Nuevas metodologías analíticas para la determinación

NUEVAS METODOLOGAS ANALTICAS

PARA LA DETERMINACIN DE ANTIOXIDANTES ALIMENTARIOS

NEW ANALYTICAL METHODOLOGIES FOR FOOD ANTIOXIDANT

DETERMINATION

Tesis Doctoral lvaro Andreu Navarro

Noviembre 2011

TTULO:Nuevas metodologas analticas para la determinacin de

antioxidantes alimentarios

AUTOR: lvaro Andreu Navarro

Edita: Servicio de Publicaciones de la Universidad de Crdoba. 2012Campus de RabanalesCtra. Nacional IV, Km. 396 A14071 Crdoba

www.uco.es/[email protected]

TTULO DE LA TESIS: NUEVAS METODOLOGAS ANALTICAS PARA LA DETERMINACIN DE ANTIOXIDANTES ALIMENTARIOS

DOCTORANDO/A: LVARO ANDREU NAVARRO

INFORME RAZONADO DEL/DE LOS DIRECTOR/ES DE LA TESIS (se har mencin a la evolucin y desarrollo de la tesis, as como a trabajos y publicaciones

derivados de la misma).

NDICE Objeto / Aim Introduccin Captulo 1: Herramientas Analticas

Captulo 2: Innovaciones en la determinacin

cromatogrfica de antioxidantes en alimentos

Captulo 3: Innovaciones en la determinacin no

cromatogrfica de antioxidantes en alimentos

Captulo 4: Nuevas investigaciones con nanopartculas de oro

Captulo 5: Discusin de resultados

Discussion of the results

Conclusiones / Conclusions Anexo

OBJETO AIM

OBJETO

INTRODUCCIN

ANTIOXIDANTES ALIMENTARIOS

Figura 1

Figura 1.

1. Antioxidantes naturales

Figura 2

Figura 2.

R1

R2

R3 R1=R2=R3=OH: cido glico R1=R2=OH, R3= - : cido protocatecuico

Flavonoides cidos hidroxibenzoicos

Gallotaninas (ej. pentagalloyglucosa)

R1

R2

cidos hidroxicinmicos

R1=OH: cido cumrico R1=R2=OH: cido cafeico

Estilbenos (ej. Resveratrol)

Figura 2.

Figura 3

Figura 3

Tabla 1.

R1

R2

R2 R2

Proantocianidinas (R1, R2=H, OH) (ej. Dmero de procianidina tipo-B,

R1=OH, R2=H)

Lignanos (ej.

Figura 3.

CATEQUINAS ANTOCIANINAS

FLAVONAS FLAVONOIDES

FLAVANONAS ISOFLAVONAS

Tabla 1.

Tabla 2

Tabla2.

2. Antioxidantes sintticos

FLUORIMETRA DE LARGA LONGITUD DE ONDA

Figura 4

Figura 4.

Figura 4

Figura 5

Figura 6

Figura 5.

Figura 6.

LUMINISCENCIA SENSIBILIZADA DE LANTNIDOS

1)

2)

3)

4)

5)

Tabla 3

Tabla3.

DISPERSIN DE LA RADIACIN

SISTEMAS DE FLUJO

Figura 7 A

Figura 7 B)

Figura 7.

Figura 8

Figura 8.

QUIMIOMETRA

Bibliografa

CAPITULO 1HERRAMIENTAS ANALTICAS

1. Estndares y reactivos

Compuesto Pureza Suministro

o

o

o o

2. Sistemas FIA

3. Instrumentacin

4. Aparatos

5. Programas informticos

CAPITULO 2

INNOVACIONES EN LA DETERMINACIN CROMATOGRFICA DE ANTIOXIDANTES EN

ALIMENTOS

-

J. Agric. Food Chem.

-

J. Sep. Sci

-

Chromatographia

-

Food. Chem

Analytical innovations in the detection of phenolics in wines

Pietro Russo, lvaro Andreu-Navarro, Mara-Paz Aguilar-Caballos, Juan-Manuel Fernndez-Romero, Agustina Gmez-Hens

Introduction.

1

118

15, 715, 18

16, 17

16

19

1, 3178,

12 2

8, 12

18

1, 311, 1317

pp

cis trans

n

1, 3, 8,

10, 12, 14

1. Materials and methods

1.1.

