been sponsored by CNPq (133325/2018-0 and 304415/2013-8) and FAPESP (2017/13137-5).

References:[1] Katzmayr MU, Klampfl CW, Buchberger W. J. Chromatogr. A 850: 355-362 (1999).[2] Brito-Neto JGA, Fracassi da Silva JA, Blanes L, Lucio do Lago CL. Electroanalysis 17: 1198-

1206 (2005).[3] Lucy CA, Wu Q. J. Chromatogr. Sci. 36: 33-36 (1998).[4] Rossi MR, Vidal DTR, do Lago CL. Food Chem. 133: 352-357 (2012).

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[PP-2]

A PROPOSAL FOR THE SENSITIVE DETERMINATION OF SEVEN NEONICOTINOIDS IN SOILS AND ENVIROMENTAL WATERS

BY MICELLAR ELECTROKINETIC CHROMATOGRAPHY

Laura Carbonell-Rozas, Francisco J. Lara, Monsalud del Olmo-Iruela and Ana M. García-Campaña

Department of de Analytical Chemistry, Faculty of Sciences, University of Granada, Granada, Spain. Email: [email protected]

1.- ObjectivesNeonicotinoids (NNIs) are a class of insecticides widely used to protect plants (i.e. crops, fruits, vegetables), livestock, and also pets from pest insect attack, acting selectively on the nicotinic acetylcholine receptors (nAChRs) in the central nervous system (CNS) of insects [1]. The main compounds belonging to this family, commercially available worldwide, are imidacloprid, thiacloprid, clothianidin, thiamethoxam, acetamiprid, nitenpyram and dinote-furan. Due to their high water solubility and relatively low molecular weight they can be easily incorporated into the plant tissues through roots or leaves and translocated to all parts of the plant, staying for a long time after application. Therefore, it is crucial to guarantee that they do not exceed the maximum residue limits (MRL) regulated by the Europe Union.

In recent years there have been a concern about the relationship between their uses and the Colony Collapse Disorder (CCD) syndrome, which involves the rapid loss of worker bees. Since 2013, three of these compounds (clothianidin, imidacloprid and thiamethoxam) have been restricted in plant protection products and treated seeds [2]. In light of these facts, there is a need for evaluating the environmental impacts of these systemic insecticides. It should be mandatory to propose efficient analytical procedures for monitoring these insecticides in environmental samples [3].

In this work, we propose for the first time the separation by MEKC-UV of seven NNIs together with the main metabolite 6-chloronicotinic acid (6-CNA). Besides, different sam-ple treatments for their efficient determination in water samples from different origins (riv-er, spring and well) as well as in soils from areas with high agricultural activity were also evaluated.

2.- MethodsA simple and efficient method has been developed and validated for the monitoring of the seven NNIs and 6-CNA. The MEKC mode was applied, using a capillary of 48.5 cm total length (50 µm i.d.) with an extended light-path (150 µm). The running electrolyte consisted of 25 mM sodium tetraborate buffer (pH 9.2) containing 120 mM of SDS and 15% of MeOH (v/v). The voltage was 27 kV with a temperature of 25ºC. The samples were dissolved in deionized water and hydrodynamically injected at 50 mbar for 12 s. UV-detection was per-formed at 220, 254 and 270 nm, depending on the analyte.

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This method was then applied to the analysis of NNIs residues in real samples. Two diff erent sample treatments were developed. For water samples, solid phase extraction (SPE) was checked using various cartridges (C18, Oasis® HLB, Oasis® HLB Prime and Strata-X), with thw best being Oasis® HLB was the best option for preconcentration and clean-up. In the case of soil samples, a simple solid-liquid extraction (SLE) was applied, using a mixture of 1:3 (v/v) ACN/dichloromethane.

3.- ResultsThe optimized MEKC-UV method was able to separate the studied NNIs in less than 12 min. On the one hand, SPE applied to water samples was an effi cient methodology for the extrac-tion, achieving an off -line preconcentration of 250-fold for eight NNIs. On the other hand, SLE was a simple sample treatment for soil samples, achieving an easy and fast procedure for the extraction of seven NNIs.

Good linearity was obtained (R2 >0.9929 for all NNIs). LODs and LOQs were less lower than 0.4 and 1.4 µg L−1 for river water and less than 2.9 and 9.5 µg kg−1 for soil samples, respectively. Satisfactory recoveries were achieved ranging from 80 to 107% for the studied NNIs in water samples of diff erent origin, and between 73 and 92% for soil samples.

Fig. 1: Electropherograms obtained by the proposed SPE-MEKC-UV method for a) a river water sample spiked with eight NNIs at 20 µg L-1. b) a blank of river water sample.

4.- ConclusionsIn comparison with previous CE based methods for monitoring of these residues in the com-parable samples [4], the proposed methodology is able to determinate more compounds in the range of the low µg/L or µg/kg, allowing the control of NNIs at relevant concentration levels using UV detection. In summary, it can be concluded that the proposed method can be considered as a powerful alternative to previously established LC and CE methods and can be satisfactorily applied in routine analysis for the monitoring of NNIs in environmental samples.

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Keywords: Neonicotinoids, micellar electrokinetic chromatography, environmental samples.

Acknowledgements: This work has been sponsored by the Spanish Ministry of Economy and Com-petitiveness (Project ref: AGL2015-70708-R). LCR thanks the Youth Employment Operational Pro-gram con-funded by the Junta de Andalucía and the European Social Fund (ESF). FJL is grateful for personal funding through the Special Research Program of the University of Granada.

References:[1] Ihara M, Matsuda M. Curr. Opin. Insect Sci. 30: 86-92 (2018).[2] Commission Implementing Regulation (EU) No 485/2013 of 24 May 2013 amending

Implementing Regulation (EU) No 540/2011, as regards the conditions of approval of the active substances clothianidin, thiamethoxam and imidacloprid, and prohibiting the use and sale of seeds treated with plant protection products containing those active substances. Official Journal of the European Union, L139, 12-26.

[3] van der Sluijs JP, Amaral-Rogers V, Belzunces LP, Bijleveld van Lexmond MF, Bonmatin JM, Chagnon M, Downs CA, Furlan L, et al. Environ. Sci. Pollut. Res. 22: 148-154 (2015).

[4] Ettiene G, Bauza R, Plata MR, Contento AM, Rios A, Electrophoresis. 33: 2969-2977 (2012).

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[PP-3]

A CHIRAL ANALYTICAL METHODOLOGY FOR COLCHICINE DETERMINATION USING NANO-LIQUID CHROMATOGRAPHY

AND AN AMYLOSE-BASED STATIONARY PHASE

Natalia Casado1, María Ángeles García1,2, Zhengjin Jiang3 and María Luisa Marina1,2


2 Institute of Chemical Reseach Andrés M. del Río (IQAR), University of Alcalá, Alcalá de Henares, Madrid, Spain

3 Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University, Guangzhou 510632, China

1.- ObjectivesTo develop a chiral analytical methodology for the antiuremic drug colchicine by nano-liquid chromatography (nano-LC), using an amylose-based stationary phase, and to apply it to the analysis of pharmaceutical formulations of this drug, which is marketed as a pure enantiomer (S-colchicine) [1].

2.- MethodsA laboratory-assembled nano-LC instrument was employed, consisting of a nano-gradient pump, an UV detector (at 220 nm) with a lab-made on-column detection cell and a four-port injection valve with a 20 nL internal loop. A lab-packed capillary column with amylose tris(3,5-dimethylphenylcarbamate) was used as the chiral stationary phase. Different param-eters, such as mobile phase composition, flow rate and operating pressure were optimized.

3.- ResultsThe best enantiomeric separation was achieved in normal phase using n-hexane/isopropanol (80/20, v/v) containing 0.2% of diethanolamine as mobile phase at a flow rate of 0.01 mL/ min at a constant pressure of 46 bar. Colchicine enantiomers were separated in less than 8 min with resolution of 2.3, the R-enantiomer eluting first (5.5 min) and then the S-enantiomer (7.4 min). The analytical characteristics of the method were evaluated, obtaining good line-arity (R2 > 0.999), recovery values (97-103%) and precision (RSD ≤ 2.6 % for elution times and RSD ≤ 2.7 % for peak areas). The method was successfully applied to the enantiomeric determination of colchicine in two different pharmaceutical formulations.

4.- ConclusionsThe developed methodology is fast and environmentally friendly, since it requires minimal amounts of organic solvents. It shows good analytical performance being able to detect the enantiomeric impurity as low as 0.1 % with respect to the active enantiomer. This fulfills the International Conference on Harmonisation (ICH) guidelines, so it can be used for the qual-ity control of colchicine pharmaceutical formulations.

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Keywords: Colchicine, nano-LC, Chiral separation, Enantiomers, amylose-based stationary phase

Acknowledgements: Authors thank the Spanish Ministry of Economy and Competitiveness for re-search project CTQ2016-76368-P. Authors also thank M. Montoya for technical assistance.

References:[1] Menendez-Lopez N, Valimana-Traverso J, Castro-Puyana M, Salgado A, Garcia MA, Marina

ML. J. Pharm. Biomed. Anal. 138: 189-196 (2017).

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[PP-4]

MODELING THE ENANTIOMERIC SEPARATION OF PHENOXY ACID HERBICIDES BY CE USING

A DUAL CYCLODEXTRIN SYSTEM

Natalia Casado1, José María Saz1, María Ángeles García1,2 and María Luisa Marina1,2



1.- ObjectivesOptimization of the separation conditions for enantiomers in CE when using two cyclodex-trins (CDs) in the separation buffer can be difficult when using a trial and error procedure. In this work, the model proposed by Dubsky et al. [1] was evaluated in the simultaneous enantiomeric separation of a mixture of phenoxy acid herbicides previously achieved by our research group using a dual CD-system consisting of hydroxypropyl-β-CD and hep-takis(2,3,6-tri-O-methyl)-β-CD [2].

2.- MethodsThe equations of Dubsky [1] established the relationship between the effective electropho-retic mobility of an analyte (mA,eff) and the total concentration of a mixture of chiral selectors (Ctot) and the molar fraction of each chiral selector (χi):

eq. (1) eq. (2) eq. (3)

3.- Results

For each CD and enantiomer, the association constant (Ki) and the electrophoretic mobility of the enantiomer-CD complex (mi) were determined using the model of Wren and Rowe [3], which is valid for CE systems using a single chiral selector. For each enantiomer, we also determined its free electrophoretic mobility (mA,f). Once these values were determined, the equations allowed us to obtain the theoretical value of mA,eff for each enantiomer, as a function of χi and Ctot in the CD mixture.

4.- ConclusionsThe results obtained allowed us to model the enantiomeric separation of the mixture of phe-noxy acid herbicides from a theoretical point of view.

Keywords: Chiral Capillary Electrophoresis, Enantiomers, Modeling, Phenoxy Acid Herbicides.

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Acknowledgements: Authors thank the Ministry of Economy and Competitiveness (Spain) for re-search project CTQ2016-76368-P.

References:[1] Dubsky P, Svobodov J, Gas B. J. Chromatogr. B 875: 30-34 (2008).[2] Valimana-Traverso J, Morante-Zarcero S, Perez-Quintanilla D, Garcia MA, Sierra I, Marina ML.

J. Chromatogr. A 1566: 146-157 (2018).[3] Wren SAC, Rowe RC. J. Chromatogr. 603: 235 (1992).

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[PP-5]

DETERMINATION OF PRIMARY AROMATIC AMINES IN KITCHENWARE BY CAPILLARY ELECTROPHORESIS

–TANDEM MASS SPECTROMETRY

Mary Ângela Favaro Perez1,2, Marisa Padula1, Carla Beatriz Grespan Bottoli2, Claudimir Lucio do Lago3 and Daniela Daniel4

1 Food Technology Institute, Ital, Campinas, Brazil. Email: [email protected] 2 Institute of Chemistry, University of Campinas (UNICAMP), Campinas, Brazil.

Email: [email protected] Institute of Chemistry, University of São Paulo, Sao Paulo (USP), Brazil4 Agilent Technologies, Inc., São Paulo, Brazil

1.- ObjectivesA capillary electrophoresis method coupled to a tandem Triple Quadrupole mass spectrom-eter (CEMS/MS) was developed and validated for determining 19 primary aromatic amines (PAAs). These amines are classified as human carcinogens and can migrate from food con-tact materials under acidic conditions. Thirty-six samples of kitchenware were analyzed.

2.- MethodsThe PAAs migration studies were carried out following European Standard EN 13130-1:2004 [1], and the technical guide [2] for the migration of PAAs.

The method was validated according to DOC-CGRE 008 guide from INMETRO [3]. Param-eters such as linearity, limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy were evaluated.

The present study analyzed 36 samples of kitchenware obtained from retail markets: 16 made of polyamide (PA), one made of polypropylene (PP), and 19 made of silicone. The PA sam-ples were: seven from Brazil, seven from China, and two from Turkey. The PP and silicone samples were from China (Fig. 1).

Fig. 1: Samples of kitchenware obtained from retail markets. First line: 16 samples of polyamide and the only sample of polypropylene (last piece). Second line: 19 samples of silicone.

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The CE-MS/MS analysis was performed by using Agilent 7100 CE system coupled to an Agilent 6430 Triple Quadrupole mass spectrometer.

3.- ResultsParameters related to the CE-MS/MS interface, background electrolyte (BGE), and mass spectrometer (MS) settings were optimized providing a separation of 19 PAAs in six minutes.

LOQ and LOD were 1.7 and 5.7 μg kg-1, respectively. Linearity was obtained over a range of 5 - 100 μg kg-1 with the coefficient of determination (R2) > 0.99 for all amines.

The precision (repeatability) in RSD ranged from 1 to 27% for 5 μg kg-1, 4 to 21% for 10 μg kg-1, and 7 to 17% for 30 μg kg-1. These values were consistent with the literature [3] We can find 21% in a range of 10-30 μg kg-1 , and 30% for 5 μg kg-1. The accuracy was 85-120% for 5 μg kg-1, 91-112% for 20 μg kg-1 and 95-106% for 40 μg kg-1. These values were also consist-ent with the literature [3] are 60-115% in a range of 20-40 μg kg-1, and 40-120% for 5 μg kg-1.

The results of specific migration for PAAs showed that two samples from Brazil and three samples from China made of PA were above the permissible limit (10 μg kg-1) for 4,4´-di-aminodiphenylmethane. Only three samples of silicone also showed the same amine above the limit permitted by legislation.

Five samples made with polyamide showed values of aniline above the limit permitted by legislation.

4.- ConclusionsA CE-MS/MS method was developed for quantification of 19 PAAs in kitchenware. Accept-able parameters for linearity, LOD, LOQ, repeatability, and accuracy were obtained during method validation.

4,4´-diaminodiphenylmethane and aniline were quantified at high concentration in the kitchenware.

The results showed that these amines need to be monitored in order to protect the health of consumers.

Keywords: primary aromatic amines, specific migration, capillary electrophoresis, mass spectrometry.

Acknowledgements: This work has been sponsored by FAPESP - São Paulo Research Foundation, Brazil (2015/26300-6 and 2014/50867-3), CNPq - National Council for Scientific and Technological Development, Brazil (465389/2014-7 and 3116712015-2), and INCTBio – The National Institute of Bioanalytical Science and Technology, Brazil.

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References:[1] European Committee for Standardization (CEN), EN 13130-1:2004, “Materials and articles

in contact with foodstuffs - plastics substances subject to limitation - part 1: guide to test methods for the specific migration of substances from plastics to foods and food simulants and the determination of substances in plastics and the selection of conditions of exposure to food simulants”, Brussels, Belgium, 2004.

[2] Simoneau, C. “Technical Guidelines on Testing the Migration of Primary Aromatic Amines from Polyamide Kitchenware and of Formaldehyde from Melamine Kitchenware”, 1st edition, European Commission. Luxembourg, 2009.

[3] INMETRO, Coordenação Geral de Acreditação. “DOQ-CGCRE-008: orientação sobre validação de métodos analíticos”. Brazil, 2016.

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[PP-6]

CHIRAL CAPILLARY ELECTROHORESIS FOR THE SEPARATION OF IVABRADINE ENANTIOMERS. QUANTITATIVE

ANALYSIS IN PHARMACEUTICAL FORMULATIONS

Natalia Casado1, Antonio Salgado2, María Castro-Puyana1,3, María Ángeles García1,3 and María Luisa Marina1,3

1 Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, University of Alcalá, Alcalá de Henares, Madrid, Spain

2 Center of Spectroscopy and Nuclear Magnetic Resonance (CERMN) Center for Support to Research in Chemistry (CAIQ), University of Alcalá, Alcalá de Henares, Madrid, Spain

3 Institute of Chemical Reseach Andrés M. del Río (IQAR), University of Alcalá, Alcalá de Henares, Madrid, Spain. Email: [email protected]

1.- ObjectivesThe aim of this work was to develop an analytical methodology by CE enabling the enantio-meric separation of ivabradine, a novel anti-ischemic and heart rate lowering drug commer-cialized as a pure enantiomer (S-ivabradine), and to apply it to the quantitative analysis of ivabradine in pharmaceutical formulations.

