Principle & Applications of Liquid ChromatographyPrinciple & Applications of Liquid Chromatographywith Mass Spectrometry (LC-MS)with Mass Spectrometry (LC-MS)

PRESENTED BY:PRESENTED BY: SANDHYA TALLASANDHYA TALLAM.PHARM (PHARMACOLOGY)M.PHARM (PHARMACOLOGY)

Principle of MSPrinciple of MS Bombardment of sample in vapor phase with Bombardment of sample in vapor phase with

high energy electron beam to converted into high energy electron beam to converted into positively charged ion species which are positively charged ion species which are separated on the basis of their mass to charge separated on the basis of their mass to charge ratio.ratio.

ContentsContents

IntroductionIntroduction Elements of LC-MSElements of LC-MS Liquid ChromatographyLiquid Chromatography InterfacesInterfaces Mass spectrometryMass spectrometry ApplicationsApplications ReferencesReferences

Hyphen is a great funHyphen is a great fun Hyphen is a lineHyphen is a line which is used to joinwhich is used to join the words of different designthe words of different design Chromatography and spectroscopyChromatography and spectroscopy Hyphened to give sensitivity and selectivityHyphened to give sensitivity and selectivity GC-MS, LC-MS and GC or LC -MS-MSGC-MS, LC-MS and GC or LC -MS-MS

IntroductionIntroduction History : Starts in early 1970’History : Starts in early 1970’s , in several laboratories the possibility s , in several laboratories the possibility

of online LCMS were investigated .of online LCMS were investigated . LC-MS : Hyphenated techniqueLC-MS : Hyphenated technique Basic concepts :Basic concepts : LCLC –To resolve complex mixture of –To resolve complex mixture of compoundscompounds InterfaceInterface – To transport & desolvation of – To transport & desolvation of sample into ion source of mass sample into ion source of mass analyser.analyser. MSMS – To ionise & analyse individual – To ionise & analyse individual resolved components , on the basis of resolved components , on the basis of their m/z ratio. their m/z ratio.

Liquid ChromatographyLiquid Chromatography

Principle – Partitioning of analyte between mobile Principle – Partitioning of analyte between mobile (liquid) & stationary (solid) phase.(liquid) & stationary (solid) phase.

RPHPLC is most commonly used in LC-MS.RPHPLC is most commonly used in LC-MS. Column is heart of LC. Column is heart of LC. Length : 5 – 25 cmLength : 5 – 25 cm ID : 3 – 5 mmID : 3 – 5 mm Particle size : 3 or 5 micron Particle size : 3 or 5 micron Material : Silica + long chain hydrocarbon.Material : Silica + long chain hydrocarbon. Liquid flow rate : 0.1 – 1.5 ml/minLiquid flow rate : 0.1 – 1.5 ml/min Column efficiency : 40,000 – 70,000 plates/meterColumn efficiency : 40,000 – 70,000 plates/meter

Mass SpectroscopyMass Spectroscopy

Mass spectrometerMass spectrometer produces a beam of ions, separates produces a beam of ions, separates them according to m/z ratio of ions.them according to m/z ratio of ions.

The "heart" of the mass spectrometer is The "heart" of the mass spectrometer is an analyzer.an analyzer. Analyzer uses electrical or magnetic fields, or Analyzer uses electrical or magnetic fields, or

combination of both, to move the ions combination of both, to move the ions from the region where they are produced from the region where they are produced to a detector . to a detector .

Need of techniqueNeed of technique

Drawbacks :Drawbacks : HPLC – On its own provide ambiguous HPLC – On its own provide ambiguous confirmation of identity of analyte.confirmation of identity of analyte. MS – Limitation in handling mixture of MS – Limitation in handling mixture of compounds.compounds. LC-MS – Benefited mutually, from high resolution LC-MS – Benefited mutually, from high resolution

separation capability of LC & high sensitive, structure separation capability of LC & high sensitive, structure specific detection capacity of mass. specific detection capacity of mass.

Elements of LC-MSElements of LC-MS

Working of an LC-MS systemWorking of an LC-MS system :

Interfacing problemInterfacing problem Problem in coupling LC-MSProblem in coupling LC-MS - Enormous mismatch between large solvent volumes from - Enormous mismatch between large solvent volumes from

LC & vacuum requirement of MS So for solving this LC & vacuum requirement of MS So for solving this problem interface is used.problem interface is used.