1.2.

p

p

cistrans

transtrans

1.3.

Figure 1

Table 1

Figure 1.

Table 1

Conditioning step

1.4.

cis trans

1.5. Estimation of LODs

20y

1.6.

2. Results and discussion

2.1.

18

Table 2

Table 2

Table 2

2.2.

2.3.

21

22, 2324

Table 1p

Table 1

2.4.

Figure 2

Figures 3 4

Figure 3

[Tb(III)], M

0.000 0.002 0.004 0.006 0.008 0.010

Area

0

20

40

60

80

100

2

1

39

5

4

711

8,12

Figure 3.

Time (min)0 5 10 15 20

IL

0

3

6

9

1

2

34 5 7

8 9 1112

Figure 4.

2.5.

Table 3

20117,

19

r

Table 4

Tabl

e 3

1.

7910

-900

0.06

45

0.0

003

0.5

0.

1 0.

9999

5

3.43

10-9

000.

1608

0

.000

3 0.

6

0.1

0.99

992

6.

2650

-200

00.

0176

0

.000

2 0.

30

0.0

9 0.

9990

15

7.

5150

-100

00.

0862

0

.000

9 1.

5

0.4

0.99

9114

10.0

50-2

000

0.05

38

0.0

004

0.7

0.

3 0.

9994

17

11.9

510

-900

0.04

54

0.0

001

0.45

0

.04

0.99

993

12

.77

300-

5000

0.00

269

0.

0000

5 -0

.1

0.1

0.

9976

114

13.8

210

0-20

000.

0140

0

.000

2 0.

4

0.2

0.99

8940

17.3

550

-200

00.

0221

0

.000

2 0.

4

0.1

0.99

9615

17.9

510

0-20

000.

0036

1

0.00

004

0.03

0

.03

0.99

8629

Tabl

e 3

(*th

e lo

wer

lim

it of

dyn

amic

rang

e fo

r eac

h ph

enol

ic is

its L

OQ

). Ita

lics:

fluor

imet

ric m

etho

d

Tabl

e 4

2.6.

Figures 5 6

Time (min)0 5 10 15 20 25 30

mAU

0

10

20

30

116 1315

324 nm

Time (min)0 5 10 15 20 25 30

mAU

0

40

80

120

160280 nm1

7 89 1012

Time (min)0 5 10 15 20 25 30

mAU

0

40

80

120 256 nm

2 3 5

14

Figure 5.

Figure 5

1Figure 6

Time (min)

0 5 10 15 20 25 30

mAU

-5

0

5

10

15

20

16

18

365 nm

Figure 6

Tables 5 6

Y Xr Y X

cis

2

Time (min)

0 5 10 15 20

IL

0

4

8

12

1

2

3 5

7

8 1112

Tabl

e 5.

Tabl

e 6.

Table 7

3, 6, 13, 14, 166, 13, 14

3

Table 7.

References

Anal. Chim. Acta 563

Food Chem. 94

J. Chromatogr., A 1038


Food Control 16



Food Chem.

Food Chem. 64

Eur. Food Res. Technol. 224

J. Agric. Food Chem. 51




Food Chem. 89



J. Sep. Sci. 29


Anal. Chem. 55

Trends Anal. Chem. 21

J. Agric. Food Chem. 54


Analyst 122

Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos

91

Usefulness of terbium-sensitized luminescence detection for the chemometric classification of wines

by their content in phenolic compounds A. Andreu-Navarro, P. Russo, M.P. Aguilar-Caballos, J.M. Fernndez-Romero and A. Gmez-Hens (*)


93

Introduction


95

1. Experimental

1.1. Wine samples

1.2. LC-separation and photometric/fluorimetric detection

96


2.1. Analytical features

Table 1

Tabl

e 1

1.

7910

-900

0.06

45

0.0

003

0.5

0.

1 0.

9999

5

3.43

10-9

000.

1608

0

.000

3 0.

6

0.1

0.99

992

6.

2650

-200

00.

0176

0

.000

2 0.

30

0.0

9 0.

9990

15

7.

5150

-100

00.

0862

0

.000

9 1.

5

0.4

0.99

9114

10.0

50-2

000

0.05

38

0.0

004

0.7

0.

3 0.