2.- MethodsA set of twenty-one cyclodextrins (CDs) and eleven amino acid-based chiral ionic liquids (CILs) were evaluated as sole chiral selectors and combined as dual chiral systems. Different experimental conditions such as the concentration of chiral selectors, the buffer composition, the pH, the temperature and the separation voltage were optimized. Moreover, NMR exper-iments were carried out to obtain information about the stoichiometry and the apparent and averaged equilibrium constants of the enantiomer-CD complexes.

3.- ResultsIvabradine enantiomers were separated in 6 min with an enantiomeric resolution of 2.7. A synergistic effect was observed when combining sulfated-γ-CD and different CILs in dual chiral systems, which significantly increased resolution (up to a value of 5.7 by adding 5 mM [TBA]+[L-Asp]- to the separation buffer). Nevertheless, no inversion in the migration order was observed under these conditions. The analytical characteristics of the optimized method using sulfated-γ-CD as chiral selector were evaluated and it was applied to the enantiomeric determination of ivabradine in a pharmaceutical formulation.

4.- ConclusionsThe developed chiral methodology shows good performance to ensure quality control of ivabradine pharmaceutical formulations,The limit of detection was <0.1 % of the enantio-meric impurity, which complies with the International Conference on Harmonisation (ICH) guidelines.

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Keywords: Chiral Capillary Electrophoresis, Enantiomers, Ivabradine, Cyclodextrins, Ionic Liquids.

Acknowledgements: Authors thank the Spanish Ministry of Economy and Competitiveness (MI-NECO) for project CTQ2016-76368-P and the Comunidad of Madrid and European funding from FSE and FEDER programs for project S2018/BAA-4393 (AVANSECAL-II-CM). M.C.P. thanks MINECO for her “Ramón y Cajal” research contract (RYC-2013-12688). Authors thank the Center for Applied Chemistry and Biotechnology (CQAB) (University of Alcalá) for the synthesis of the CILs.

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[PP-7]

ENANTIOSEPARATION OF AMINO ACIDS USING AN O-[2- (METHACRYLOYLOXY)-ETHYLCARBAMOYL]-10,11-

DIHYDROQUINIDINE-SILICA HYBRID MONOLITHIC COLUMN BY NANO-LC. DETERMINATION OF NORVALINE

AND TRYPTOPHAN IN FOOD SUPPLEMENTS

Dongsheng Xu1,2,3, Elena Sánchez-López1,4, Qiqin Wang2,3, Zhengjin Jiang2,3 and María Luisa Marina1,4


2 Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University, Guangzhou, China

3 Department of Pharmacy and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of traditional Chinese Medicine and New Drug Research, Jinan University, Guangzhou, China

4 Institute of Chemical Reseach Andrés M. del Río (IQAR), University of Alcalá, Alcalá de Henares, Madrid, Spain. Email: [email protected]

1.- ObjectivesAmino acids (AAs) play a crucial role in living organisms. Differences in properties of D- and L-AAs have been widely reported both in protein and non-protein AAs. Thus, it is im-portant to control the presence of both enantiomers. In this work, an analytical methodology was developed by nano-LC for the enantiomeric separation of protein and non-protein AAs using an O-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroquinidine (MQD)-silica hybrid monolithic column synthesized by our research team [1]. The developed methodolo-gy was applied to the determination of L-norvaline and L-tryptophan in food supplements.

2.- MethodsAn MQD-silica hybrid monolithic column (15 cm length x 100 µm internal diameter) was employed. The nano-LC was a laboratory self-assembled instrument with UV-Vis detection. Twenty-seven 9-fluorenylmethoxycarbonyl (FMOC)-AAs (19 protein + 8 non-protein) were analyzed.

3.- ResultsDifferent parameters such as mobile phase apparent pH, acetonitrile content, and buffer con-centration, were optimized to allow the best enantioresolution. Nineteen AAs were enantio-merically discriminated, 11 being baseline separated. The method showed good performance in the determination of L-norvaline and L-tryptophan in food supplements. Levels of L-nor-valine and L-tryptophan were in agreement with labelled contents except for one sample that did not show presence of L-norvaline, contrary to the labelled indication.

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4.- ConclusionsThe developed strategy based on the use of the MQD-silica hybrid monolithic column has shown to be useful for the enantiomeric separation of FMOC-AAs and for the determination of L-norvaline and L-tryptophan in food supplements.

Keywords: amino acids, chiral separation, nano-LC, quinidine monolithic column.

Acknowledgements: M.L.M and E.S.L. thank the Ministry of Economy and Competitiveness (Spain) for research projects CTQ2013-48740-P and CTQ2016-76368-P. They also thank the Comu-nidad of Madrid (Spain) and European funding from FEDER program for research project S2013/ABI-3028 (AVANSECAL-CM) and for D.X. and E.S.L.´s contracts.

References:[1] Xu D, Wang Q, Sanchez-Lopez E, Jiang Z, Marina ML. J. Chromatogr. A 1593: 63-72 (2019).

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[PP-8]

GLYCOPEPTIDE ANALYSIS BY ON-LINE TiO2 SOLID-PHASE EXTRACTION CAPILLARY

ELECTROPHORESIS-MASS SPECTROMETRY

Estela Giménez, Montserrat Mancera-Arteu, Nejsi Lleshi, Fernando Benavente and Victoria Sanz-Nebot

Department of Chemical Engineering and Analytical Chemistry, Institute for Research on Nutrition and Food Safety (INSA·UB), University of Barcelona, Barcelona, Spain. Email: [email protected]

1.- ObjectivesThis study describes an on-line solid-phase extraction capillary electrophoresis-mass spec-trometry (SPE-CE-MS) method, using titanium dioxide (TiO2) nanoparticles for the selec-tive purification and preconcentration of glycopeptides, obtained from enzymatic digests of glycoproteins, analyzed in bottom-up proteomic approaches.

2.- MethodsRecombinant human erythropoietin (rhEPO), bovine alpha-acid glycoprotein (AGP) and apolipoprotein C3 (APO-C3) standard glycoproteins were subjected to enzymatic digestion with trypsin. For TiO2-SPE-CE-MS, a single-frit particle-packed microcartridge was inte-grated in-line, near the entrance of the separation capillary, and no valves were necessary for the operation. First, the sorbent was conditioned with binding buffer. After loading the sample (~100 µL) and several washing steps, retained glycopeptides were eluted and glyco-peptide glycoforms were separated and detected by CE-MS.

3.- ResultsThe O126-glycopeptide from rhEPO was used to optimize the methodology. Under the opti-mized conditions, the microcartridge lifetime was around 10 analyses and the repeatability was acceptable (%RSD values of 9-11% and 6-11% for migration times and peak areas, respectively). The method was linear between 0.5 and 50 mg·L-1 for O126 glycoforms con-taining NeuAc. The LOD was 0.25 mg·L-1 which was 100 times lower than by CE-MS. The methodology was later tested to include APO-C3 and AGP, thereby demonstrating its applicability for the analysis of glycopeptides with different glycan composition and nature.

4.- ConclusionsThe established TiO2-SPE-CE-MS method made possible, to substantially increase detec-tion sensitivity of glycopeptides of the studied glycoproteins in comparison to regular CE-MS, without compromising separation between glycoforms. Its application to study other glycoproteins deemed as relevant biopharmaceuticals or biomarkers for a wide variety of diseases is also possible.

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Keywords: glycopeptides; capillary electrophoresis; mass spectrometry; on-line solid-phase extraction.

Acknowledgements: This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (RTI2018-097411-B-I00) and the Cathedra UB Rector Francisco Buscarons Úbeda.

References:[1] Pont L, Pero-Gascon R, Gimenez E, Sanz-Nebot V, Benavente F. Anal. Chim. Acta 1079: 1-19

(2019).

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[PP-9]

DISPERSIVE MICRO SOLID PHASE EXTRACTION USING MODIFIED MAGNETITE NANOPARTICLES

WITH IONIC SURFACTANTS

Yolanda Martín-Biosca1, M. Pérez-Baeza1, R. Muñoz-Espí2, L. Escuder-Gilabert1, S. Sagrado1,3 and M. J. Medina-Hernández1

1 Department of Analytical Chemistry, University of Valencia, Burjassot, Spain. Email: [email protected]

2 Institute of Material Science (ICMUV), University of Valencia, Paterna, Spain3 Interuniversity Institute of Molecular Recognition Research and Technological

Development (IDM), Polytechnic University of Valencia, University of Valencia, Valencia, Spain

Hemimicelle/admicelle solid phase extraction (SPE) has been widely used for sample prepa-ration. It uses surfactant monolayers (hemimicelles) or surfactant bilayers (admicelles) ad-sorbed onto an oppositely charged solid support as sorbent materials. Hemimicelles and admicelles possess regions of different polarities. The surfactant hydrocarbon chains of hem-imicelles provide hydrophobic interactions for hydrophobic analytes, while the presence of ionic groups in admicelles allows the electrostatic interactions with ionic analytes. Therefore, they allow the adsolubilization of analytes of different nature. In hemimicelles/admicelles dispersive solid phase extractions the solid support can affect the formation process of hem-imicelle/admicelle SPE which determines the loading capacity. The use of magnetic nano-particles (NPs) as solid support offers several advantages such as high extraction capacity, large breakthrough volumes, simple analyte desorption and easy regeneration of sorbents. Among them, magnetite-NPs show great advantages due to their strong magnetism, low toxicity, high surface area, easy surface modification and low cost.

1.- ObjectivesThe objective of this communication is to study the potential of hemimicelles and admicelles of sodium dodecylsulphate (SDS) and cetyltrimethyl ammonium bromide (CTAB) adsorbed onto magnetite-NPs for preconcentration of low amounts of cationic and anionic compounds.

2.- MethodsIn this communication, a comprehensive study about the parameters that affect the extraction recovery such as the ratio of the amount of magnetite-NPs to the ionic surfactant (SDS and CTAB), extraction time, sample pH, ionic strength, desorption conditions and sample vol-ume for different analytes has been carried out.

3.- ResultsFor basic pH, magnetite-NPs present negative charge, therefore electrostatic interaction and CTAB adsorption occurs. On the other hand,, acid pH is required for SDS adsorption. The

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concentration of the surfactant determines the formation of hemicelles and admicelles and subsequently the net charge of the sorbent. For the extraction of anionic and cationic com-pounds CTAB and SDS admicelles respectively provide recoveries close to 100%.

4.- ConclusionsSample pH is a key parameter that determines the net charge of magnetite-NPs and therefore the nature of the surfactant to be used. Surfactant concentration is also an important variable to be considered since admicelles are required to extract ionic compounds

Keywords: Dispersive solid phase extraction; Magnetite-NPs; CTAB; SDS; Acidic and basic compounds.

Acknowledgements: This work has been supported by the Project CTQ2015-70904-R (MINECO/FEDER, UE).

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[PP-10]

ASSESSMENT OF A HPLC METHOD WITH IN-LINE DIODE-ARRAY, FLUORESCENCE AND ELECTROCHEMICAL

DETECTORS FOR THE SIMULTANEOUS AND RELIABLE DETERMINATION OF PARABENS IN COSMETIC PRODUCTS

L. Abad , S. Lucas, M.T. Sevilla, M.J. Gismera and J.R. Procopio

Department of Analytical Chemistry and Instrumental Analysis, Faculty of Sciences, Autonomous University of Madrid, Madrid Spain. E-mail: [email protected]

1. - ObjectivesParabens are a group of substances that are widely used in cosmetics and personal care products to prevent the growth of microbes. However, the continued use of parabens may constitute a potential risk for human health. Parabens were regulated in the European Union (EU) in 2009 as preservatives in cosmetic products [1]. Isopropylparaben (iPP), isobutylpar-aben (iBP), phenylparaben, benzylparaben (BzP) and pentylparaben were banned in the EU in 2014. This regulation also set the maximum concentration limits for butylparaben (BP) or propylparaben (PP) as 0.14%, for methylparaben (MP) or ethylparaben (EP) as 0.4%, and for the mixture of these four ingredients in 0.8%, wherein the sum of the individual concen-tration of BP and PP cannot exceed 0.14 % [2]. In this context, adequate analytical tools are needed to establish compliance with the current standards in order to protect the health of consumers.

In this work, it is described a HPLC method with three in-line detection systems: diode-array (DAD), fluorescence (FLD) and electrochemical (ECD) detector, for the simultaneous deter-mination of MP, EP, iPP, PP, BP and BzP in cosmetic products.

2. - MethodsThe spectroscopic and electrochemical properties of the target analytes are studied to deter-mine out the optimal detection conditions. Absorbance and fluorescence spectra of parabens are obtained at pH values ranging between 2 to 8. The electrochemical behaviour is also studied by cyclic voltammetry using disposable screen-printed carbon electrodes (SPCEs). Hydrodynamic curves are obtained in order to establish the suitable detection potential. The hydrodynamic curves and electrochemical detection in amperometric mode are carried out using a thin-layer flow cell with SPCEs. The chromatographic conditions on a C-18 reversed-phase column are optimized using DAD. In order to obtain the best separation con-ditions of parabens, acetonitrile percentages ranging from 35% to 75% are assessed under isocratic conditions. The influence of the pH of the mobile phase and the flow rate on resolu-tion and retention time is also studied.

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3. – ResultsAccording to the spectroscopic results, DAD operates at 257 nm and wavelengths of ex-citation and emission of 257 nm and 315 nm, respectively, are set for FLD. From the hy-drodynamic curves, a +1.0 V (vs. Ag/AgCl) potential is chosen as the optimum to operate the ECD. Adequate resolutions and retention times are obtained employing a mobile phase consisting of 35:65 (v/v) acetonitrile: 0.01 M phosphate buffer (pH 2) at a flow rate of 1.5 mL min-1. Under these chromatographic and detection conditions, the separation of the six target analytes is achieved in less than 18 minutes. Figure 1 shows the typical chromatograms obtained in the optimized separation and detection conditions. The method shows high pre-cision (RSD <6%) for all detection modes. The limits of quantification, in the range of 3 to 426 µg·L-1, allow the determination of parabens according to the European legislation [2]. More sensitive and selective detection is obtained using FL and EC detection modes. The HPLC method with DAD, FLD and ECD is used to quantify parabens in cosmetic products. To validate the proposed method, the analyzed samples were spiked and good recoveries were obtained. Most of the studied samples present some interference for DAD and FLD. Therefore, the more selectivity of ECD makes the identification and quantification of para-bens more reliable.

Fig. 1 Typical chromatograms of MP (a), EP (b), iPP (c), PP (d), BP (e), BzP (f) in the optimized separation and detection conditions.

4. - ConclusionsThe proposed chromatographic method with in-line detectors (DAD-FLD-ECD) is suitable for the determination of MP, EP, iPP, PP, BP and BzP. The limits of detection and quantifica-tion at the microgram per litre are far below the established levels in the European legislation. The use of more selective detectors is required due to the complexity of cosmetic products matrices.

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Keywords: Cosmetic products; HPLC; In-line detectors; Parabens; Screen-printed carbon electrodes

References:[1] Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November

2009 on cosmetic products. Official Journal of the European Union 22.12.2009.[2] Commission Regulation (EU) No 1004/2014 of 18 September 2014 amending Annex V to

Regulation (EC) No 1223/2009 of the European Parliament and of the Council on cosmetic products. Official Journal of the European Union 26.9.2014.

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[PP-11]

ENANTIOMERIC DETERMINATION OF THE AGROCHEMICAL PROTHIOCONAZOLE

BY CHIRAL CAPILLARY ELECTROHORESIS

Sara Jiménez-Jiménez1, María Castro-Puyana1,2, María Ángeles García1,2, and María Luisa Marina1,2.



1.- ObjectivesThis work was sought to develop a capillary electrophoresis (CE) methodology to achieve the fast enantiomeric separation of the antifungal agent prothiconazole ((R,S)-[2-(1-chlorocyclopropyl)-3-(2-chlorophenyl)-2-hydroxypropyl]-1,2-dihydro-3H-1,2,4-triazole-3-thione), and to apply to its quantitation in commercial agrochemical formulations.

2.- MethodsAmong the 11 cyclodextrins (CDs) evaluated as chiral selectors, sulfated-γ-CD and heptakis-2,3,6-tri-O-methyl-β-CD showed superior chiral discrimination power The lat-ter CD gave rise to shorter analysis times than sulfated-γ-CD. This CD was selected to develop a chiral methodology for the determination of prothioconazole. The effects of CD concentration, temperature and separation voltage on the enantiomeric resolution was investigated.