Problem while interfacingProblem while interfacing Necessity to remove solvent. Necessity to remove solvent. Difficulty in vaporising nonvolatile solvent. Difficulty in vaporising nonvolatile solvent. Degradation of thermally labile compound. Degradation of thermally labile compound.

Interfaces Used in LC-MSInterfaces Used in LC-MS Moving beltMoving belt Thermospray interfaceThermospray interface Particle beam interfaceParticle beam interface Atmospheric pressure ionisation (API)Atmospheric pressure ionisation (API) - with ESI- with ESI - with APCI- with APCI Capillary inletCapillary inlet Continuous flow – fast atom bombardmentContinuous flow – fast atom bombardment Direct liquid introductionDirect liquid introduction

Moving belt interfaceMoving belt interface

Important reason for it’s success is compatibility with wide Important reason for it’s success is compatibility with wide range of chromatographic conditions ; also EI, CI & FAB range of chromatographic conditions ; also EI, CI & FAB methods employed easily.methods employed easily.

Limitations :1) Complex mechanical device Limitations :1) Complex mechanical device 2) Renewal of belt & belt memory are 2) Renewal of belt & belt memory are

troublesome. troublesome.

Thermospray interfaceThermospray interface

Particle beam interfaceParticle beam interface

Column effluent is nebulised pneumatically into near Column effluent is nebulised pneumatically into near atmospheric pressure desolvation chamber atmospheric pressure desolvation chamber

Role of momentum separator Role of momentum separator

Momentum separatorMomentum separator

Electrospray IonisationElectrospray Ionisation

Unique feature – Desolvation & ionization process occur together in the Unique feature – Desolvation & ionization process occur together in the ion source at atmospheric pressure .ion source at atmospheric pressure .

Nebulisation is done by using strong electric potential difference of 3kv & Nebulisation is done by using strong electric potential difference of 3kv & NN22 flow. flow.

Nebulisation occur at only flow rate of below 10 microlitre/min.Nebulisation occur at only flow rate of below 10 microlitre/min.

Electro Spray Ionization (ESI)Electro Spray Ionization (ESI)

3 stages of ESI : 3 stages of ESI : - Formation of charged droplet - Formation of charged droplet - Solvent evaporation & droplet fission - Solvent evaporation & droplet fission - Formation of gas phase ion - Formation of gas phase ion

Atmospheric Pressure Chemical Atmospheric Pressure Chemical Ionisation (APCI)Ionisation (APCI)

It’s based on solvent mediated CI by ion-molecule reactions It’s based on solvent mediated CI by ion-molecule reactions initiated by electron produced in the Corona discharge needle.initiated by electron produced in the Corona discharge needle.

Solvent vapor act as reagent gas.Solvent vapor act as reagent gas.

3030

APC

I

Mass AnalyserMass Analyser

Linear quadrupole mass analyserLinear quadrupole mass analyser Quadrupole – ion trap mass analyserQuadrupole – ion trap mass analyser Time of flight Time of flight Quadrupole - time of flight Quadrupole - time of flight FT –ICR - MSFT –ICR - MS

Linear Quadrupole Mass AnalyserLinear Quadrupole Mass Analyser

Mass scanning is done by varying RF & DC frequencies & Mass scanning is done by varying RF & DC frequencies & keeping ratio constant .keeping ratio constant .

Advantages :- easy in use.Advantages :- easy in use. - electric voltage easy & rapidly varied.- electric voltage easy & rapidly varied.

Quadrupole – Ion Trap Mass Quadrupole – Ion Trap Mass AnalyserAnalyser

Ions are introduced to the trap in a pulsed mode & stabilised Ions are introduced to the trap in a pulsed mode & stabilised by the He gas by the He gas

Function of ion trap is to retain unsorted ion temporarily then Function of ion trap is to retain unsorted ion temporarily then they are released to detector sequentially scanning the electric they are released to detector sequentially scanning the electric field.field.