9994

17

11.9

510

-900

0.04

54

0.0

001

0.45

0

.04

0.99

993

12

.77

300-

5000

0.00

269

0.

0000

5 -0

.1

0.1

0.

9976

114

13.8

210

0-20

000.

0140

0

.000

2 0.

4

0.2

0.99

8940

17.3

550

-200

00.

0221

0

.000

2 0.

4

0.1

0.99

9615

17.9

510

0-20

000.

0036

1

0.00

004

0.03

0

.03

0.99

8629

Tabl

e 1

Com

poun

d Re

tent

ion

time

(min

) D

ynam

ic ra

nge*

(n

g m

L-1 )

Slop

e

SD

y-

inte

rcep

t S

D

r2 LO

D

(ng

mL-

1 )tra

ns-R

esve

ratro

l 22

.86

10-7

500

0.13

782

0.

0000

3 0.

2

0.08

0.

9999

2 Q

uerc

etin

23

.52

200-

3500

0.

097

0.

001

-5

2

0.99

9560

ci

s-Re

sver

atro

l 23

.67

100-

5000

0.

0595

0

.000

3 -1

.7

0.7

0.

9999

35

Kae

mpf

erol

24

.84

50-2

000

0.08

92

0.0

005

3.1

0.

5 0.

9999

18

(*th

e lo

wer

lim

it of

dyn

amic

rang

e fo

r eac

h ph

enol

ic is

its L

OQ

). Ita

lics:

fluor

imet

ric m

etho

d

Innovaciones en la determicin cromatogrfica de antioxidantes en alimentos

99

2.2. Statistical and Preliminary data exploration

Table 2

Table 1.

Table 2. Phenolic content in the analyzed samples

Table 2. Continuation.

102

2.3. Factor Analysis using Principal Components

Table 3


103

Table 3

Table 3. Features of Factor Analysis a

a) Extraction method using Principal Component Analysis with Kaiser Normalization, b) using Principal Component Analysis


105

Table 3

2.4. Discriminant analysis

106

Table 4.

Tabl

e 4.

(I) d

ata

from

Win

e Va

riet

y

Tabl

e 4.

(II)

dat

a fr

om G

eogr

aphi

cal O

rigi

n

Tabl

e 5.


111

Table 5

Figure 1

Figure 1.

112

3. Conclusions

Acknowledgement


113

References


115

Luminescent determination of flavonoids in orange juices by liquid chromatography with post-column

derivatization with aluminium and terbium A. Andreu-Navarro, J.M. Fernndez-Romero, A. Gmez-Hens(*)

Captulo 2

116

Introduction


117

Figure 1

Figure 1.

Captulo 2

118

1. Experimental

1.1. Instrumentation

1.2. Reagents


119

1.3. Manifold and Procedure

Figure 2

1.4. Analysis of orange juice samples

Captulo 2

120

Figure 2.


2.1. Study of the luminescence reaction


121

Figure 3

Figure 3.A

Figure 3.B

Figure 3.C

Captulo 2

122

Figure 3.

300 350 400 450 5000

500

1000

2000

3000

4000

3

4

1

Excitationwavelenght, nm ( em= 545 nm)

2

(A)

300 350 400 450 5000

500

1000

2000

3000

4000

3

4

1

2

400 500 550 600 6500

500

1000

2000

3000

4000

4

3

1

Emissionwavelenght, nm (

2

(C)

400 500 550 600 6500

500

1000

2000

3000

4000

4

3

1

Emissionwavelenght, nm ( ex = 450 nm)

2

(C)

0

500

1000

2000

3000

4000

4

3

1

ex= 360 nm)

2

(B)

400 450 500 550 600 6500

500

1000

2000

3000

4000

4

3

1

Emissionwavelenght, nm (

(B)

400 450 500 550 600 650

Lum

ines

cenc

e In

tens

ity, a

.u.