3.- ResultsProthioconazole enantiomers were separated in 4.5 min with a resolution of 2.8. The analyti-cal characteristics of the method were evaluated and showed good performance for the deter-mination of prothioconazole. The method was applied to the quantitation of prothioconazole in a commercial agrochemical formulation.

4.- ConclusionsThis is the first time that prothioconazole enantiomers were separated by Capillary Electro-phoresis. The developed method enables the fast quantitation of this antifungal in commer-cial agrochemical formulations.

Keywords: Chiral Capillary Electrophoresis, Enantiomers, Cyclodextrins, Prothioconazole, Agro-chemical Formulations.

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Acknowledgements: Authors thank financial support from the Spanish Ministry of Economy and Competitiveness for research project CTQ2016-76368-P. M.C.P. also thanks this Ministry for her “Ramón y Cajal” research contract (RYC-2013-12688). S. Jiménez-Jiménez thanks the University of Alcalá for her research grant. Authors thank A. Martín and L. Cortés for technical assistance.

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[PP-12]

STUDY OF COMBINED TOXICITY OF DULOXETINE AND ECONAZOLE ON NON-TARGET ORGANISMS USING CHIRAL CAPILLARY ELECTROPHORESIS

Jesús Valimaña-Traverso1, Georgiana Amariei1, Karina Boltes1,2, María Ángeles García1,3 and María Luisa Marina1,3

1. Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, University of Alcalá, Alcalá de Henares, Madrid, Spain. Email: [email protected]

2. Madrid Institute for Advances Studies of Water (IMDEA Agua), Scientific Technological Park, Alcalá de Henares, Madrid, Spain

3. Institute of Chemical Reseach Andrés M. del Río (IQAR), University of Alcalá, Alcalá de Henares, Madrid, Spain

1.- ObjectivesThe aim of this work was to evaluate the EC50 toxicity parameters of duloxetine and econa-zole and their mixtures on two non-target organisms (the aquatic plant, Spirodela polyrhiza and the microcrustacean Daphnia magna) using chiral Capillary Electrophoresis (CE) for the determination of the real concentrations of these emergent contaminants.

2.- MethodsA CE methodology enabling the simultaneous enantiomeric separation of duloxetine and econazole in 7.5 min with enantiomeric resolutions higher than 6.5 was employed in this work to determine real (and not nominal) concentrations of both drugs in order to calculate toxicity parameters. Results for toxicity parameters were corroborated by measuring chlo-rophyll fluorescence emission for the aquatic plant and by evaluating the oxidative stress using fluorescence images in the case of the microcrustacean. Combination indexes were used to obtain information on the type and level of interactions of the drug mixture with the organisms.

3.- ResultsEC50 values allowed us to include both drugs and their mixtures within the group of very toxic compounds for both aquatic organisms. Econazole shows greater toxicity than dulox-etine on Spirodela polyrhiza while the toxicity of duloxetine on Daphia magna was higher than that of econazole. A strong synergism was observed at 48 h exposure time and any effect level, which demonstrated the high toxicity of the drug mixture compared with the individual drug solutions on Daphnia magna. For Spirodela polyrhiza, the combined effect of drugs revealed slight to moderate antagonism above the EC50 value and additivity below EC50, showing a different profile which must be considered in risk assessment for aquatic environments.

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4.- ConclusionsThe results obtained demonstrated the potential of CE to evaluate the toxicity of emerging contaminants on non-target organisms. This is the first time that the combined toxicity of both drugs on Daphnia magna and the toxicity of duloxetine, econazole and their mixtures on an aquatic plant, were evaluated.

Keywords: Chiral Capillary Electrophoresis, Enantiomers, Cyclodextrins, Duloxetine, Econazole, Toxicity, Non-target Organisms.

Acknowledgements: M.L.M., M.A.G, J.V.T. thank the Ministry of Economy and Competitiveness (Spain) for project CTQ2016-76368-P. J.V.T. thanks the same Ministry for his predoctoral contrat (BES-2014-070532). G.A. and K.B. thank financial support from the Dirección General de Univer-sidades e Investigación of the Comunidad of Madrid (Spain), Research Network S2013/MAE-2716.

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[PP-13]

IN-SOURCE FRAGMENTATION FOR IDENTIFICATION OF MODIFIED LYSINES

BY CE-ESI(+)-TOF-MS IN BIOLOGICAL MATRICES

Maricruz Mamani-Huanca, Ana Gradillas, Ángeles López-Gonzálvez and Coral Barbas

Centre for Metabolomics and Bioanalysis (CEMBIO), Chemistry and Biochemistry Department, Faculty of Pharmacy, University CEU San Pablo, Campus Monteprincipe, Boadilla del Monte, Madrid, Spain. E-mail: [email protected]

1.- BackgroundAmong the 20 proteinogenic amino acids, L-lysine (Lys) residue is one of the most heavily modified. These modified Lys residues are cleaved from the proteins and usually appear as “unknowns” during the annotation of compounds in untargeted metabolomics. Lys modifica-tions play key roles in the mechanisms of aggregation and toxicity of proteins. Those mod-ified residues are also key drivers in regulating many biological processes that range from growth and proliferation to pathological conditions, such as neurodegeneration and cancer.

2.- ObjectivesIn our work, we present a method for identification of modified L-lysines using character-istic fragment ions from in-source fragmentation (ISF) based on CE-ESI(+)-TOF-MS. We present a structure-based fragmentation study with special attention to the mechanisms of the fragmentation reactions.[1] We also explore how complex matrices influence on the frag-mentation behavior.

3.- MethodsStandards solution (25 mg/mL) of a set of modified L-lysines were prepared with 0.2 mM of methionine sulfone. Human urine, plasma, macrophages, neutrophils and Leishmania sp samples were used to explore fragmentation behaviour. All samples were analysed using a CE-ESI-TOF system in normal polarity in a fused-silica capillary using an optimal modified method developed in CEMBIO[2].

4.- ResultsSetting the fragmentor voltage at 100 and 200 V, a full fragmentation spectra was obtained yielding an in-house library of fragments for modified L-lysines along with their protonated pseudomolecular ions [M+H]+, adducts and multimers. Based on the ISF pattern observed, a comprehensive study of the mechanisms involved in the fragmentation reactions was per-formed. With this interpretation we have contributed to the knowledge in the mechanisms involved in the generation of characteristic fragment ions with high diagnostic value. The results obtained were used to design a workflow for the identification of known and unknown modified L-lysines present in human plasma based on the diagnostic ions. Selected modified L-LysH+ were further tested in different biological matrices, analyzed under our optimal conditions, to explore how the matrix influences the fragmentation behavior. In all studied

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samples the fragmentation patterns were highly reproducible; however, variations in the ion signal intensity were observed due to the co-elution with other compounds in the matrix.

5.- ConclusionsWe have developed an analytical procedure and a strategy to identify modified L-LysH+ in biological samples based on experimental characteristic fragment ions generated by sup-ported theoretical fragmentation mechanisms. We have also proved that the fragmentation is independent of the biological matrix.

Keywords: diagnostic ion, in-source fragmentation, modified lysines.

References:[1] Demarque DP, Crotti AEM, Vessecchi R, Lopes JLC, Lopes NP. Nat. Prod. Rep. 33: 432-455

(2016).[2] Godzien J, Armitage EG, Angulo S, Martinez-Alcazar MP, Alonso-Herranz V, Otero A, Lopez-

Gonzalvez A, Barbas C. Electrophoresis 36: 2188-2195 (2015).

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[PP-14]

ENANTIOMERIC DETERMINATION OF ECONAZOLE AND SULCONAZOLE IN PHARMACEUTICAL

FORMULATIONS BY CE USING HYDROXYPROPYL-β-CD COMBINED WITH CHIRAL IONIC LIQUIDS

Sandra Salido-Fortuna1, María Castro-Puyana1,2 and María Luisa Marina1,2



1.- ObjectivesThe aim of this work was to optimize the enantiomeric separation of econazole and sulcona-zole by CE using dual chiral systems composed of hydroxypropyl-β-cyclodextrin (HP-β-CD) and chiral ionic liquids (CILs) and to apply the developed methods to the enantiomeric quantitation of these drugs in pharmaceutical formulations.

2.- MethodsFollowing previous results of our research group showing the discrimination power of dual chiral systems composed of HP-β-CD and CILs for econazole and sulconazole [1], the enan-tiomeric separation of these drugs was optimized. Tetrabutylammonium-L-lysine ([TBA][L-Lys]) or tetrabutylammonium-L-glutamic acid ([TBA][L-Glu]) were used as CILs. The effect of the buffer concentration, nature and concentration of CIL, temperature and applied voltage was investigated in order to optimize enantiomeric resolutions and analysis time.

3.- ResultsEnantiomeric resolutions of 3.6 in 12 min for econazole and 2.4 in 18 min for sulcona-zole were obtained. Analytical characteristics of the developed methodology were evaluated showing a good performance for the determination of these drugs in pharmaceutical formula-tions with limits of detection of 1.3 and 1.6 mg/L for econazole and sulconazole, respectively.

4.- ConclusionsThe combination of HP-β-CD and [TBA][L-Lys] as a dual chiral system in CE enabled us to develop analytical methodologies for the enantiomeric determination of econazole and sulconazole in pharmaceutical formulations. The results showed a good agreement between the label claims and amounts found for these drugs in the final products

Keywords: Chiral Capillary Electrophoresis; Cyclodextrin; Chiral Ionic Liquids; Econazole; Enan-tioseparation; Sulconazole; Quantitation; Pharmaceutical formulations.

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Acknowledgements: Authors thank the Spanish Ministry of Economy and Competitiveness (MI-NECO) for project CTQ2016-76368-P and the Comunidad of Madrid and European funding from FSE and FEDER programs for project S2018/BAA-4393 (AVANSECAL-II-CM). M.C.P. thanks MINECO for her “Ramón y Cajal” research contract (RYC-2013-12688). Authors thank the Center for Applied Chemistry and Biotechnology (CQAB) (University of Alcalá) for the synthesis of the CILs. Authors also thank C. Macías and J. Gila for technical assistance.

References:[1] Salido-Fortuna S, Greno M, Castro-Puyana M, Marina ML. J. Chromatogr. A 2019. DOI:

10.1016/j.chroma.2019.460375.

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[PP-15]

EVALUATION OF SELECTIVE POLYPHENOL INDEXES IN WINES BASED ON CAPILLARY ELECTROPHORESIS

WITH ELECTROCHEMICAL DETECTION

Alberto Sánchez Arribas1, Mónica Moreno1, Noelia Blázquez1, Eugenia Alonso1, Esperanza Bermejo1, Antonio Zapardiel2 and Manuel Chicharro1

1 Department of Analytical Chemistry and Instrumental Analysis, Autonomous University of Madrid, Madrid, Spain. Email: [email protected]

2 Department of Analytical Sciences, National University of Education at the Distance (UNED), Madrid, Spain

1.- ObjectivesThe main goal of this work was the application of class-selective electrochemical polyphe-nol indexes for the characterization of the polyphenol content of wine and related products. Therefore, the electrophoretic separation and subsequent electrochemical detection of repre-sentative polyphenols was studied. Under appropriate experimental conditions, the selective co-migration of structure-related polyphenols, namely flavonoids, hydroxycinnamic acids, and benzoic acids, was able to be achieved.

2.- MethodsCapillary electrophoresis separations were carried out using a P/ACE-MDQ system (Beck-man Coulter). An electrochemical cell [1] was coupled to this equipment, which enabled am-perometric detection. A glassy carbon electrode was employed as working electrode whereas silver and platinum wires served as reference and counter electrodes, respectively. Sample treatment consisted of proper dilution and filtration before introduction into the electrophore-sis equipment.

3.- ResultsThe composition and nature of the separation buffer was studied using a model polyphenol mixture (caffeic, coumaric, ferulic, gallic, sinapic, and syringic acids, and catechin and rutin, 10 mM each). The best separation conditions were attained when using 20 mM acetic acid buffer pH 5.0 as background electrolyte combined with a separation voltage of 25.0 kV at 25ºC.

Hydrodynamic voltammograms were built using the electropherograms of individual polyphenols obtained under these conditions at increasing detection potentials. Increasing amperometric signals were obtained from +0.10-0.20 V up to +0.75 V for all of the tested compounds, therefore +0.70 V was set as detection potential as a compromise between sen-sitivity and low background signal.

Wine samples were subsequently analysed and representative signals of each target polyphenol classes were observed. The stability and reproducibility in both the migration times and the analytical signals corresponding to samples analysed up to date were fair,

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with calculated RSD values below 8.3 % in all cases. In addition, preliminary studies were conducted for the evaluation of the recovery, showing good recovery values (95-102 %) for all polyphenols tested. Therefore, the estimation of total flavonoids (tF), total hydroxycin-namic acids (tHC) and total hydroxybenzoic acids (tHB) indexes was accomplished based on these results.

Fig. 1 Representative electropherogram of model polyphenol mixture under selected separation and detection conditions.

4.- ConclusionsThe proposed analytical approach based on the combination of capillary electrophoretic sep-aration and electrochemical detection has proved its ability to differentiate between some polyphenolic groups based on their characteristic chemical structure. This promoted the esti-mation of electrochemical polyphenol indexes in a fast, selective and reliable way. The ana-lytical information obtained can supplement that provided by traditional spectrophotometric or chromatographic methodologies for the characterization of wines and related products. Currently there is work in progress focused in the comparison of the information offered with this methodology and the polyphenol content estimated using spectrophotometric methods described for wines (Folin-Ciocalteu or the Total Phenolic Index).

Keywords: Electrochemical detection; Flavonoids; Phenolic acids; Polyphenol index; Wine.

Acknowledgements: The authors wish to thank Spanish Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER) for the financial support (Grant

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CTQ2015-64505-R). N. Blázquez (PEJ-2017-TL/IND-7302) and E. Alonso (PEJ-2018-TL/IND-11539) thank for their contracts funded by Comunidad de Madrid and Fondo Social Europeo.

References:[1] Sánchez-Arribas A, Martínez‐Fernández M, Moreno M, Bermejo E, Zapardiel A, Chicharro M.

Electrophoresis 35: 1693-1700 (2014).

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[PP-16]

A NOVEL APPROACH TO SCREEN FOR NATURAL EXTRACTS WITH INHIBITORY ACTIVITY AGAINST

MICROBIAL CARNITINE MONOOXYGENASE USING CAPILLARY ELECTROPHORESIS

Júlia Boldú, Carolina Simó and Virginia García-Cañas

Laboratory of Molecular Nutrition and Metabolism, Institute of Food Science and Research (CIAL), Spanish National Research Council (CSIC), Madrid, Spain. E-mail: [email protected]

1.- ObjectivesElevated levels of trimethylamine N-oxide (TMAO) in plasma has been recently proposed as a risk factor in cardiovascular disease and has shown to have prognostic value in various studies [1]. TMAO is generated via a metaorganismal pathway that starts with conversion of dietary precursors (choline, L-carnitine, etc.) into trimethylamine (TMA) by gut microbiota, followed by oxidation by host liver enzymes. The discovery of novel dietary compounds able to interfere with the microbial TMA formation has attracted much attention in recent years. However, to date there have been no reports of dietary compounds or natural extracts able to inhibit the activity of carnitine monooxygenase, a microbial enzyme that features the conversion of L-carnitine to TMA. The main goal of the present work was the development of a methodology that combines resazurin-based microplate assay with a CE-UV method for the determination of carnitine monooxygenase inhibitory potential of natural extracts.

2.- MethodsIn this work, a TMA producer strain of Klebsiella pneumoniae, isolated from human gut, was used. The fluorescent signal derived from bacterial growth was measured using Synergy HT microplate reader (BioTek Instruments), and TMA accumulation in bacterial samples was derivatized with 2,4′-dibromoacetophenone reagent and analyzed using a P/ACE 5010 CE system equipped with a diode array UV-Vis detector (Beckman Coulter).

3.- ResultsThe analytical methodology was based on the combined use of a microdilution method with resazurin reagent for real-time monitoring of bacterial viability and a CE-UV method to de-termine the accumulation of TMA in samples. During the optimization of the methodology, different parameters affecting the fluorescence signal and CE analysis were investigated. The optimized conditions were successfully applied to assay the carnitine monooxygenase inhibitory potential of a collection of natural extracts.

4.- ConclusionsThe proposed methodology that combines a microplate fluorescence assay and CE-UV to simultaneously monitor the bacterial growth and metabolic rate for TMA production has

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shown to be a suitable procedure to determine carnitine monooxygenase inhibitory potential of natural extracts.

Keywords: Trimethylamine; gut microbiota; natural extracts; capillary electrophoresis.

Acknowledgements: This work was funded by the Spanish Ministry of Science, Innovation and Universities (project AGL2017-89055-R).