ApplicationsApplications Quantitative bioanalysisQuantitative bioanalysis Clinical application – TDMClinical application – TDM Pharmacokinetic studyPharmacokinetic study Drug metabolismDrug metabolism Proteomics Proteomics Analysis of pestisidesAnalysis of pestisides Drug discovery & drug develpomentDrug discovery & drug develpoment Analysis of steroidsAnalysis of steroids Food safety analysis Food safety analysis Stability testing & impurity profilingStability testing & impurity profiling

Quantitative BioanalysisQuantitative Bioanalysis

Methods for quantitative measurement of a drug, Methods for quantitative measurement of a drug, drug metabolites or chemicals in biological fluids.drug metabolites or chemicals in biological fluids.

Determination of : Determination of : Resperine (by ESI or APCI )Resperine (by ESI or APCI ) Risperidone & 9-OH Risperidone ( by ESI ) Risperidone & 9-OH Risperidone ( by ESI ) Lovastatin , Simvaststin .Lovastatin , Simvaststin .

Clinical Application - TDMClinical Application - TDM The ability to accurately measure drug levels in whole The ability to accurately measure drug levels in whole

blood is vital for effective quantification of drugs.blood is vital for effective quantification of drugs. With the help of MS-MS its easy to distinguish parent With the help of MS-MS its easy to distinguish parent

drug molecule from metabolites by use of molecular mass .drug molecule from metabolites by use of molecular mass . Determination of :Determination of :

Sirolimus , TacrilimusSirolimus , Tacrilimus Amphetamines (by LC-APCI-MS)Amphetamines (by LC-APCI-MS) LSD & Cocaine (by LC-ESI-MS) LSD & Cocaine (by LC-ESI-MS)

Pharmacokinetic StudiesPharmacokinetic Studies These studies tell us how quickly a drug will be These studies tell us how quickly a drug will be

cleared from the hepatic blood flow, and organs of cleared from the hepatic blood flow, and organs of the body.This is done by detection of the drug in the the body.This is done by detection of the drug in the body matrices such as blood & urine.body matrices such as blood & urine.

With the help of MS-MS we can program the detector With the help of MS-MS we can program the detector to select out certain ions to fragment .to select out certain ions to fragment .

eg. Cefixime, Adefovir, Zolmitriptaneg. Cefixime, Adefovir, Zolmitriptan

Drug MetabolismDrug Metabolism Easier to characterize metabolites (previously Easier to characterize metabolites (previously

difficult task)difficult task) Its done by comparing directly the spectraIts done by comparing directly the spectra of the drug with those of it’s metabolites.of the drug with those of it’s metabolites. Common biotransformation (eg. oxidaton, Common biotransformation (eg. oxidaton,

reduction,hydrolysis etc.) detected from molecular reduction,hydrolysis etc.) detected from molecular mass of metabolite. mass of metabolite.

eg. Zolpidem (by ESI ) eg. Zolpidem (by ESI ) Quercetin (by ESI) Quercetin (by ESI)

ProteomicsProteomics

Its the large-scale study of protein, particularly their Its the large-scale study of protein, particularly their structures and functions .structures and functions .

Important technique :Important technique : peptide mass fingerprinting(by nano ESI-MS)peptide mass fingerprinting(by nano ESI-MS) Peptide sequence analysis – this is not possible by Peptide sequence analysis – this is not possible by

MS, needs MS-MS. MS, needs MS-MS. accurate mass,affinity or sequence tags- accurate mass,affinity or sequence tags-

(by FT-ICR-MS)(by FT-ICR-MS)

Analysis of PestisidesAnalysis of Pestisides

Suitability of LC-MS ionisation mode for various class Suitability of LC-MS ionisation mode for various class of pestisidesof pestisides

Drug Discovery & Drug Drug Discovery & Drug DevelopmentDevelopment

Drug development steps : drug discovery, preclinical Drug development steps : drug discovery, preclinical development , clinical development , manufacturing .development , clinical development , manufacturing .

LC-MS needed in every step, LC-MS needed in every step, It help in checking proper progress in the synthesis of It help in checking proper progress in the synthesis of

new chemical entity.new chemical entity.