123

2.2. Optimization of Variables

Table 1

Table 1. Variables

Variables Range studied Optimum

value

Instrumental variables

Excitation wavelength, nm Emission wavelength, nm Ex/em slits PMT gain, V

200 600 400 600 2.5 15

400 - 800

360 545

10/10 500

Chromatographic variables

HPP flow-rate, mL/min Injection volume, L Elution mode Temperature, C [HAc], M pH

0.5 3.0 20 500 Gradient

15 35

0.05 0.25 2.5 6.0

0.7 200

-

25 C

0.15 4.0

Derivatizing variables

LPP flow-rate, mL/min L1 reactor length, cm [Al(III)], mM [Tb(III)], mM [HAc], M pH [SDS], mM

0.2 1.5 50 300

0.2 -20.0 0.1 10.0

0.05 0.25 1.5 6.0 0.01 0.5

0.5 100

15.0 5.0

0.15 4.0 0.1

Captulo 2

124

Chromatographic variables

Post-column derivatization variables


125

Figure 4

Figure 4.A

Figure 4.B

Figure 4.C

2.3. Analytical features

Table 2

Captulo 2

126

Figure 4.


127

2.4. Application of the method

Figure 5

Fig. 5.A Fig. 5.BFig. 5.C

Table 3

Tabl

e 2.

Ana

lyte

R

eten

tion

time

(min

) Eq

uatio

n (1

) r2

y/

x(2)

Li

near

rang

e (n

g/m

L)

Det

ectio

n lim

it (n

g/m

L)

RSD

%(3

) lo

w

leve

l hi

gh

leve

l N

arin

gin

10.2

0 y=

(5.0

0

0.02

) x +

5.63

3

.54

0.99

55

0,24

6.

0 -1

700

1.0

3.9

3.8

Hes

perid

in

11.2

0 y=

(0.5

6

0.01

) x +

0.2

0

0.57

0.

9936

0,

04

9.8

1

700

2.7

3.7

4.7

Que

rcet

in

16.8

0 y=

(59.

08

0.2

7) x

2

0.66

10

.5

0.99

57

0,40

2.

1

2000

1.

0 1.

3 4.

6 N

arin

geni

n 21

.20

y=(2

1.95

0

.14)

x +

0.3

1

11.4

0.

9958

0,

65

5.2

15

00

1.5

4.3

4.1

Kae

mpf

erol

22

.50

y= (2

5.34

0

.07)

x -

0.28

6

.32

0.99

83

0,93

2.

5

2000

0.

8 2.

6 3.

3 y

x

Tabl

e 3.


129

Figure 5.

130

3. Conclusions

Acknowledgements


131

References

Chu, Y.H.; Chang, C.L.; Hsu, H.F.; J. Sci. Food Agricul. 2000, 80, 561-566


133

Long-Wavelength Fluorescence detection of Flavonoids in Orange Juices using Liquid

Chromatography

A. Andreu-Navarro, J.M. Fernndez-Romero, A. Gmez-Hens

Captulo 2

134


135

Introduction

figure 1

Captulo 2

136

Figure 1.

1. Experimental

O

O

R 2 O

O H

R 1

R 3 R 4

O

O

R 2 O

O H

R 1

R 3 R 4

O

O

R 2 O

O H

R 1

R 3 R 4

O

O

R 2 O

O H

R 1

R 3 R 4

O

O

R 2 O

O H

R 1

R 3 R 4


137

HPLC.

Post-column reaction system.

Figure 2

Table 1

Captulo 2

138

Figure 2

Table 1.

Time (min) % Acetic buffer (0.15 mol L-1, pH 4.0)

% Acetonitrile % Methanol

FIGURE 2

LPP

D1w2

w1

C18 MC

A C

HPP

SD

HPIV

L2

PC

FLD

B

L1

D2


139


Figure 3

Captulo 2

140

Figure 3.

600 620 640 660 680 700

0

400

800

1200

1600

2000

Fluo

resc

ence

inte

nsity

Emission wavelength (nm) ( ex= 585 nm)

(3)

(1) (2)

(4)

(5)

(6)


141

Table 2

Table 2. Type of Variables

Variables Range studied Optimum value

Captulo 2

142

Figure 4

Figure 4a

Figure 4b


143

Figure 4c

Figure 5a

Table 2

Table 3

Captulo 2

144

Figure 4.

(a)

0

100

200

300

400

500

600

[Cresyl violet] (mol L-1)

PEA

K A

REA

S

(1) (2)

(3)

1 4 7 10 13 16 19

(b)

0

200

400

600

800

1000

0.05 0.1 0.15 0.2 [Ce(IV)] (mmol L-1)

(1) (2)

(3)

PEA

K A

REA

S

(c)

0

500

1000

1500

2000

2500

0,01 0.5 1 1.5 2 [CTAB] (mmol L-1)

PEA

K A

REA

S


145

Figure 5.