References:[1] Nowinski A, Ufnal M. Nutrients 46: 7-12 (2018).

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[PP-17]

FIRST PROPOSAL BASED ON CAPILLARY ELECTROPHORESIS FOR THE ANALYSIS OF FIPRONIL AND METABOLITES. APPLICATION TO EGG SAMPLES

M. Mar Aparicio-Muriana1, Ivona Lhotská2, Francisco J. Lara1 and Ana M. García-Campaña1

1 Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Avda. Fuente Nueva s/n, 18071, Granada, Spain. Email: [email protected]

2 Charles University, Faculty of Pharmacy in Hradec Králové, Prague, Czech Republic

1.- ObjectivesFipronil is an insecticide belonging to the phenylpyrazole family mainly used in insect-con-trol products for agriculture or veterinary medicine. It targets the gamma-aminobutyric acid (GABA) receptor, damaging the insect central nervous system and producing hyperexcita-tion and insect death [1]. In fact, the use of fipronil is connected to with honeybee mortality through the Colony Collapse Disorder. Thus, its utilization has been prohibited in food-pro-ducing animals. Some fipronil degradation products (e.g. fipronil-sulfone) have higher tox-icological significance than the parent compound [2]. Accordingly, in the European Union (EU), the maximum residue limit (MRL) for fipronil is defined as the sum of fipronil and its sulfone degradation product. Fipronil has recently been involved in a European health alert due to the presence of high residue levels of the pesticide in fresh hen eggs (144 times greater than the MRL which is fixed at 5 µg·kg-1 [3]) as a result of an illegal use. Taking these facts into account, it is crucial to monitor pesticide residues to ensure they do not exceed the regu-lated MRL. For that purpose, a large number of analytical procedures have been proposed for the determination of fipronil, most of them based on High Performance Liquid Chromatog-raphy with different detector systems [4,5]. The aim of this work is to evaluate the feasibility of capillary electrophoresis (CE) for the determination of fipronil and its primary degradation products in hen egg samples.

2.- MethodsA simple and efficient method has been developed and validated for the determination of fipronil and fipronil-sulfone. Micellar electrokinetic chromatography (MEKC) mode was eventually applied, using a capillary of 48.5 cm total length (50 µm i.d.). The background electrolyte was a solution of 50 mM ammonium perfluorooctanoate (APFO) pH 9.0 contain-ing 10% (v/v) of methanol as organic modifier. Voltage and temperature were set at 25 kV and 25°C respectively. Samples were dissolved in 20 mM SDS and injected hydrodynami-cally at 50 mbar for 20 s. UV-detection was performed at 280 nm for both analytes. After-ward, the proposed method was applied to the determination of fipronil and fipronil-sulfone in real samples. A simple procedure based on salting-out assisted liquid-liquid extraction (SALLE) was considered as sample treatment. Type and volume of the extraction solvent,

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type of salt and mechanical shaking time were evaluated. Acetonitrile and ammonium sulfate were selected as the extraction solvent and salt respectively.

3.- ResultsThe optimized MEKC-UV method was able to separate fi pronil, fi pronil-sulfone and fi pronil-sulfi de in less than 12 min. However, the analysis of a blank egg sample showed an endoge-nous interferent peak at the same migration time of fi pronil-sulfi de, so it could not be quanti-fi ed using this extraction method. From a regulatory point of view, the EU sets the MRL for fi pronil as the sum of fi pronil and fi pronil sulfone.

Good linearity was obtained (R2>0.991 for both analytes). LODs were 90 µg·kg-1 for fi pronil and 150 µg·kg-1 for fi pronil-sulfone while LOQ were 300 µg·kg-1 for fi pronil and 500 µg·kg-1 for fi pronil-sulfone. Satisfactory recoveries were achieved ranging from 83 to 88% and pre-cision, calculated as relative standard deviation of peak areas, was lower than 14% in all cases.

Fig. 1: Electropherogram obtained by the proposed MEKC-UV method. Peaks: 1: fi lpronil-sulfi de; 2: fi pronil; 3: fi pronil-sulfone

4.- ConclusionsIn the present study we have developed the fi rst methodology for the separation of fi pronil and two degradation products, fi pronil-sulfi de and fi pronil-sulfone by CE coupled to diode array spectrophotometry detection. Thus, a pioneer MEKC-UV method using APFO as surfactant has been developed for the determination of fi pronil and its main metabolite, fi pronil-sulfone in egg samples. Validation parameters such as precision and accuracy were investigated to ensure the feasibility of the method. Satisfactory results for sensitivity were suitable for regulatory purposes when extra preconcentration or more sensitive detectors, such as mass spectrometry (MS), are required. The background electrolyte (BGE) was selected to be com-patible with MS detection. In summary, it can be concluded that the proposed method paves the way for further capillary electrophoretic studies, e.g. exploring the coupling of CE with MS, which may allow the determination of these pesticides at even lower concentrations.

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Keywords: Fipronil, micellar electrokinetic chromatography, food safety.

Acknowledgements: This work has been sponsored by Spanish Ministry of Economy and Com-petitiveness (Project ref: AGL2015-70708-R) and EU Funds. MMAM is grateful for a predoctoral grant from The Ministry of Science, Innovation and Universities (FPU17/03810). FJL is grateful for personal funding through the Special Research Program of the University of Granada.

References:[1] Gunasekara AS, Truong T, Goh KS, Spurlock F, Tjeerdema RS. J. Pestic. Sci. 32: 189-199 (2007).[2] Zhao X. J. Pharmacol. Exp. Ther. 314: 363-373 (2005).[3] European Commission EU Pesticides database, (2019). http://ec.europa.eu/food/plant/pesticides/

eu-pesticides-database/public/?event=pesticide.residue.CurrentMRL&language=EN (accessed April 4, 2019).

[4] Hadjmohammadi MR, Nikou SM, Kamel K. Acta Chim. Slov. 53: 517-520 (2006).[5] Cid YP, Ferreira TP, Medeiros DMVC, Oliveira RM, Silva NCC, Magalhaes VS, Scott FB. Quim.

Nova. 35: 2063-2066 (2012).

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[PP-18]

PSA FROM URINE: CHALLENGES FOR CE ANALYSIS OF ITS ISOFORMS

Diana Navarro-Calderón, Raquel Saez-Brox, Sergio López-Duque, Ángel Puerta, José Carlos Díez-Masa and Mercedes de Frutos

Institute of Organic Chemistry (IQOG-CSIC), Madrid, Spain. Email [email protected]

1.- ObjectivesSeveral approaches were followed by different teams to find new more selective prostate cancer markers. Capillary electrophoresis (CE) allows the separation of several peaks (proteoforms) translational modifications (PTMs) may alter the size and/or charge of the PSA proteoforms, that should be reflected in the CE profile of this glycoprotein.

We seek an economically affordable method for PSA proteoform analysis to be implemented in hospitals using routine available equipment. CE with ultraviolet detection was chosen. Due to low abundance of PSA in blood and ethical restrictions to obtain seminal plasma, urine was chosen for PSA analysis.

The objective of this communication is to present several challenges that we have faced to analyze isoforms of PSA from urine by CE-UV.

2.- MethodsTo analyze PSA isoforms by CE-UV, the glycoprotein must be purified from the urine. Stud-ies shown in this presentation were performed with actual urine samples from donors, as well as with model systems (solutions of commercial PSA). The effect of factors such as urine storage conditions, pre-treatment steps, affinity purification, and PSA concentration on the PSA recovery and CE profile were studied.

3.- ResultsPurification of PSA from urine required pretreatment steps and affinity chromatography pu-rification. Factors involved on each of the steps influence the PSA recovery as well as the CE profile of the PSA isoforms.

4.- ConclusionsCE analysis of PSA isoforms from urine required careful control of each of the steps involved on sampling, handling and purification.

Keywords: Prostate specific antigen, PSA, urine, CE, glycosylation, isoforms,

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Acknowledgements: This work was supported by the Comunidad of Madrid and European funding from FSE and FEDER programs (project S2018/BAA-4393, AVANSECAL-II-CM). D. N-C and R. S-B acknowledge contracts in the frame of the YGI Plans financed by the ESF and the YEI.

References:[1] Garrido-Medina R, Diez-Masa JC, de Frutos M. Electrophoresis 32: 2036-2043 (2011).[2] Farina-Gomez N, Puerta A, Gonzalez M, Diez-Masa JC, de Frutos M. J. Chromatogr. A

1443: 254-261 (2016).

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[PP-19]

PAPER-BASED ELECTROCHEMICAL PLATFORMS FOR EMERGING CONTAMINANT ANALYSIS: PRECONCENTRATION

AND DETECTION OF DICLOFENAC AND AZITHROMYCIN

E. Costa-Rama1, T. Magalhães1, O. Amor-Gutiérrez2, H.P.A. Nouws1, M.C. Blanco-López2, C. Delerue-Matos1 and M.T. Fernández-Abedul2

1 REQUIMTE/LAQV, Instituto Superior de Engenharia do Porto, Politecnico do Porto, Porto, Portugal. Email: [email protected]

2 Department of Physical Chemistry and Analytical Chemistry, University of Oviedo, Oviedo, Spain. Email: [email protected]

1.- ObjectivesIn the last few years, pharmaceuticals such as antibiotics and anti-inflammatory drugs, have become important environmental contaminants due to their massive use and their persis-tence. Furthermore, conventional wastewater treatment plants are not specifically designed for their elimination. Consequently, they are widely distributed in the environment and, al-though found at very low concentrations, their presence has been associated with damage on human and animal health.

Diclofenac (DCF) is a common anti-inflammatory drug that is broadly used as analgesic for acute joint inflammation and mild to moderate pain. Azithromycin (AZI) is a semisynthetic antibiotic derived from erythromycin and is widely prescribed to treat infections of the res-piratory tract, skin and soft tissues. DCF and AZI are frequently found in environmental waters, which is a serious problem since several studies associate harmful effects to different organisms after their exposure to trace levels of these substances.

Therefore, the development of innovative devices that allows the analysis of DCF and AZI in a cost- and time-efficient way is an important challenge. Moreover, the portability of these devices is also a highly-interesting characteristic since they are aimed for environmental analysis.

2.- MethodsIn this context, a simple electroanalytical device for the detection of DCF and AZI was developed by combining a paper-based carbon working electrode (WE) with gold-plated metallic wires as pseudoreference- and counter electrodes [1,2]. The WE’s area was wax-delimited and the metallic wires of a standard header connector were used.

The electrochemical behaviour of DCF and AZI on this paper-based device was studied. To improve the sensitivity, several preconcentration strategies were evaluated. The procedure for preconcentrating the pharmaceuticals on the paper-based WE was very simple: an aliquot of the analyte solution was deposited onto the WE and left to dry. The volumes of the aliquots and the drying temperature were optimized in order to achieve the best analytical signal.

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3.- ResultsAs can be seen in Fig. 1, preconcentration provides a signifi cant increase in the current inten-sity of the anodic peak for both pharmaceuticals (32.5 µA vs. 2.1 µA for DCF (75 µM) and 35.6 µA vs. 7.1 µA for AZI (250 µM)). Once the procedure was optimized, linear calibration plots were obtained between 0.10 and 100 µM for DCF and between 5 and 1000 µM for AZI. The preconcentration step provides a huge increase in the sensitivity of electrochemical detection and the limits of detection were ca. 800-fold lower for DCF (70 nM vs. 55 µM) and 49-fold lower for AZI (4 µM vs. 205 µM) respectively. The developed platform and pre-concentration procedure were used for the determination of both pharmaceuticals in spiked tap water.

Fig. 1: LSVs recorded in: (a) 10 µL of a 75-µM DCF solution; (b) 10 µL of 0.1 M PB pH 7.0 after preconcentration of 40 µL of a 75-µM DCF solution; (c) 10 µL of a 250-µM AZI solution;

(d) 10 µL of 0.1 M PB pH 7.0 after preconcentration of 20 µL of a 250-µM AZI solution.

4.- Conclusions

A user-friendly paper-based device for the electrochemical analysis of DCF and AZI was developed. The porous matrix of the paper was explored for on-site preconcentration which considerably improved the sensitivity of the analysis without the need of external preconcen-tration systems that increase the fi nal cost of the analysis and the amount of waste generated. Moreover, the preconcentration procedure allowed to decouple the sample from the detection volume, as well as the possibility to change the medium. Due to the versatility and simplicity of the platform, another advantage of its design is that multiplexed platforms with independ-ent electrochemical cells can be easily be constructed.

Keywords: Paper-based devices, Electroanalysis, Preconcentration, Pharmaceuticals, Contamination.

Acknowledgements: The authors would like to thank the EU and the Fundação para a Ciência e a Tecnologia (FCT) / UEFISCDI / FORMAS for funding, in the frame of the collaborative interna-tional consortium REWATER fi nanced under the ERA-NET Cofund WaterWorks2015 Call. This ERA-NET is an integral part of the 2016 Joint Activities developed by the Water Challenges for a Changing World Joint Programme Initiative (Water JPI). This work was also supported by the EU and FCT (project FOODnanoHEALTH, Portugal2020, Norte-01-0145-FEDER-000011 and project UID/QUI/50006/2019) and by the Spanish Ministry of Economy and Competitiveness (MINECO,

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project CTQ2014-58826-R). Estefanía Costa-Rama also thanks the Government of Principado de Asturias and Marie Curie-Cofund Actions for the post-doctoral grant “Clarín-Cofund” ACA17-20.

References:[1] Amor-Gutierrez O, Costa Rama E, Costa-Garcia A, Fernandez-Abedul MT. Biosens. Bioelectr.

93: 40-45 (2017).[2] Costa-Rama E, Nouws HPA, Delerue-Matos C, Blanco-Lopez MC, Fernandez-Abedul MT. Anal.

Chim. Acta. 1074: 89-97 (2019).

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[PP-20]

IONIC LIQUID AND MULTI-WALLED CARBON NANOTUBES FOR THE EXTRACTION OF CARBAMATE PESTICIDES FROM WATER SAMPLES PRIOR TO THEIR DETERMINATION BY CAPILLARY ELECTROPHORESIS

Jihane Ben Attig1,2,3, Latifa Latrous3, Mohammed Zougagh2,4 and Ángel Ríos1,2

1 Department of Analytical Chemistry and Food Technology, Faculty of Chemical Science and Technology, University of Castilla-La Mancha, E-13071 Ciudad Real, Spain. Email: [email protected]

2 Regional Institute for Applied Chemistry Research (IRICA), E-13071 Ciudad Real, Spain

3 Department of Chemistry, Faculty of Sciences of Tunis, University of Tunis El Manar, University Campus of El Manar II, Tunis, Tunisia

4 Department of Analytical Chemistry and Food Technology, Faculty of Pharmacy, University of Castilla-La Mancha, Albacete, Spain

1.- ObjectivesThe aim of this study is the development of a novel methodology to extract N-methylcarba-mate pesticides with ionic liquid and multi-walled carbon nanotubes (ILs-MWCNTs) from water samples prior to their separation and quantification by capillary electrophoresis with diode array detector (CE-DAD).

2.- MethodsThe wide use of carbamates causes the contamination of aquatic environments. Their trace-state determination, in various aqueous media, requires a pre-concentration step ffollowed by analysis often conducted by high-performance liquid chromatography with ultraviolet visi-ble detector (HPLC-UV), high-performance liquid chromatography with mass spectrometry detector (HPLC LC-MS) or CE-DAD.

Fig. 1: Scheme of the general procedure for temperature controlled ionic liquid dispersive liquid and MWCNTs microextraction

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The methodology of this work was as follows: a volume of 100 µL of 1-hexyl-3- methylim-idazolium hexafluorophosphate ([C6MIM] [PF6]) and 10 mg of MWCNTs was added into a 10 mL volumetric flask plus 10 mL of ultra-pure water. This solution was spiked with each carbamate sample at a concentration of 10 μg mL-1. The mixture was sonicated for 15 min. and the flask was heated to 90°C for 20 minutes. heated to 90°C for 20 min. The flask was then cooled on ice bath for 20 min and a cloudy solution was formed. This solution was centrifuged for 15 min at 3000 rpm. The upper aqueous phase was removed with a syringe, and the drop of ionic liquid containing the analytes was dissolved in 3 mL of methanol. After evaporation of methanol under nitrogen stream, the residue was redissolved in buffer borate. Finally, the extract was injected into the CE system. All these procedures can be seen in Fig. 1.

3.- ResultsIn this work, several parameters have been optimized as can be seen in Table1.

Table 1. Operating conditions for extraction and separation of NMCs by CE-DAD.