Analysis of SteroidsAnalysis of Steroids

Doping analysis in sports Doping analysis in sports Detection of Stanozolol , methandrostenolone in Detection of Stanozolol , methandrostenolone in

equine & human urine by LC-ESI-MS. equine & human urine by LC-ESI-MS. Detection of testosterone by LC-ESI-MS in Detection of testosterone by LC-ESI-MS in

Human athletes .Human athletes .

Food Safety AnalysisFood Safety Analysis

LC-MS imp in quantification & confirmation of LC-MS imp in quantification & confirmation of identity of food contaminants (eg. Sea food)identity of food contaminants (eg. Sea food)

LC-MS of antibiotic & antibacterial compound:LC-MS of antibiotic & antibacterial compound:

Stability Testing & Impurity Stability Testing & Impurity ProfilingProfiling

In stability testing : to determine the identity, chemical In stability testing : to determine the identity, chemical structure of the API and quantification levels as well as structure of the API and quantification levels as well as presence of degradants, excipients and impurities .presence of degradants, excipients and impurities .

eg. determination of degradants ineg. determination of degradants in --.Trimethoprim - Artesunate.Trimethoprim - Artesunate In impurity profiling : detection of minor components In impurity profiling : detection of minor components

in presence of major components.in presence of major components. eg. In identification of eg. In identification of Polar impurity in mosapridePolar impurity in mosapride

LC/MS in Clinical Labs LC/MS in Clinical Labs Neonatal screeningNeonatal screening

Specialist triple quard applicationSpecialist triple quard application Therapeutic drug monitoringTherapeutic drug monitoring

To replace immuno assaysTo replace immuno assays Drugs of abuseDrugs of abuse

LC/MS in the Environmental FieldLC/MS in the Environmental Field WaterWater

Identification and quantization of pollutantsIdentification and quantization of pollutants Pesticides, antibioticsPesticides, antibiotics

Food Food chemical contaminantschemical contaminants antibioticsantibiotics natural toxinsnatural toxins

Animal feedsAnimal feeds contaminants, illegal substancescontaminants, illegal substances

LC/MS in Other Industries LC/MS in Other Industries OrganometallicsOrganometallics

structure structure DetergentsDetergents

Quality Control, competitors productsQuality Control, competitors products PolymersPolymers

molecular weight, structuremolecular weight, structure

ReferencesReferences Frank Settle. Handbook of Frank Settle. Handbook of Instrumental TechniquesInstrumental Techniques for Analytical for Analytical

ChemistryChemistry 1 1stst ed.647-659. ed.647-659. Willard, Merritt, Dean, Settle. Willard, Merritt, Dean, Settle. Instrumental MethodsInstrumental Methods of Analysisof Analysis .7 .7thth ed. ed.

618-620.618-620. Skoog, Holler,Nieman. Skoog, Holler,Nieman. Principles of InstrumentalPrinciples of Instrumental AnalysisAnalysis,5,5thth ed.1992 ed.1992; ; 501-511:528-531:738-739.501-511:528-531:738-739. Christian Garry. Christian Garry. Analytical ChemistryAnalytical Chemistry. 6. 6thth ed. 608-610 ed. 608-610 Wilfried M. A. Niessen . Liquid Chromatography – Mass Spectroscopy . Wilfried M. A. Niessen . Liquid Chromatography – Mass Spectroscopy .

33rdrd ed. 80-120 , 164- 180 . ed. 80-120 , 164- 180 . J. Flarakos*, W. Luoa, M. Aman, D. Svinarov, Quantification of

risperidone and 9-hydroxyrisperidone in plasmaand saliva from adult and pediatric patients by liquidchromatography–mass spectrometry .Journal of Chromatography A, 1026 (2004) 175–183

ReferencesReferences N. Lindeg°ardh a,b*, A.M. Dondorp a,b, P.

Singhasivanon a, Validation and application of a liquid chromatographic–mass spectrometric method for determination of artesunate in pharmaceutical samples. Journal of Pharmaceutical and Biomedical Analysis 45 (2007) 149–153.

www.pubmedcentral.nih.govwww.pubmedcentral.nih.gov www.sciencedirect.comwww.sciencedirect.com www.agilent.com/chemwww.agilent.com/chem www.waters.comwww.waters.com www.interscience.wiley.comwww.interscience.wiley.com