Time (min)

Fluo

resc

ence

inte

nsity

0

400

800

1200

1600

2000

0

400

800

1200

1600

2000

(1)

(3)

(b)

0

0

400

800

1200

1600

2000

0

400

800

1200

1600

2000

0

400

800

1200

1600

2000

0

400

800

1200

1600

2000

(2)

(4)

(5)

(a)

0 2 4 6 8 10 12

(3) (2)

(4)

(5) (6)

Captulo 2

146

Table 4,

Figure 5b

Tabl

e 3.

N

Anal

yte

Rete

ntio

n tim

e

SD (m

in)(1

) Eq

uatio

n (2

) R2

y/

x

(p-v

alue

) (3

)

Line

ar

rang

e (n

g m

L-1 )

Dete

ctio

n lim

it (n

g m

L-1 )

RSD%

(4)

14

8

Tabl

e 4.

Nat

ural

Juic

e 1

Nat

ural

Juic

e 1

(Nav

el)

Nat

ural

Juic

e 2

(Nav

el)

Nat

ural

Juic

e 3

(Cle

men

tina)

Com

mer

cial

Juic

e 1

Co

mm

erci

al Ju

ice

2

Anal

ytes

Co

nc.F

ound

( S

D) (1

)

Reco

very

(%)

Conc

. Fou

nd

( S

D) (1

)

Reco

very

(%)

Conc

. Fou

nd

( S

D) (1

)

Reco

very

(%)

Conc

. Fou

nd

( S

D) (1

)

Reco

very

(%)

Conc

. Fou

nd

( S

D) (1

)

Reco

very

(%)

(1) C

once

ntra

cion

in

g m

L-1 (

SD=

stand

ard

devi

atio

n). (

2) R

ecov

erie

s af

ter a

dditi

on o

f 20.

0 an

d 10

0.0

ng m

L-1 ,

first

and

seco

nd a

dditi

on,

resp

ectiv

ely.


149

3. Conclusions

Acknowledgements

Captulo 2

150

References

CAPITULO 3

INNOVACIONES EN LA DETERMINACIN NO CROMATOGRFICA DE ANTIOXIDANTES EN

ALIMENTOS

-

Anal. Chim. Acta

-

Anal. Chim. Acta

Bibliografa

Determination of Antioxidant Additives in Foodstuffs

by Direct Measurement of Gold Nanoparticle Formation using Resonance Light Scattering Detection

A. Andreu-Navarro, J.M. Fernndez-Romero, A. Gmez-Hens Department of Analytical Chemistry, Faculty of Sciences, University of Crdoba, Marie Curie Annex Building. Campus of Rabanales, E-14071 Crdoba, Spain

Introduction

(Figure 1)

Figure 1

1. Experimental

Figu

re 1

.

Figure 2

Figure 2.


Figure 3.A

Figure 3.B

Figure 3.C

Figure 3.A

Figu

re 3

.

Table 1

Table 1. Type

of variable Variable Range

studied Optimum

value

Instrumental

Stopped-flow device

Chemical

Figure 4.A

Figure 4.B)Figure 4.C

Figure 4.D

Figure 4.

Table 1

Table 2

Table 3

3. Conclusions

Acknowledgements

Tabl

e 2.

Anal

yte

Equa

tion

(1)

r2

y/x

(2)

Line

ar r

ange

()

Det

ecti

on L

imit

()

RS

D%

(3)

Tabl

e 3.

Sam

ple

N

Com

mer

cial

N

ame

Food

stuf

f Ty

pe

Dilu

tion

Fact

or

Anal

yte

Refe

renc

e M

etho

d Pr

opos

ed M

etho

d

References

Innovaciones en la determinacin no cromatogrfica de antioxidantes en alimentos

175

Determination of polyphenolic content in beverages using laccase, gold nanoparticles and long

wavelength fluorimetry


Captulo 3

176

Introduction


177

Figure 1

Figure 1

Captulo 3

178

Fig 1.


179

1. Experimental

Trametes versicolor

Captulo 3

180

Figure 2

Figure 2.


181

Figure 3.

Captulo 3

182


Figure 3

Figure 3


183

Figure 3

.