Parameter Optimal conditionsExtraction procedure

IL volume 100µLSorbent Material MWCNTs

Sorbent mass 10 mgElution solvent DCM

Elution solvent volume 3CE method

Buffer composition 15 mM borate solution[1] Buffer pH 11.47

Separation voltage 20 kVCapilllary dimension 75µm i.d x 80 cmCapillary temperature 25°C

Injection time 10 s Injection pressure 50 mbar

Wavelength 209 nm

Under the optimized conditions, the LODs and LOQs were in the range of 0.025–0.73 and 0.04–2.4 ng mL−1, respectively. The calibration curves were linear (R2 ≥ 0.9996) over the concentration ranges from 1 to 1000 ng mL−1. The recoveries of N-methylcarbamate pesticides were from 76.4 ± 1.7 up to 106.5 ± 3.5%. The method was applied to water samples with different characteristics to evaluate the performance under real conditions.

4.- ConclusionsA simple, rapid, and environment-friendly method based on ionic liquids (ILs), mul-ti-walled carbon nanotubes (MWCNTs) and CE-DAD for the monitoring of N-methylcar-bamate pesticides in water was developed. The present method provides several advantages when compared with other previously described previously methods, in terms of precision,

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sensitivity and recoveries. The limits of quantifications are below the values reported in Di-rective 1126/2014/EC [2] for these types of samples.

Keywords: Capillary electrophoresis, ionic liquid, multi-walled carbon nanotubes, N-methylcarba-mate pesticides, water samples.

Acknowledgements: We thanks to the Spanish Ministry of Economy and Competitiveness (MINE-CO) and JJCC Castilla-La Mancha and European Commission for funding this work with Grants CTQ2016-78793-P and SBPLY/17/180501/ 000333, respectively.

References:[1] Cheng X, Wang Q, Zhang S, Zhang W, He P, Fang Y. Talanta 71:1083–1087 (2007).[2] REGLAMENTO (UE) No 1126/2014 DE LA COMISIÓN de 17 de Octubre de 2014.

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[PP-21]

DEVELOPMENT OF A CE-UV METHOD FOR MONITORING MARKERS OF SPOILAGE IN APPLE

JUICE CAUSED BY YEAST AND BACTERIA

Fabiana Amaral da Silva1, and Ana Valéria Colnaghi Simionato1,2

1 Institute of Chemistry - Unicamp, Campinas, Brazil. Email: [email protected] Institute of Science and Technology in Bionalytics - Unicamp, Campinas, Brazil

1. - ObjectivesDevelopment of a novel micellar electrokinetic capillary chromatography (MEKC) method with direct UV detection in order to detect and quantify five potential markers of spoilage in apple juice. These include guaiacol (2-methoxyphenol), 2,6-dichlorophenol, 2,6-dibro-mophenol (all of them synthesized by the spoilage bacteria of the genus Alicyclobacillus); In addition, 4-vinylguaiacol (2-methoxy-4-vinylphenol), synthesized by yeasts, and diacetyl (2,3-butanedione) synthesized by lactic acid bacteria on apple juice samples were also determined.

2. - MethodsFruit juices are susceptible to the contamination by spoilage microorganisms, such as the thermo acidophilic bacteria Alicyclobacillus acidoterrestris, which produces the off-flavor molecules guaiacol (2-methoxyphenol), 2,6 –dichlorophenol and 2,6-dibromophenol [1]. These impart a unpleasant medicinal taste in juices, occasionally resulting in product recall. Fermentative yeasts and lactic acid bacteria also possess the ability to survive under acidic conditions typically found in the juice matrix. Lactic acid bacteria synthesize metabolites such as lactic acid, acetoin and diacetyl [2], the last one associated with an undesirable but-tery flavor in apple juice. Yeasts may produce metabolites as ethanol, 3-methyl-1-butanol and the off-flavor 4-vinylguaiacol [3]. The monitoring of diacetyl and 4-vinylguaiacol is of spe-cial interest in the brewing industry, in order to avoid unpleasant organoleptic properties in beer [4]. Spoilage is associated with economic losses to the fruit juice and beverage industry. Therefore, proposal of simple methods to detect the formation of these metabolites is crucial to avoid further juice contamination.

Considering that four out of five molecules under study are phenols and exhibit pKa val-ues ranging between 6.67 (2,6-dibromophenol) and 9.98 (4-vinylguaiacol), a strong alka-line background electrolyte (BGE) was applied in order to charge the molecules as anions, allowing their countermigration separation. The selected electrolyte was sodium tetraborate (pKa 9.14) due to adequate buffering capacity at the selected analysis pH. Sodium dodecyl sulfate (SDS) was added to improve separation of the phenolic compounds, and to improve separation of diacetyl (pKa 15.98), which is not charged in alkaline medium. However, the subsequent addition of sulfated β-cyclodextrin (S-β-CD) to the BGE improved the results. Its interaction with diacetyl resulted in a product with excellent UV absorption, which

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was absent without the reagent. The BGE was optimized by studying parameters such as pH and buffer composition, SDS and S-β-CD. The experiments were performed with an Agilent 7100 CE system equipped with DAD detection, using an uncoated fused-silica cap-illary (Agilent Technologies) with 66.4 cm total length, 58 cm effective length, and 50 µm I.D. The optimum analysis conditions were achieved with UV detection monitoring at 208 nm, applied voltage of 25kV, capillary temperature of 20ºC, hydrodynamic injection of 10 s (50 mbar), and BGE composed of 40 mmol/L sodium tetraborate, 25 mmol/L SDS, and 5 mmol/L S-β-CD at pH 10.

3. - ResultsCE analyses under optimized conditions were performed in less than 25 min, as shown in Figure 1. The partition of the analytes into the micelles seems to be the primary factor gov-erning their mobility under these electrophoretic conditions. Diacetyl was the first detected species (tm = 6.098 min) since it was most hydrophilic molecule and exhibited the lowest interaction with the micelles within the compounds under study. It also interacts with the negatively charged S-β-CD, which contributes to slowing its migration in respect to the EOF. Guaiacol is the second to migrate (tm = 11.219 min), followed by 2,6-dichlorophenol (tm = 18.665 min) and 2,6-dibromophenol (tm = 19.998 min). The last detected analyte was 4-vi-nylguiacol (tm = 21.977 min) which was the most hydrophobic compound of the mixture (presence of a vinyl group), strongly interacting with the micelles. The analytical conditions resulted in resolutions above 2.5 and peak efficiencies ranging from 1.54 to 3.54 × 105.

Fig. 1: electropherogram illustrating the separation of analytical standards of the five analytes in the following order: diacetyl (2 mmol/L), guaiacol (1 mmol/L), 2,6-dichlorophenol (1 mmol/L), 2,6-dibro-

mophenol (1 mmol/L) and 4-vinylguaiacol(1.6 mmol/L). Analysis conditions: uncoated fused-silica capillary, 66.4 cm total length, 58 cm effective length, and 50 µm I.D. UV detection at 208 nm, applied voltage of 25kV, capillary temperature of 20ºC, hydrodynamic injection of 10 s (50 mbar). BGE com-

posed of 40 mmol/L sodium tetraborate, 25 mmol/L SDS, and 5 mmol/L S-β-CD at pH 10.

4. - ConclusionsThe proposed method enables separation and detection of potential markers of apple juice spoilage. The next steps to be pursued are the method validation in the blank sample matrix and working on a sample stacking strategy in order to reach lower limits of detection.

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Keywords: Alicyclobacillus acidoterrestris; CE-UV; diacetyl; 2,6-dichlorophenol, 2,6-dibromophe-nol; fruit juice spoilage; guaicol; MEKC; 4-vinylguaicol.

Acknowledgements: This work has been sponsored by the Coordination for the Improvement of Higher Education Personnel (CAPES). The authors would like to acknowledge São Paulo Research Foundation (FAPESP) and The Brazilian National Council for Scientific and Technological Devel-opment (CNPq) as well.

References:[1] Steyn CE, Cameron M, Witthuhn RC. Intern. J. Food Microbiol. 147(1): 1-11 (2011).[2] Splittstoesser DF. J. Food Protect. 45(9): 874-877 (1982).[3] Sperber WH, Doyle MP. Compendium of the Microbiological Spoilage of Foods and Beverages,

Springer (2009).[4] Havkin-Frenkel D, Dudai N. Biotechnology in Flavor Production, Wiley Blackewell (2016).

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[PP-22]

LAB-ON-A-CHIP SYSTEMS BASED ON ACTIVE TRANSPORT USING GRAPHDIYNE MICROMOTORS

Víctor de la Asunción-Nadal1, Kaisong Yuan1,2, Beatriz Jurado-Sanchez1, and Alberto Escarpa1


2 Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University, Guangzhou, China

V. de la Asunción and K. Yuan contributed equally.

1.- ObjectivesHerein we propose the use of nanomaterial-based tubular micromotors for on-chip operation. To this end, the fi rst aim was to develop a simplifi ed method (i.e. template-assisted electro-chemical deposition) for the fabrication of 2D carbon-based material micromotors such as graphdiyne (GDY). Along with the 2D material, ferromagnetic nickel (Ni) was added to impart the micromotors with inherent magnetic properties for further guidance. Diff erent hydrogen peroxide catalytic metals, such as platinum (Pt), manganese dioxide (MnO2) or copper/palladium (Cu/Pd) alloy were used for propulsion. In a second objective, propulsion performance (in terms of speed and towing force) of each set of micromotors was tested in off -chip and on-chip applications in order to select the best option for an intended application in real domains.

2.- Methods and ResultsFor the achievement of the fi rst objective, template electrosynthesis of the micromotors was performed using graphdiyne as outer layer while diff erent catalysts were employed as an inner layer: platinum/nickel bilayer, manganese dioxide and palladium/copper alloy. Cyclic voltammetry was performed to deposit the GDY layer inside the pores (⌀ = 5 μm) of a gold-sputtered polycarbonate membrane; whereas amperometry was adopted for the electrodeposition on the inner catalytic layers. Raman spectra were taken of both pristine graphdiyne and hollow graphdiyne tubes. Overlaying both the pristine and electrodeposited graphdiyne we can assume the material was electrodeposited without suff ering major struc-tural changes, as the D/G ratio remains constant. Along with Scanning Electron Microscopy (SEM), Energy-Dispersive X-ray spectroscopy (EDX) microanalysis was performed to iden-tify the diff erent materials used as catalysts (Pt, MnO2, Pd/Cu) or magnetic material (Ni). Finally, Atomic Force Microscopy (AFM) was performed in order to characterize the outer layer morphology, showing a coarse layered arrangement (see Fig. 1).

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Fig. 1: Micromotor characterization. From left to right: Raman spectroscopy, scanning electron microscopy (SEM) with electron-dispersive X-ray analysis (EDX) (elements depicted)

and atomic force microscopy (AFM).

Following the second objective, “off -chip” micromotor performance was tested using diff erent hydrogen peroxide concentrations ranging from 0.5% to 5.0%. Micromotor movement was recorded and traced showing diff erent patterns such as circular, fl ower-like and linear motion traces. The highest speed was recorded for Pt catalyst being 2.7 times higher than Pd/Cu and 3.9 times higher than MnO2 on average using a peroxide concentration of 5.0% and 1% SDS. Considering the high speeds achieved with this catalyst, platinum micromotors were used for further experiments. Performance of GDY-Pt motors was tested in diff erent common clinical samples: blood serum and whole saliva; and food media: whole milk and red wine (see Fig. 2). Despite the complexity of the media, effi cient propulsion was observed in all cases.

Fig. 2: Micromotor performance. Left: Speeds for diff erent catalysts and hydrogen peroxide concentra-tions. Right: Operation in diff erent biological/food media. Scale bar = 10 μm..

Micromotor performance was tested inside microfl uidic channels. In this sense, the most important features to consider are maneuverability and propulsion in the miniaturized envi-ronment, especially against the current. We studied the performance of a micromotor inside a microfl uidic channel with a back fl ow. Flow speed was measured by tracking small tracer

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particles; the maximum flow speed was 4.8 times higher than the micromotor speed. None-theless, the apparent micromotor speed relative to the channel walls was only 22% lower than that of the motors swimming in a still drop. Regarding the maneuverability, Ni-contain-ing motors were used for this application. Applied magnetic fields were used to orient the motors along the long axis of the channel. According to our results, micromotors were able to follow the path avoiding bubbles without stopping its motion. In our experiment, after crossing the main channel, the micromotor gets to a three-way junction, where the chosen path can be switched by changing the direction of the magnetic field (see Fig. 3).

Fig. 3: Micromotor on-chip performance. Top: Motor crossing a channel. Bottom: Motor changing its path.

3.- ConclusionsWe synthesised micro-sized swimmers using a novel 2D material with promising properties. Such motors were characterized using techniques such as SEM, EDX, Raman microscopy or AFM. These motors were prepared using a range of ferromagnetic and catalytic materials, and its operation was demonstrated in different media. Finally, the on-chip operation was studied showing good propulsion without significant speed loss in an almost 5-fold speed back flow.

Keywords: micromotors, graphdiyne, lab-on-a-chip.

Acknowledgements: K. Yuan acknowledges the Spanish Ministry of Science, Innovation and Uni-versities for his pre-doctoral contract (RYC-2015-17558, co-financed by EU). V. de la Asunción-Na-dal acknowledges the FPI fellowship received from the University of Alcalá (Plan Propio de Inves-tigación UAH). B. J-S acknowledges support from the Spanish Ministry of Science, Innovation and Universities (RYC-2015-17558, co-financed by EU) and from the University of Alcalá (CCG2018/EXP-018). AE acknowledges financial support from the Spanish Ministry of Science, Innovation and Universities (CTQ2017-86441-C2-1-R) and the TRANSNANOAVANSENS program (S2018/NMT-4349) from the Community of Madrid.

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[PP-23]

LAB-ON-A-CHIP OPERATIONS PERFORMED BY MAGNETIC CARBON-BASED MICROMOTORS

Roberto Maria-Hormigos1, Beatriz Jurado-Sánchez1,2, and Alberto Escarpa1,2


2 Chemical Research Institute “Andrés M. del Río”, University of Alcalá, Alcalá de Henares, Madrid, Spain

1.- ObjectivesWe propose the development of magnetic carbon-based micromotors for lab-on-a-chip (LOC) operations. Firstly, highly rough carbon black tubular engines are used for fluores-cence detection of dopamine. Secondly, carbon nanonotubes-based micromotors modified with lectins and an inner antibiofouling layer are used for selective transport of sugar-modi-fied particles (as cell mimics) in human plasma.

2.- MethodsCarbon black‐Ni‐PtNP (CB‐Ni‐PtNP) micromotors were prepared by direct deposition of the carbon nanomaterial followed by electrodeposition of the magnetic (Ni) and catalytic (Pt-NPs) layers. Multiwalled carbon nanotubes-Ni-PtNP-protective polymeric layer (MWCNTs‐Ni‐PtNPs-OPD) were prepared by direct electroreduction of the carbon nanomaterial fol-lowed by the electrodeposition of the metallics and poly-o-phenylenediamine (OPD) layers. The deposition or electrochemical reduction takes place into 5 μm-diameter conical pores of a polycarbonate membrane as templated. Then, the resulting micromotors are released, cleaned and their morphology, chemical composition and distribution, catalysis activity and magnetic guidance are characterized.

Sodium cholate is used to reduce superficial tension of the media which promote the constantly formation of oxygen bubbles in the inner catalytic surface of the micromotors. These bubbles are capable of propelling the micromotors. Bubbles are formed by catalytic decomposition of hydrogen peroxide, which is added to the solutions. Magnetic guidance is performed using external neodymium magnets, which permit control the micromotors directionality inside the LOC platforms to perform different operations.

3.- ResultsCarbon black micromotors are employed for rhodamine 6G (Rh6G) adsorption on their sur-face in the first reservoir of the LOC platform (made of polydimethylsiloxane, PDMS), then micromotors are guided to the second reservoir of the LOC where dopamine is added. Rh6G absorbed in the micromotors carbon surface are release to the media. Rh6G is a molecule with a strong fluorescence. That fluorescence is quenched at the micromotors surface in the first reservoir and then recover in the second one when it is released, the monitoring of this fluorescence can be used for dopamine detection.

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MWCNTs-Ni-PtNPs-OPD micromotors are functionalized with wheat germ agglutinin (WGA) or concavaline A (ConA) lectins by activation of the carboxylic groups present in the carbon outer layer of the micromotors followed by covalent bonding between the carbox-ylic functions and the lectins. Then, lectin-modified micromotors are swimming in the first micromotor reservoir where they interact with polystyrene particles modified with sugars. Lectins recognize specific particles that are modified with the sugar that they are selective for. Then, the particles attached to the micromotor surface are transported between LOC reservoirs.

Fig. 1: LOC operations. Left part, dopamine indirect detection in the second reservoir (bottom images) by rhodamine 6G adsorption-desorption and fluorescence “ON-OFF-ON” scheme on CB-micromotors. Right part, polystyrene-sugar particles recognition-transport-isolation by lectin functionalized MWCNTs

micromotors in raw human plasma.