Captulo 3

184

0 200 400 600 800 10000,12

0,14

0,16

0,18

0,20

0,22

Fluo

resc

ence

inte

nsity

, a.u

.

Time, s

(1) (3)(2) (4)

FIGURE 3

Figure 3.

Table 1


185

Figure 4.A

Figure 4.B

Figure 4.C

Captulo 3

186

Table 1.

Table 1

Table 2


187

Figure 4.

0

50

100

150

200

250

0 10 20 30 40 50 60

Tim

e, s

[Au NPs], mol L-1

0

100

200

300

400

500

0 0,5 1 1,5 2

Tim

e, s

[Laccase], u.a. mL-1

0

100

200

300

400

500

600

0 5 10 15 20 25

Tim

e, s

[Indocyanine Green], mol L-1

Cap

tulo

3

Tabl

e 2

Ana

lytic

al fe

atur

es o

f the

met

hod

Anal

yte

Equa

tion

(1)

r2

y/x

(2)

Line

ar ra

nge

(m

ol L

-1)

Dete

ctio

n Li

mit

(m

ol L

-1)

RSD%

(3)

low

leve

lhi

gh le

vel

Cate

chol

y=

(246

2

) x +

(4

2)

0,99

80.

330.

08

5 0.

014.

45.

5Hy

droq

uino

ne

y=(5

83

3) x

(

4

2)

0.99

90.

500.

05

2 0.

013.

65.

6Hy

drox

yhyd

roqu

inon

e y=

(92.

8

0.7)

x +

(13.

3

0.8)

0.

999

0.11

0.09

5

0.03

3.5

1.8

Galli

c Ac

id

y=(2

27

2) x

+ (1

5

3)

0.99

90.

760.

13

5 0.

043.

95.

4Py

roga

llol

y=(1

63

1) x

+ (2

3

2 0,

999

0.54

0.17

5

0.04

3.5

4.6

Tabl

e 3

Sam

ple

Com

mer

cial

na

me

Food

stuf

f ty

pe

Ref

eren

ce M

etho

d Pr

opos

ed M

etho

d Co

nc. F

ound

(SD

)(1)

Conc

. Fou

nd (

SD)(1

) Re

cove

ry %

(2)

1s

t 2n

d

1 Su

nny

Del

ight

O

rang

e ju

ice

394

80

34

4

116

90.0

10

0.2

2 Sa

lobr

ea

Gra

pe ju

ice

166

2

159

17

89

.9

97.1

3

Alte

za T

ea

Gre

en te

a 31

2

24

8

91.5

99

.9

4 Tw

inin

gs T

ea

Cinn

amon

tea

58

2

48

6

90.4

97

.3

(1) P

olyp

heno

ls m

easu

red

as G

A, c

once

ntra

tion

in m

g L-

1 fo

r jui

ces

and

mg

g-1 f

or te

as (2

) Rec

over

ies

afte

r the

firs

t (0,

2 m

ol

L-1 )

and

seco

nd a

dditi

on (1

m

ol L

-1),

resp

ectiv

ely.


189

Table 3

3. Conclusions

Captulo 3

190

References

Singleton, V. L.; Rossi, J. A.; Am. J. Enol. Viticult. 1965, 16, 144-158.

.


191

CAPITULO 4NUEVAS INVESTIGACIONES CON

NANOPARTICULAS DE ORO

1. Introduccin

2. Objetivos

3. Conocimientos adquiridos

Figura 1

Fig 1.

Figura 2

Figura 2, lnea A

Figura 2, lnea B)

Figura 3

Figura 2

Figura 3.

Figura 3

Figura 4

Figura 5

Figura 4.

Figura 5

Figura 5.

Des

plaz

amie

nto

del m

nim

o SP

R

Figura 6

Figura 6

4. Trabajo experimental

1. Lnea de base

2. Asociacin 3. Disociacin 4. Regeneracin 1. Lnea de base

Paso:

Des

plaz

amie

nto

del m

nim

o S

PR

Figura 7

(Figura 7.1) (Figura 7.2)

Figura 7.

Central EM-LAB, University Hospital Regensburg (2010) 300 nm

Tamao = 20 nmAbs = 529 nm

Abs

orba

ncia

(nm)

Figura 8

Bibliografa

CAPITULO 5DISCUSIN DE LOS RESULTADOS

DISCUSSION OF THE RESULTS

Introduccin

1. Metodologas basadas en separacin cromatogrfica, derivatizacin postcolumna y deteccin fluorescente

Tabla 1.

Tabla 2

Tabla 2.

Tabla 3

Tabla 3.

Tabla 4

Tabla 4

Tabla 5

Tabla 5

Tabla 6

Tabla 6

2. Metodologas automticas no cromatogrficas basadas en el uso de la tcnica de flujo detenido y nanopartculas

Tabla 7

Tabla 7

Introduction

1. Methodologies based on chromatographic separation, post-column derivatization and fluorescence detection

Table 1

Table 1.

Table 2

Table 2.

Table 3

Table 3.

Table 4

Table 4.

Table 5

Table 5.

Table 6

Table 6.

2. Automatic non-chromatographic methodologies based in the used of stopped-flow technique and nanoparticles.

Table 7

Table 7.

Bibliografia References

912

CONCLUSIONESCONCLUSIONS

CONCLUSIONES

CONCLUSIONS

Analytical Innovations in the Detection of Phenolicsin Wines

PIETRO RUSSO, LVARO ANDREU-NAVARRO, MARA-PAZ AGUILAR-CABALLOS,JUAN-MANUEL FERNNDEZ-ROMERO, AND AGUSTINA GMEZ-HENS*

Department of Analytical Chemistry, Marie Curie Annex building, Campus of Rabanales, Universityof Crdoba, E-14071 Crdoba, Spain

A liquid chromatographic method with online photometric and luminescent detection for thedetermination of 18 phenolic compounds in wines is reported. Photometric detection is performed atfour wavelengths, namely, 256, 280, 320, and 365 nm, using a diode array detection system. Theluminescent detection is achieved by means of a postcolumn derivatization reaction of 10 of thesecompounds with terbium(III) in the presence of synergistic agents, such as ethylenediaminetetraaceticacid (EDTA) and n-octyltriphosphine oxide (TOPO). A micellar medium provided by the surfactantssodium dodecyl sulfate and Triton X-100 was used for the determination of the luminescent chelatesat ex 317 and em 545 nm. The long wavelength emission of lanthanide chelates can minimizeinterferences from background sample matrix, which usually emit at shorter wavelengths. The analyticalfeatures of the photometric and fluorometric methods, such as dynamic ranges of the calibrationgraphs, detection limits, and precision data, have been obtained. The practical usefulness of thedeveloped methods is demonstrated by the analysis of Spanish and Italian wine samples (red, ros,oloroso, and white), which were diluted and directly injected into the chromatographic system. Theaccuracy of both methods was checked by assaying a recovery study, which was performed at threedifferent analyte levels for each type of sample.

KEYWORDS: Phenolic compounds; postcolumn derivatization; terbium-sensitized luminescence; winesamples

INTRODUCTION

Phenolics are a wide group of compounds constituted byphenolic aldehydes, hydroxybenzoic and hydroxycinnamic acids,catechins, flavonols, and stilbenes, in their monomeric form orconjugated to some species, such as tartaric acid in the case ofcinnamic acids (1), among others. These compounds are presentin wines because they are secondary metabolites of plants. Thecomposition of phenolics and their concentration depend ongrape variety, geographical origin, soil type, collection system,and grape processing. These compounds are responsible of thesensory properties of the wines, and, also, they are anticarci-nogenic and have an anti-inflammatory action when they areregularly ingested. A particular example of the importance ofmonitoring phenolic concentration to control the quality of winesis the presence of aromatic aldehydes, formed by a group ofvolatile compounds that are extracted from wood lignin duringthe winemaking process. The presence of these aldehydes inwines is an indicator of fermentation and aging in oak barrels,their absence being indicative of counterfeit aged wines. Vanillinand syringaldehyde are the most abundant aromatic aldehydesin wines.

The occurrence of phenolics has been extensively studied byliquid chromatographic methods (118). In most of them,conventional reversed-phase columns, constituted by packedmicroparticulate bonded silica, have been used (15, 715, 18),which generally feature separations of 1425 compounds inalmost 1 h or 32 phenolics in 90 min, which makes routineanalysis of these compounds very tedious. The use of othermaterials such as mesoporous silica has given rise to monolithiccolumns, which operate at higher flow rates with lower back-pressures than conventional columns (16, 17). Thus, they allowthe analysis of samples using direct injection or low sampledilutions, because the cleaning and regeneration of the columncan be done more quickly than in the conventional ones due tothe high flow rates afforded. Monolithic columns have been usedfor the determination of 15 phenolics in red and white winesamples (16) with separation times around 30 min and, also,for the direct analysis of cider samples (19).