4.- ConclusionsIn conclusion, we have described the successful integration of CN-based micromotors on-board of chips for complex molecular operations. The rich outer surface chemistry of CB has been utilized for the specific absorption of Rh6G towards novel fluorescence on-chip strategies. The potential of the new dynamic loading and unloading micromotor stations has been illustrated for Rhodamine 6G based-detection operations. The high towing force of lec-tin MW-based micromotors has been utilized for the selective separation of sugar modified polystyrene particles within the different reservoirs of a LOC. The micromotors were mod-ified with an inner OPD layer for highly efficient capture and transport of target particles in complex biomedia such as raw human plasma. The new protective layer avoids the fouling of the Pt catalyst by plasma proteins. This unsolved problem can hamper the application of similar tubular micromotors for direct assays in raw samples. Such new micromotor engi-neering holds considerable potential for novel point-of-site devices based on active transport, simultaneous and selective bacteria separation and culture, and many more. Such concept can be extended for the selective separation of bacteria and other relevant cells containing the target sugar residues in its cell wall. The developed platforms do not require an external mechanism or pumps for its efficient operation, holding thus considerable promise for the creation of portable new generation analytical systems.

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Keywords: Micromotors; carbon nanomaterials; Lab-on-a-chip; active transport.

Acknowledgements: This work has been sponsored by the Spanish Ministry of Science, Innova-tion and Universities for funding (BES-2015-072346, RYC-2015-17558, co-financed by EU and CTQ2017-86441-C2-1-R), Community of Madrid (TRANSNANOAVANSENS-CM, P2018/NMT-4349) University of Alcalá (CCGP2017-351 EXP/040 and CCG2018/EXP-018).

References:[1] Maria-Hormigos R, Jurado-Sanchez B, Escarpa A. Nanoscale, 9, 19, 6286-6290 (2017).

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[PP-24]

ELECTROCHEMICAL MICROFLUIDIC CHIP COUPLED TO MAGNETOIMMUNOASSAY FOR PROCALCITONIN

DETECTION IN CLINICAL SAMPLES

María Moreno-Guzmán1, Águeda Molinero-Fernández2, Miguel Ángel López2,3, and Alberto Escarpa2,3

1 Department of Chemistry in Pharmaceutical Sciences, Analytical Chemistry, Faculty of Pharmacy, Universidad Complutense de Madrid. Avda. Complutense, s/n, E-28040 Madrid, Spain. E-mail: [email protected]

2 Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, University of Alcalá. Ctra. Madrid-Barcelona, Km. 33.600, E-28871 Alcalá de Henares, Madrid, Spain

3 Chemical Engineering and Chemical Research Institute “Andres M. Del Río”, University of Alcalá, Alcalá de Henares, Madrid, Spain

1.- ObjectivesIn this work, we propose a magneto-immunosensing approach using an electrochemical mi-crofluidic chip for determination of procalcitonin (PCT), a well-established biomarker for sepsis in clinical samples.

2.- MethodsInmunoanalytical methodology was developed on the basis of an enzyme-linked immuno-sorbent assay (ELISA) sandwich scheme, where biotinylated capture antibody was immo-bilized on streptavidin-functionalized magnetic beads (MBs) and detection antibody was labelled with horseradish peroxidase (HRP) enzyme. Once molecular recognition of PCT analyte has been produced, the extent of the affinity reaction is evaluated by the addition of the enzymatic substrate and electrochemical mediator whose reduction on the electrode surface is directly related to the activity of the enzyme tracer.

MBs-immunocomplex were previously deposited into the sample reservoir (SR) of the mi-crofluidic chip (Figure 1). Microchannels, buffer reservoirs (BR), and detection reservoir (DR) were filled with running buffer (PBST) while enzymatic reservoir (ES) was filled with a mixture of 45 µL of 1 mM HQ plus 5 µL of 50mM H2O2. To place the MBs in the longitu-dinal channel, a voltage of 1500V was applied between reservoirs SR and DR, using 3 pulses of 25 seconds, while other reservoirs were left floating. Magnetic particles were retained within the microchannel by the aid of a magnet situated on the top. After a washing step with PBST from RB (10 s applying 1500 V from RB to DR) the enzymatic substrates were injected and pumped to cross through the particles bed (1000 s applying 1500 V from ES to DR reservoirs).

Amperometric measurements were performed at -0.2 V on the gold working electrode situated at the end of the microchannel. Amperometric signals, calculated as the difference between the

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steady-state and the background currents, were fitted to the four-parameter logistic equation using the software SigmaPlot 10.0.

Fig. 1: Electrochemistry measurement by using flow immunosensor. Image of magnetic beads inserted inside the channel, viewed from the microscope.

3.- ResultsThe developed approach exhibited excellent analytical characteristics. The linear range was from 0.15 to 20 ng/mL, and limit detection was 0.05 ng/mL which permits PCT detection at the levels required for sepsis diagnosis.

Precision was studied for microfluidic immunosensors gave an intra-assay and inter-assay precision of 5.0% and 9.0% for 0.1 ng ml-1 PCT, respectively.

Specificity of the immunosensor for PCT analysis was checked in presence of a large excess of C-reactive protein (CRP) (16 μg/ml) (another biomarker usually determined for sepsis diagnosis), heparin, EDTA and citrate (as other relevant molecules largely contained in blood tubes). As observed, there were no significant differences between the currents found in ab-sence or in the presence of these compounds. This demonstrated the excellent selectivity of the immunosensor for PCT detection.

In order to evaluate the suitability of the developed immunosensors for PCT determination in clinical samples, different calibration curves were carried out in human serum and plas-ma from healthy people. The matrix effects was evaluated by comparison of the calibration curve parameters obtained in PBS buffer with those obtained in both kind of matrices. The non-existence of matrix effects was proved since no statistically significant differences (p < 0.05) were observed.

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4.- ConclusionsA electrochemical microfluidic chip coupled to magnetoimmunoassay for PCT determina-tion in clinical samples was successfully developed with good analytical characteristics.

Furthermore, the advantages of the microfluidic platforms with higher surface/volume ratio, provided a reduction in analysis time, easy automation and miniaturization, make of this ap-proach a potential tool as a point-of-care (POC) device for the diagnosis of sepsis.

Keywords: Procalcitonin, magneto-immunosensor, microfluidic chip, sepsis.

Acknowledgements: This work was supported by the Spanish Ministry of Economy and Competi-tiveness (CTQ2017-86441-C2-1-R), the TRANSNANOAVANSENS program (S2018/NMT-4349) from the Community of Madrid and La Caixa Impulse program (CI017-00038) (A.E, MA.L.G, M.M.G and A.M.F.). A.M.F. acknowledges the FPU fellowship from the Spanish Ministry of Edu-cation Culture and Sports.

References:[1] Hervas M, Lopez MA, Escarpa A. The Analyst 136: 2131-2138 (2011).[2] Hervas M, Lopez MA, Escarpa A. Trends Anal. Chem. 31: 109-128 (2012).[3] Sandquist M, Wong HR. Expert Rev. Clin. Immunol. 10: 1349-1356 (2014).

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[PP-25]

METAL-ORGANIC FRAMEWORKS IN POLYMER MONOLITHS FOR ON-LINE SOLID-PHASE EXTRACTION

CAPILLARY ELECTROPHORESIS OF POLYCYCLIC AROMATIC HYDROCARBON DERIVATIVES

Héctor Martínez Pérez-Cejuela1, Fernando Benavente2, Ernesto Francisco Simo-Alfonso1, and Jose Manuel Herrero-Martinez1

1 Department of Analytical Chemistry, University of Valencia, Valencia. Spain. Email: [email protected]

2 Department of Chemical Engineering and Analytical Chemistry, Institute for Research on Nutrition and Food Safety (INSA-UB), University of Barcelona, Barcelona, Spain

1.- ObjectivesIn this study, a metal-organic framework (MOF) named HKUST-1 was synthesized in situ in a polymer monolith. The MOF was explored as a sorbent for the analysis of polycyclic ar-omatic hydrocarbons derivatives by on-line solid-phase extraction capillary electrophoresis (SPE-CE) [1,2].

2.- MethodsThe hybrid sorbent was prepared after by grinding a polymeric monolith followed by in situ layer-by-layer self-assembly to graft the MOF on the polymer surface. The sorbent was used to build fritless particle-packed microcartridges that were integrated in-line near the entrance of the separation capillary. No valves were necessary for operation in the MOF-SPE-CE mode. The modified capillaries were employed for the analysis of nitrated and oxygenated polycyclic aromatic hydrocarbons (PAHs). These compounds are emitted mainly by com-bustion sources and post-emission transformation of PAHs. After loading a large volume of sample (∼50-100 μL), the capillary was rinsed to remove non-retained molecules and filled with background electrolyte. The analytes were preconcentrated and then eluted in a small volume of an appropriate solution (~25-50 nL) before separation and ultraviolet detection.

3.- ResultsThe different steps of the SPE-CE method were optimized to analyze dilute solutions of PAHs. The developed MOF-affinity system gave satisfactory repeatability and acceptable reusability. Linearity and limits of detection were also investigated. The developed method was applied to the analysis of environmental water samples.

4.- ConclusionsWe have demonstrated that hybrid MOF materials can be regarded as promising sorbents to prepare microcartridges for SPE-CE. These MOF sorbents expand the applicability of SPE-CE beyond the typical sorbents based on immobilized metal affinity resins with copper or nickel.

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Keywords: capillary electrophoresis; MOF, on-line solid-phase extraction; PAH.

Acknowledgements: This work has been sponsored by projects PROMETEO/2016/145 (Valencian Government, Spain), RTI2018-095536-B-I00 and RTI2018-097411-B-I00 (Ministry of Science, In-novation and Universities, Spain).

References:[1] Yu Y, Ren Y, Shen W, Deng H, Gao Z. TrAC, Trends Anal. Chem. 50: 33-41 (2013).[2] Pont L, Pero-Gascon R, Gimenez E, Sanz-Nebot V, Benavente F. Analytica Chimica Acta 1079:

1-19 (2019).

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[PP-26]

CE-MS METABOLOMICS STUDY OF H9C2 CARDIAC CELLS SUBMITTED TO OXIDATIVE STRESS

Mónica Forca Lima1,2, Aline Mara dos Santos3, Coral Barbas1, Ana Valeria Colnaghi Simionato2, and Francisco Javier Rupérez1

1 Center for Metabolomics and Bioanalysis (CEMBIO, University CEU-San Pablo, Madrid, Spain. Email: [email protected]

2 Institute of Chemistry, State University of Campinas (UNICAMP), Caminas, SP, Brazil

3 Institute of Biology, State University of Campinas (UNICAMP), Caminas, SP, Brazil

1.- ObjectivesCardiac hypertrophy followed by heart failure is related to one of the biggest causes of death in the world. This disease corresponds to an increase in cardiac muscle mass and occurs as a response to a physiological or pathological overload. In acute myocardial infarction, the healthy region of cardiac muscle takes on more work due to the cellular death of the region affected by the infarct. This response may result in heart failure, arrhythmia, and sudden death. The aim of this research project is to perform an untargeted metabolomics inves-tigation of H9C2 cell line cultured in vitro, submitted to oxidative stress (with hydrogen peroxide), beta-adrenergic stimulation (using isoproterenol–ISO) and the use of doxorubicin (DOX) using capillary electrophoresis (CE), coupled to mass spectrometry (MS).

2.- MethodsH9C2 cells were grown in the presence of either H2O2, ISO, and DOX, and after harvesting, the methanolic extract obtained from cells was thawed on ice and shaken vigorously using vortex. Each extract (60 µL) was mixed with an aqueous solution containing 0.1 mol L-1 of formic acid and 0.2 mmol L-1 of methionine sulfone internal standard (IS). In parallel, 200 µL of each cell groth culture medium was mixed with acetonitrile, formic acid and IS. All samples were transferred to a 30 kDa porosity filter and centrifuged. Quality Control (QC) samples were made by mixing aliquots of each sample, and treated under identical conditions.

The filtrates were analysed on a CE instrument coupled to a TOF/MS equipped with sheath liquid (Agilent Technologies, Santa Clara CA, USA). Separation was carried out using a fused-silica capillary with normal polarity voltage. The MS was operated in positive polar-ity, with a full scan from 80 to 1000 m/z. Samples were injected by pressure and stacked by applying the BGE under pressure. The raw data collected by the CE-MS was cleaned of background noise and unrelated ions by the MFE algorithm. Features were simultaneously multialigned across samples by means of recursive analysis (MassHunter Profinder, Agilent Technologies). Each compound was described by mass, migration time and abundance.

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Multivariate and univariate analysis was carried out to evaluate samples classification, and to find out the compounds responsible for such classification. The significant compounds were identified comparing their accurate mass and corrected migration time with an in-house library, built from authentic standards.

3.- ConclusionsCE/MS has provided a perfect tool to evaluate differences in metabolites related to amino acid, polyamine and nucleotide metabolic routes for all conditions. The relationship between the variations within cells (endometabolome) and outside cells (exometabolome) also permits the evaluation of mechanisms for uptake and excretion of metabolites in different conditions

Keywords: Metabolomics; Cell culture; Cardiac Hypertrophy; Capillary Electrophoresis / Mass Spectrometry

Acknowledgements: MFL received funding from Airbus Defense and Space through the CLX-2 program developed with Comando da Aeronáutica (COMAER) and the Brazilian Government

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[PP-27]

COMPARATIVE CHARACTERIZATION OF THE FC DOMAIN N-GLYCOSYLATION IN MONOCLONAL ANTIBODY AND FUSION

PROTEIN THERAPEUTICS BY CGE-LIF AND UHPLC-FL

Andras Guttman1,2, Marton Szigeti2, Akos Szekrenyes3, Marcin Moczulski4, Jean Charles Berliet4 and Stephen Lock5

1 Sciex, Brea, CA, USA.2 Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen,

Hungary, H-4032 3 Mabxience, Leon, Spain4 Sciex, Darmstadt, Germany5 Sciex, Warrington, United Kingdom Contact: [email protected]

Comprehensive characterization of the N-linked carbohydrates of therapeutic antibodies and Fc-fusion proteins provides essential information about this important critical quality attrib-ute. The most frequently used high performance liquid phase separation methods for carbo-hydrate analysis are ultrahigh pressure liquid chromatography (UHPLC) and capillary elec-trophoresis (CE). Both methods require derivatization of the released sugars prior to analysis to support optical detection (commonly fluorescent). In case of CE, charged fluorophores should be used to support the electromigration process. One of the most frequently used fluorophores for carbohydrate labeling in CE is aminopyrenetrisulfonate (APTS) requiring a simple reductive amination reaction. This tag features excellent fluorescent characteris-tics and provides uniform labeling at the reductive end of the sugars only, i.e., one fluoro-phore per oligosaccharide. Similar reductive amination-based approach is used for UHPLC analysis most of the time, however, in this instance the charge on the fluorophore is not of high importance. In most cases 2-aminobenzamide (2-AB) label is used for chromatogra-phy based analysis of carbohydrates. In this poster, the separation of fluorophore labeled N-linked carbohydrates, released from the monoclonal antibody therapeutics of adalimumab (Humira®) and the Fc-Fusion protein of etanercept (Enbrel®) and three partitioned glycan libraries are compared with the use of capillary gel electrophoresis with laser induced flu-orescence (CGE-LIF) detection and UHPLC with fluorescence (FL) detection. In all cases, standard industry sample preparation and separation parameters were used for the analysis.

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[PP-28]

DEVELOPMENT OF ISOLATION TECHNIQUES AND CESI-MS METHODS FOR THE CHARACTERISATION

OF NANOMATERIAL PROTEIN CORONAS

Klaus Faserl1, Andrew Chetwynd2, Iseult Lynch2, Herbert Lindner1, Christopher Loessner3, Marcin Moczulski3, Jean Charles Berliet3 and Stephen Lock4

1 University of Innsbruck, Innsbruck, Austria2 University of Birmingham, Birmingham, U.K3 Sciex, Darmstadt, Germany.4 Sciex, Warrington, United Kingdom Contact: [email protected]

The protein corona is the layer of proteins which adsorb to the surface of a nanoparticle.1 Nano LC-MS/MS is the most common platform for protein corona characterisation.

This poster describes a new workflow to isolate the NP corona for CESI-MS/MS analysis. This protein isolation workflow allowed detection up to 86% more proteins than convention-al methods.

CESI-MS/MS analysis throughput was 3x greater than nano LC-MS/MS. This was because CESI-MS/MS had no detectable carryover, unlike nano LC-MS/MS which needed multiple blank runs to clean the column.

In conclusion, CESI-MS/MS allowed rapid and robust characterisation of the protein corona.

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[PP-29]

METHOD OPTIMIZATION AND EVALUATION FOR RNA PURITY ANALYSIS USING CE-LIF TECHNOLOGY

Tingting Li1, Christopher Loessner2, Marcin Moczulski2, Jean Charles Berliet2, Stephen Lock3

1 Sciex, Brea, CA, USA.2 Sciex, Darmstadt, Germany3 Sciex, Warrington, United Kingdom Contact: [email protected]

Capillary Electrophoresis (CE) has been demonstrated to be one of the most powerful tech-niques for analysis of a wide variety of molecules. The PA800 Plus Pharmaceutical Analysis System from SCIEX is an outstanding instrument, specifically designed and optimized for capillary gel electrophoresis (CGE) separation, which is widely used for the separation of biologics such as protein, peptide, nucleic acids, etc.

Compared to traditional slab-gel based electrophoresis methods, CGE offers superior resolu-tion, shorter analysis time, automated operation and exceptional sensitivity when combined with a laser induced fluorescence detector. Compared to chip-based CE systems, the PA800 Plus provides open chemistry, which enables flexibility for method modification and optimi-zation to generate optimal results for each specific project.

Recent advances in gene therapy research have gained promise in the utility of gene thera-peutics compounds. The ability to quantify RNA purity and quality is critical to ensure the safety and efficacy of these molecules. In this study, a CE-LIF fast separation method which was used to evaluate total RNA quality1 was optimized to achieve higher resolution; and the method was then evaluated for RNA purity determination. The resolution of this method was optimized for RNA size ranging from 200 bases to 6583 bases and evaluated by spiking a 1.2kb positive RNA marker into several commercially available RNA ladders. A calibration curve was generated from the RNA ladders which could then be used to estimate the size of unknown sample peaks. In addition, assay repeatability, linearity, LOQ, LOD were also evaluated in this study. The optimized method could be used as a RNA platform analytical method for RNA analysis or further modified to suit more specific user criteria.

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[PP-30]

SIMPLIFY MODIFICATION MAPPING AT THE INTACT PROTEIN LEVEL WITH ON-LINE SEPARATION

AND ANALYSIS BY CE-MS

Christopher Loessner2, Marcin Moczulski2, Jean Charles Berliet2 and Stephen Lock1

1 Sciex, Warrington, United Kingdom2 Sciex, Darmstadt, Germany. Contact: [email protected]

Performing on-line, high-resolution separation of proteoforms prior to mass spectrometry (MS) can be difficult. Often, one must either implement an immunocapture enrichment strat-egy and/or lengthy cation exchange, pre-fractionation steps prior to MS analysis. In this presentation, we demonstrate the use of capillary electrospray – mass spectrometry (CE-MS) methods for high-resolution separation of intact proteoforms.

We will use protein standards to demonstrate how CESI-MS [which is the integration of cap-illary electrophoresis (CE) and electrospray ionization (ESI) into a single process in a single device 1] can separate intact proteins and how it differs from separations obtained by stand-ard reverse phase LC-MS analysis. We then move on to examples where this approach has been applied to analysis of biological and pharmaceutical samples where CE-MS has been used to separate and identify proteoforms where charge differences aid the separation by CE.

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LIST OF AUTHORS

Name Affiliation E-mail Phone

Abad, L. Autonomous University of Madrid [email protected]

Adamo, Cristina B. University of Campinas

Aguilar, Carme Rovira i Virgili University [email protected]

Airado-Rodríguez, Diego University of Granada

Aldirbashi, Osama Newborn Screening Ontario, Children’s Hospital for Eastern Ontario

Alharthi, Sarah Oklahoma State University

Alonso, Eugenia Autonomous University of Madrid

Álvarez-Rivera, Gerardo Foodomics Laboratory, CIAL, CSIC

Alves de Piloto-Fernandes, Anna Maria São Francisco University

Alves Santana, Michele University of São Paulo [email protected] 55113091-3781

Amariei, Georgiana University of Alcalá

Amor-Gutiérrez, O. University of Oviedo

Amplatz, Benno Medical University of Innsbruck

Angulo, Santiago Centre for Metabolomics and Bioanalysis (CEMBIO), University CEU San Pablo

Aparicio-Muriana, M. Mar University of Granada [email protected] +34 958 24 23 85

Arce, Lourdes Institute of Fine Chemistry and Nanochemistry, University of Córdoba [email protected]

Arroyo-Manzanares, Natalia University of Murcia [email protected]

Asunción Nadal, Víctor de la University of Alcalá [email protected]

Aydogan, Cemil Oklahoma State University

Ballesteros-Vivas, Diego Foodomics Laboratory, CIAL, CSIC

Barbas, Coral Centre for Metabolomics and Bioanalysis (CEMBIO), University CEU San Pablo

Barbosa, José Institute for Research on Nutrition and Food Safety (INSA.UB), University of Barcelona

Beauchamp, Michael Brigham Young University

Beloborodov, Stanislav S. York University

Ben Attig, Jihane University of Castilla-La Mancha; Regional Institute for Applied Chemistry Research (IRICA); University of Tunis El Manar

[email protected] +34 926 29 53 00

Benavente, Fernando Institute for Research on Nutrition and Food Safety (INSA.UB), University of Barcelona [email protected] +34 93 403 91 16

Benito, Selma University of Alcalá; Networking Biomedical Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN)

Berezovski, Maxim V. University of Ottawa

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Berliet, Jean Charles Sciex [email protected]

Bermejo, Esperanza Autonomous University of Madrid

Bernardo-Bermejo, Samuel University of Alcalá [email protected] +34 91 885 49 71

Bernier, Michel Translational Gerontology Branch, National Institute on Aging, National Institutes of Health

Bilkova, Zuzana University of Pardubice [email protected]

Blanco-Covián, L. University of Oviedo

Blanco-López, M. C. University of Oviedo

Blázquez, Noelia Autonomous University of Madrid

Boldú, Júlia Institute of Food Science and Research (CIAL), Spanish National Research Council (CSIC)

Boltes, Karina University of Alcalá; Madrid Institute for Advanced Studies of Water (IMDEA Agua)

Bonnabry, P. Geneva University Hospitals (HUG)

Borrull, Francesc Rovira i Virgili University

Bramall, N. Jet Propulsion Laboratory, California Institute of Technolo-gy

Bressan, Lucas P. University of Campinas

Britz-McKibbin, Philip McMaster University [email protected]

Bueno Duarte, Gustavo Henrique Institute of Chemistry - Unicamp

Calull, Marta Rovira i Virgili University [email protected]

Carbonell-Rozas, Laura University of Granada [email protected] +34 958 24 23 85

Carlo, Michele Del University of Teramo

Carrão, Daniel B. Universidade de São Paulo; Colorado State University [email protected]

Carvalho Pimenta, Daniel Instituto Butantan

Casado, Natalia University of Alcalá [email protected] +34 91 885 63 89

Castro Costa, Brenda Maria de University of Campinas

Castro-Puyana, María Chemical Research Institute “Andrés M. del Río” (IQAR), University of Alcalá [email protected]

Ceolin Mariano, Douglas Oscar Centre for Metabolomics and Bioanalysis (CEMBIO), University CEU San Pablo [email protected]

Cerovsky, Vaclav Institute of Organic Chemistry and Biochemistry, The Czech Academy of Sciences

Chakraborty, Pranesh Newborn Screening Ontario, Children’s Hospital for Eastern Ontario

Channon, Rob B. Colorado State University

Chapman, Jeff MicroGEM International [email protected] +1-714-745-9495

Chetwynd, Andrew University of Birmingham

Chicharro, Manuel Autonomous University of Madrid

25th LACE Symposium



Cifuentes, Alejandro Foodomics Laboratory, CIAL, CSIC [email protected] +34 91 001 79 55

Claudino, Mario Angelo Universidade São Francisco

Cleaveland, Natalie University of Michigan

Coelho, Aline Guadalupe University of Campinas

Colnaghi Simionato, Ana Valéria University of Campinas [email protected] 55 19 35213145

Compagnone, Dario University of Teramo

Costa-Rama, E. University of Oviedo; REQUIMTE/LAQV, Instituto Superior de Engenharia do Porto, Politécnico do Porto

[email protected]

Cruces-Blanco, Carmen University of Granada

Daniel, Daniela Agilent Technologies [email protected]

Delerue-Matos, C. REQUIMTE/LAQV, Instituto Superior de Engenharia do Porto, Politécnico do Porto

Della Pelle, Flavio University of Teramo [email protected]

deMello, Andrew J. ETH Zürich [email protected]

DiBattista, Alicia McMaster University

Díez-Masa, José Carlos Institute of Organic Chemistry (IQOG-CSIC) [email protected] +34 91 258 74 94

Domínguez-Vega, Elena Amsterdam Institute for Molecules, Medicines and Systems, Vrije Universiteit; Center for Proteomics and Metabolomics, Leiden University Medical Center

[email protected]

El Rassi, Ziad Oklahoma State University [email protected] 405-744-5931

Escarpa, Alberto Chemical Research Institute “Andrés M. del Río” (IQAR), University of Alcalá [email protected] +34 91 885 49 95

Escuder-Gilabert, L. University of Valencia

Faccio, Andrea Centre for Metabolomics and Bioanalysis (CEMBIO), University CEU San Pablo; University of São Paulo

Falck, D. Center for Proteomics and Metabolomics, Leiden University Medical Center

Faserl, Klaus Medical University of Innsbruck

Favaro Perez, Mary Ângela Food Technology Institute, Ital; University of Campinas [email protected] 55 19 3521 2067,

55 19 3521 3023

Fernández-Abedul, M. T. University of Oviedo [email protected] +34 985 10 29 68

Forca Lima, Mónica Centre for Metabolomics and Bioanalysis (CEMBIO), University CEU San Pablo; University of Campinas


Foret, Frantisek Institute of Analytical Chemistry of the Czech Academy of Sciences; Central European Institute of Technology, Masaryk University

[email protected]

Fracassi da Silva, José Alberto University of Campinas [email protected] 13083-861

Frutos, Mercedes de Institute of Organic Chemistry (IQOG-CSIC) [email protected] +34 91 258 75 11

Furter, Jasmine S. University of Basel [email protected] +41 61 207 1052

25th LACE Symposium



G. Crevillén, Agustín National University of Education at the Distance (UNED) [email protected] +34 91 398 73 67

Gámiz-Gracia, Laura University of Granada

Ganewatta, Nisansala Oklahoma State University

García, María Ángeles Chemical Research Institute “Andrés M. del Río” (IQAR), University of Alcalá [email protected]

García-Campaña, Ana M. University of Granada [email protected]

García-Cañas, Virginia Institute of Food Science and Research, Spanish National Research Council (CSIC) [email protected]

Giménez, Estela Institute for Research on Nutrition and Food Safety (INSA.UB), University of Barcelona [email protected] +34 93 403 97 78

Gismera, M. J. Autonomous University of Madrid

Gonçalves Mora, Aline Universidade São Francisco

González-López, A. University of Oviedo

Gottardo, Rossella University of Verona

Gradillas, Ana Centre for Metabolomics and Bioanalysis (CEMBIO), University CEU San Pablo

Greño, Maider University of Alcalá [email protected] +34 91 885 49 71

Grespan Bottoli, Carla Beatriz University of Campinas

Gstöttner, C. Center for Proteomics and Metabolomics, Leiden University Medical Center

Gunasekara, Dulan University of Kansas

Guttman, Andras Research Institute of Biomolecular and Chemical Engineering, University of Pannonia; University of Debrecen

[email protected] +36 (88) 624-063

Guzman, Norberto A. Princeton Biochemicals, Inc. [email protected] +1-908-510-5258

Haselberg, Rob Amsterdam Institute for Molecules, Medicines and Systems, Vrije Universiteit

Hauser, Peter C. University of Basel [email protected] +41 61 207 1003

Henry, Charles S. Colorado State University [email protected]

Hernández-Mesa, Maykel University of Granada [email protected]

Hernández-Rodríguez, Juan F. University of Alcalá [email protected]

Herrero, Miguel Foodomics Laboratory, CIAL, CSIC

Herrero-Martínez, José Manuel University of Valencia [email protected] +34 96 354 40 62

Holzner, Gregor ETH Zürich

Ibáñez, Elena Foodomics Laboratory, CIAL, CSIC

Janussi, Sabrina Universidade São Francisco

Jaramillo, Elizabeth NASA, Jet Propulsion Laboratory, California Institute of Technology [email protected]

Jesus, Dosil P. de University of Campinas [email protected]

Jiang, Zhengjin Jinan University

Jiménez-Jiménez, Sara University of Alcalá

25th LACE Symposium



Junger, Ayandra S. University of Campinas

Jurado-Campos, Natividad Institute of Fine Chemistry and Nanochemistry, University of Córdoba

Jurado-Sánchez, Beatriz Chemical Research Institute “Andrés M. del Río” (IQAR), University of Alcalá

Juskova, Petra Institute of Analytical Chemistry of the Czech Academy of Sciences; ETH Zürich [email protected]

Kasicka, Vaclav Institute of Organic Chemistry and Biochemistry, The Czech Academy of Sciences

[email protected] +420-220 183 239

Kehl, F. Jet Propulsion Laboratory, California Institute of Technolo-gy

Kennedy, Robert T. University of Michigan [email protected] 1-734-615-4363

Konášová, Renáta Academy of Sciences of the Czech Republic

Kota, Arun K. Colorado State University

Koval, Dušan Academy of Sciences of the Czech Republic [email protected]

Krylov, Sergey N. York University [email protected] +1 (416)736-2100

Krylova, Svetlana M. York University [email protected]

Kupcik, Rudolf University of Pardubice [email protected]

Lago, Claudimir Lucio do University of São Paulo [email protected] + 55 11 3091-3828

Lahouidak, Samah University of Castilla-La Mancha; Regional Institute for Applied Chemistry Research (IRICA); Laboratoire d’Ingénieries des Procédés de l’Energie et de l’Environnement, ENSA, B.P.

[email protected]

Lara, Francisco J. University of Granada

Latrous, Latifa University of Tunis El Manar

Le, An T. H. York University

Lhotská, Ivona Charles University

Li, Tingting Sciex

Lindner, Herbert H. Medical University of Innsbruck [email protected] +43 512 9003 70310

Lleshi, Nejsi Institute for Research on Nutrition and Food Safety (INSA.UB), University of Barcelona

Lock, Stephen Sciex [email protected]

Loessner, Christopher Sciex

López, Miguel Ángel Chemical Research Institute “Andrés M. del Río” (IQAR), University of Alcalá

López-Duque, Sergio Institute of Organic Chemistry (IQOG-CSIC)

López-Gonzálvez, Ángeles Centre for Metabolomics and Bioanalysis (CEMBIO), University CEU San Pablo [email protected]

Lucas, S. Autonomous University of Madrid

Lucio-Cazaña, Francisco Javier University of Alcalá

Lunte, Susan M. University of Kansas [email protected]

25th LACE Symposium



Lynch, Iseult University of Birmingham

Madrid, R.E. FACET, National University of Tucumán [email protected]

Magalhães, T. REQUIMTE/LAQV, Instituto Superior de Engenharia do Porto, Politécnico do Porto

Mamani-Huanca, Maricruz Centre for Metabolomics and Bioanalysis (CEMBIO), University CEU San Pablo [email protected]

Mancera-Arteu, Montserrat Institute for Research on Nutrition and Food Safety (INSA.UB), University of Barcelona

María-Hormigos, Roberto University of Alcalá [email protected]

Marina, María Luisa Chemical Research Institute “Andrés M. del Río” (IQAR), University of Alcalá [email protected] +34 91 884 39 35

Martín-Biosca, Yolanda University of Valencia Yolanda.martin @uv.es

Martínez Pérez-Cejuela, Héctor University of Valencia [email protected] +34 96 354 40 62

McGown, Linda B. Rensselaer Polytechnic Institute [email protected] +1 (518)276-3681

Medina-Hernández, M. J. University of Valencia

Mendiola, Jose A. Foodomics Laboratory, CIAL, CSIC

Menger, Ruth F. Colorado State University

Moczulski, Marcin Sciex [email protected]

Moga, Ancuta University of Valencia

Molinero-Fernández, Águeda University of Alcalá [email protected]

Monincova, Lenka Institute of Organic Chemistry and Biochemistry, The Czech Academy of Sciences

Mora, M. Fernanda NASA, Jet Propulsion Laboratory, California Institute of Technology [email protected]

Moreira da Silva Lima, Taíssa University of Campinas

Moreno, Mónica Autonomous University of Madrid

Moreno-González, David University of Granada

Moreno-Guzmán, María Complutense University of Madrid [email protected]

Mozer Sciani, Juliana Universidade São Francisco

Muñoz-Espí, R. Institute of Material Science (ICMUV), University of Valencia

Nanni, P.I. FACET, National University of Tucumán

Navarro-Calderón, Diana Institute of Organic Chemistry (IQOG-CSIC)

Noell, Aaron NASA, Jet Propulsion Laboratory, California Institute of Technology

Nouws, H.P.A. REQUIMTE/LAQV, Instituto Superior de Engenharia do Porto, Politécnico do Porto

Novotny, Jakub Institute of Analytical Chemistry of the Czech Academy of Sciences; University of Pardubice

[email protected]

Núñez-Bajo, E. University of Oviedo

Oliveira Carvalho, Patricia de São Francisco University [email protected] 55 11 24548298

25th LACE Symposium



Oliveira, Anderson R. M. de Universidade de São Paulo

Olmo-Iruela, Monsalud del University of Granada

Ouimet, Claire University of Michigan

Pacheco, Marta University of Alcalá [email protected]

Padula, Marisa Food Technology Institute, Ital

Palacio, Covadonga University of Verona

Pérez-Alcaraz, Alberto Rovira i Virgili University [email protected] +34 997 55 86 29

Pérez-Baeza, M. University of Valencia

Pérez-Míguez, Raquel University of Alcalá

Pero-Gascon, Roger Institute for Research on Nutrition and Food Safety (INSA.UB), University of Barcelona [email protected] +34 93 403 91 23

Piana de Andrade, Victor AC Camargo Cancer Center [email protected] 55 11 21895000

Poppi, Ronei J. University of Campinas

Procopio, J.R. Autonomous University of Madrid

Puerta, Ángel Institute of Organic Chemistry (IQOG-CSIC) [email protected] +34 91 562 29 00

Ríos, Ángel University of Castilla-La Mancha; Regional Institute for Applied Chemistry Research (IRICA)


Robinson-Fuentes, Virginia A. Universidad Michoacana de San Nicolas de Hidalgo [email protected] +52 1 4432191452

Rodríguez-Gómez, Rocío Institute of Fine Chemistry and Nanochemistry, University of Córdoba

Rohrbasser, C. Pharmelp

Rojas, Daniel University of Alcalá; University of Teramo

Roth, S. College of Engineering and Architecture of Fribourg

Rudaz, Serge University of Geneva [email protected] +41.22.379.65.72

Rupérez, Francisco Javier Centre for Metabolomics and Bioanalysis (CEMBIO), University CEU San Pablo [email protected] +34 91 372 47 53

Saez-Brox, Raquel Institute of Organic Chemistry (IQOG-CSIC)

Sagrado, S. Interuniversity Institute of Molecular Recognition Research and Technological Development (IDM), Polytechnic University of Valencia, University of Valencia

Salgado, Antonio Center of Spectroscopy and Nuclear Magnetic Resonance (CERMN), Center for Support to Research in Chemistry (CAIQ), University of Alcalá

Salghi, R. Laboratoire d’Ingénieries des Procédés de l’Energie et de l’Environnement, ENSA, B.P.

Salido-Fortuna, Sandra University of Alcalá

Sánchez Arribas, Alberto Autonomous University of Madrid [email protected]

Sánchez-López, Elena Chemical Research Institute “Andrés M. del Río” (IQAR), University of Alcalá

25th LACE Symposium



Sánchez-Martínez, J. David Foodomics Laboratory, CIAL, CSIC

Santos, Aline Mara dos University of Campinas

Sanz-Nebot, Victoria Institute for Research on Nutrition and Food Safety (INSA.UB), University of Barcelona

Sarr, S. O. University Cheikh Anta Diop of Dakar

Saul, David MicroGEM International

Saz, José María University of Alcalá

Schappler, Julie University of Geneva

Schmidt Filho, Jayr AC Camargo Cancer Center

Senard, T. Center for Proteomics and Metabolomics, Leiden University Medical Center

Sevilla, M. T. Autonomous University of Madrid

Sierra, Tania University of Alcalá [email protected]

Silva, Fabiana Amaral da University of Campinas [email protected] 55 19 35213014, 55 19 35213145

Simó, Carolina Institute of Food Science and Research (CIAL), Spanish National Research Council (CSIC)

Simó-Alfonso, Ernesto Francisco University of Valencia

Singh, Nagendra S. Translational Gerontology Branch, National Institute on Aging, National Institutes of Health

Šolínová, Veronika Academy of Sciences of the Czech Republic

Somsen, Govert W. Amsterdam Institute for Molecules, Medicines and Systems, Vrije Universiteit [email protected]

Soriano, M. Laura Regional Institute for Applied Chemistry Research (IRICA); University of Castilla- La Mancha

[email protected]

Stanton, Jo University of Otago

Stavrakis, Stavros ETH Zürich

Suárez-Montenegro, Zully Foodomics Laboratory, CIAL, CSIC

Szekrenyes, Akos Mabxience

Szigeti, Marton University of Debrecen

Tagliaro, Franco University of Verona

Tavares da Costa, E. Jet Propulsion Laboratory, California Institute of Technolo-gy

Tavares, Marina F. M. University of São Paulo

Tejada-Casado, Carmen University of Granada

Timmins, James Helix Diagnostics, Inc.

Tumova, Tereza Institute of Organic Chemistry and Biochemistry, The Czech Academy of Sciences; University of Chemistry and Technology

25th LACE Symposium



Valbuena, Laura Institute of Fine Chemistry and Nanochemistry, University of Córdoba

Valdés, Alberto Foodomics Laboratory, CIAL, CSIC

Valimaña-Traverso, Jesús University of Alcalá

Vázquez, Luis Institute of Materials Science of Madrid (CSIC)

Vázquez-Garcidueñas, María Soledad Universidad Michoacana de San Nicolas de Hidalgo

Vázquez-Marrufo, Gerardo Universidad Michoacana de San Nicolas de Hidalgo

Vergara-Barberán, María University of Valencia

Veuthey, J.-L. University of Geneva

Vizioli, Nora M. University of Buenos Aires; Nacional Council of Scientific and Technical Research (CONICET)

[email protected]

Voeten, Robert L.C. Amsterdam Institute for Molecules, Medicines and Systems, Vrije Universiteit; TI-COAST

Vorlet, O. College of Engineering and Architecture of Fribourg

Wainer, Irving W. Translational Gerontology Branch, National Institute on Aging, National Institutes of Health

Wang, Qiqin Jinan University

Wang, Tong Ye York University

Wang, Wei Colorado State University

Warnakula, Indika University of Kansas

Weinberger, Robert CE Technologies, Inc. [email protected]

Wijesinghe, Manjula University of Kansas

Willis, Peter NASA, Jet Propulsion Laboratory, California Institute of Technology

Woolley, Adam T. Brigham Young University

Wuhrer, M. Center for Proteomics and Metabolomics, Leiden University Medical Center

Xu, Dongsheng University of Alcalá; Jinan University

Yuan, Kaisong University of Alcalá; Jinan University [email protected]

Zapardiel, Antonio National University of Education at the Distance (UNED)

Zavala-Sánchez, Xiomara Universidad Michoacana de San Nicolas de Hidalgo

Zougagh, Mohammed University of Castilla-La Mancha; Regional Institute for Applied Chemistry Research (IRICA)

[email protected]




Authors Index

25th LACE Symposium


A

Abad, L. 178Adamo, Cristina B. 127Aguilar, Carme 61, 144Airado-Rodríguez, Diego 130Aldirbashi, Osama 65Alharthi, Sarah 36Alonso, Eugenia 189Álvarez-Rivera, Gerardo 115Alves de Piloto-Fernandes, Anna Maria 69Alves Santana, Michele 157Amariei, Georgiana 183Amor-Gutiérrez, O. 73, 199Amplatz, Benno 79Angulo, Santiago 71Aparicio-Muriana, M. Mar 194Arce, Lourdes 103Arroyo-Manzanares, Natalia 103Asunción-Nadal, Víctor de la 208Aydogan, Cemil 36

B

Ballesteros-Vivas, Diego 115Barbas, Coral 71, 151, 185, 219Barbosa, José 140Beauchamp, Michael 76Beloborodov, Stanislav S. 54Ben Attig, Jihane 202Benavente, Fernando 63, 112, 140, 174, 217Benito, Selma 153Berezovski, Maxim 112, 140Berliet, Jean Charles 221, 222, 223, 224Bermejo, Esperanza 189Bernardo-Bermejo, Samuel 153Bernier, Michel 71Bilkova, Zuzana 41Blanco-Covián, L. 73Blanco-López, M.C. 73, 199Blázquez, Noelia 189Boldú, Júlia 192Boltes, Karina 183

Bonnabry, P. 43Borrull, Francesc 61, 144Bramall, N. 45Bressan, Lucas P. 76, 127Britz-McKibbin, Philip 65Bueno-Duarte, Gustavo Henrique 69

C

Calull, Marta 61, 144Carbonell-Rozas, Laura 160Carlo, Michele Del 89Carrão, Daniel B. 119Carvalho Pimenta, Daniel 151Casado, Natalia 163, 165, 170Castro Costa, Brenda Maria de 76Castro-Puyana, María 99, 106, 142, 153,

170, 181, 187Ceolin Mariano, Douglas Oscar 151Cerovsky, Vaclav 51Chakraborty, Pranesh 65Channon, Rob B. 119Chapman, Jeff 108Chetwynd, Andrew 222Chicharro, Manuel 189Cifuentes, Alejandro 115Claudino, Mario Angelo 151Cleaveland, Natalie 34Coelho, Aline Guadalupe 76Colnaghi Simionato, Ana Valéria 69, 205,

219Compagnone, Dario 89Costa-Rama, E. 73, 199Cruces-Blanco, Carmen 130

D

Daniel, Daniela 167Delerue-Matos, C. 73, 199Della Pelle, Flavio 89DeMello, Andrew J. 46DiBattista, Alicia 65Diez-Masa, Jose Carlos 92, 138, 197Domínguez-Vega, Elena 67, 106

25th LACE Symposium


E

El Rassi, Ziad 36Escarpa, Alberto 60, 89, 94, 122, 148, 208,

211, 214Escuder-Gilabert, L. 101, 176

F

Faccio, Andrea 71Falck, D. 67Faserl, Klaus 79, 222Favaro Perez, Mary Ângela 167Fernández-Abedul, M.T. 73, 199Forca Lima, Mónica 219Foret, Frantisek 41Fracassi da Silva, José Alberto 76Frutos, Mercedes de 92, 138, 197Furter, Jasmine S. 49

G

Gámiz-Gracia, Laura 110Ganewatta, Nisansala 36García-Campaña, Ana M. 110, 130, 160, 194García-Cañas, Virginia 85, 192García, María Ángeles 142, 163, 165, 170,

181, 183G. Crevillen, Agustín 94Giménez, Estela 174Gismera, M.J. 178Gonçalves Mora, Aline 151González-López, A. 73Gottardo, Rossella 124Gradillas, Ana 185Greño, Maider 99, 142Grespan Bottoli, Carla Beatriz 167Gstöttner, C. 67Gunasekara, Dulan 87Guttman, Andras 114, 221Guzman, Norberto A. 33

H

Haselberg, Rob 106Hauser, Peter C. 49

Henry, Charles 119Hernández-Mesa, Maykel 110, 130Hernández-Rodríguez, Juan F. 122Herrero-Martínez, José Manuel 63, 217Herrero, Miguel 115Holzner, Gregor 46

I

Ibáñez, Elena 115

J

Janussi, Sabrina 151Jaramillo, Elizabeth 146Jesus, Dosil P. de 127Jiang, Zhengjin 163, 172Jiménez-Jiménez, Sara 181Junger, Ayandra S. 127Jurado-Campos, Natividad 103Jurado-Sánchez, Beatriz 148, 208, 211Juskova, Petra 41

K

Kasicka, Vaclav 51Kehl, F. 45Kennedy, Robert T. 34Konášová, Renáta 136Kota, Arun K. 119Koval, Dušan 136Krylova, Svetlana M. 54Krylov, Sergey N. 54Kupcik, Rudolf 41

L

Lago, Claudimir Lucio do 157, 167Lahouidak, Samah 133Lara, Francisco J. 110, 160, 194Latrous, Latifa 202Le, An T. H. 54Lhotská, Ivona 194Lindner, Herbert 79, 222Li, Tingting 223Lleshi, Nejsi 174

25th LACE Symposium


Lock, Stephen 221, 222, 223, 224Loessner, Christopher 222, 223, 224Lopez-Duque, Sergio 197López-Gonzálvez, Ángeles 185López, Miguel Ángel 60, 214Lucas, S. 178Lucio-Cazaña, Francisco Javier 153Lunte, Susan M. 87Lynch, Iseult 222

M

Madrid, R.E. 73Magalhães, T. 199Mamani-Huanca, Maricruz 185Mancera-Arteu, Montserrat 174Maria-Hormigos, Roberto 211Marina, María Luisa 99, 106, 142, 153, 163,

165, 170, 172, 181, 183, 187Martín-Biosca, Y. 101, 176Martínez Pérez-Cejuela, Héctor 217McGown, Linda B. 59Medina-Hernández, M.J. 101, 176Mendiola, Jose A. 115Menger, Ruth F. 119Moczulski, Marcin 221, 222, 223, 224Moga, Ancuta 63Molinero-Fernández, Águeda 60, 214Monincova, Lenka 51Mora, Fernanda M. 45, 146Moreira da Silva Lima, Taíssa 76Moreno-González, David 110Moreno-Guzmán, María 60, 214Moreno, Mónica 189Mozer Sciani, Juliana 151Muñoz-Espí, R. 176

N

Nanni, P.I. 73Navarro-Calderon, Diana 197Noell, Aaron 146Nouws, H.P.A. 73, 199Novotny, Jakub 41Núñez-Bajo, E. 73

O

Oliveira, Anderson R. M. de 119Oliveira-Carvalho, Patricia de 69Olmo-Iruela, Monsalud del 110, 160Ouimet, Claire 34

P

Pacheco, Marta 148Padula, Marisa 167Palacio, Covadonga 124Pérez-Alcaraz, Alberto 61, 144Pérez-Baeza, M. 176Pérez-Míguez, Raquel 106Pero-Gascon, Roger 112, 140Piana-de Andrade, Victor 69Poppi, Ronei J. 127Procopio, J.R. 178Puerta, Angel 92, 138, 197

R

Ríos, Ángel 97, 133, 202Robinson-Fuentes, Virginia A. 82Rodríguez-Gómez, Rocío 103Rohrbasser, C. 43Rojas, Daniel 89, 122Roth, S. 43Rudaz, Serge 43Rupérez, Francisco Javier 71, 151, 219

S

Saez-Brox, Raquel 197Sagrado, S. 101, 176Salgado, Antonio 170Salghi, R. 133Salido-Fortuna, Sandra 99, 187Sánchez Arribas, Alberto 189Sánchez-López, Elena 153, 172Sánchez-Martínez, J. David 115Santos, Aline Mara dos 219Sanz-Nebot, Victoria 112, 140, 174Sarr, S.O. 43Saul, David 108Saz, Jose María 165

25th LACE Symposium


Schappler, J. 43Schmidt-Filho, Jayr 69Senard, T. 67Sevilla, M.T. 178Sierra, Tania 94Silva, Fabiana Amaral da 205Silva, José Alberto F. da 127Simó-Alfonso, Ernesto Francisco 63, 217Simó, Carolina 85, 192Singh, Nagendra S. 71Šolínová, Veronika 136Somsen, Govert W. 106Soriano, M. Laura 133Stanton, Jo 108Stavrakis, Stavros 46Suárez-Montenegro, Zully 115Szekrenyes, Akos 221Szigeti, Marton 114, 221

T

Tagliaro, Franco 124Tavares da Costa, E. 45Tavares, Marina F. M. 71Tejada-Casado, Carmen 110Timmins, James 81Tumova, Tereza 51

V

Valbuena, Laura 103Valdés, Alberto 115Valimaña-Traverso, Jesús 183Vázquez-Garcidueñas, Ma. Soledad 82Vázquez, Luis 89Vázquez-Marrufo, Gerardo 82Vergara-Barberán, María 63Veuthey, J.-L. 43Vizioli, Nora M. 84Voeten, Robert L.C. 106Vorlet, O. 43

W

Wainer, Irving W. 71Wang, Qiqin 172Wang, Tong Ye 54Wang, Wei 119Warnakula, Indika 87Weinberger, Robert 81Wijesinghe, Manjula 87Willis, Peter 45, 146Woolley, Adam T. 76Wuhrer, M. 67

X

Xu, Dongsheng 172

Y

Yuan, Kaisong 208

Z

Zapardiel, Antonio 189Zavala-Sánchez, Xiomara 82Zougagh, Mohammed 133, 202

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25th LACE - FGUA · Daniel Rojas Tizón ... José Alberto Fracassi da Silva. 25th LACE Symposium Alcalá de Henares, Madrid, Spain, September 29th - October 2nd, 2019 15 16:10 - 16:30