Diode array detection (1, 317) has been extensively usedfor the development of liquid chromatography methods, whereasfluorometric (8, 12) and mass spectrometry (2) detection systemshave been used to a lesser extent. Most fluorometric methodsproposed are based on measurements of the intrinsic fluores-cence of some phenolics. Although these methods featuregenerally lower detection limits than photometric methods, the

* Author to whom correspondence should be addressed (telephone+ 34-957218645; fax +34-957-218644; e-mail [email protected]).

1858 J. Agric. Food Chem. 2008, 56, 18581865

10.1021/jf073206t CCC: $40.75 2008 American Chemical SocietyPublished on Web 02/22/2008

Analytical Methods

Usefulness of terbium-sensitised luminescence detection for the chemometricclassification of wines by their content in phenolic compounds

A. Andreu-Navarro, P. Russo, M.P. Aguilar-Caballos, J.M. Fernndez-Romero, A. Gmez-Hens

Department of Analytical Chemistry, Marie Curie Annex Building, Campus of Rabanales, University of Crdoba, E-14071 Crdoba, Spain

a r t i c l e i n f o

Article history:Received 4 May 2009Received in revised form 2 August 2010Accepted 9 August 2010

Keywords:Phenolic compoundsLC-photometry and fluorimetryTerbium(III)Factor Analysis (FA)Linear discriminant analysis (LDA)Wine samples

a b s t r a c t

Amethod for wine classification based on the phenolic compound content, wine variety and geographicalarea is described. The method involves the use of the results obtained from the analysis of fifteen samplesof Italian and Spanish wines from different geographical origins [Sicilia (Italy) and Crdoba (Spain)] usingliquid chromatography (LC) with photometric and fluorimetric detection, in which eighteen phenolicswere determined: gallic acid, protocatechuic acid, p-hydroxybenzoic acid, salicylic acid, vanillic acid, caf-feic acid, syringic acid, catechin, vanillin, p-coumaric acid, syringaldehyde, epicatechin, ferulic acid, rutin,trans- and cis-resveratrol, quercetin and kaempferol. Photometric measurements were performed select-ing four wavelengths (256, 280, 320 and 365 nm), using a diode-array detection system. The fluorimetricdetection was achieved by measuring the sensitised luminescence provided by the chelates formedbetween each analyte and terbium (III). All samples were commercial wines bought in local marketsand analysed immediately after they were opened. The pattern data matrix was constructed by the con-centration of each analyte present in wine, which was determined by the most adequate method, namelyLC-photometric or LC-fluorimetric method. This data matrix was subjected to different algorithms inorder to classify and characterise the wine samples adequately. Supervised (LDA) and un-supervised(FA) pattern recognition methods were used. The wine pattern generation with LC separation and dualdetection approach to determine eighteen phenolic compounds and the chemometric treatment providean appropriate way with recognition and prediction rates. The values obtained for these rates were 100%when fluorimetric detection was used. These results can be considered satisfactory, which proves theusefulness of the selected variables.

! 2010 Elsevier Ltd. All rights reserved.

1. Introduction

In recent years, there has been an increasing interest in thedevelopment of chemometric applications in order to establishthe variety, geographical origin, manipulation and other techno-logical features, which define the taste of foods and beverages fromagricultural origin (Ariyama & Yasui, 2006; Marcos, Fischer, Rea, &Hill, 1998; Sanz, Prez, Herrera, Sanz, & Juan, 1995; Smith, 2005).The reasons for this interest are: (1) laws enforce labelling of thegeographical origin of the foodstuff and beverages in many coun-tries due to demands of more information from consumers andimprovement of their domestic production; (2) producers have be-gun to advertise their brands of high-quality products by includinggeographical origin and other features in the label for economicreasons; (3) prevention of frauds and adulteration on the label,production and commercialisation. Thus, the development of

methods giving acceptable information is highly desirable for con-sumers, producers and administrative authorities.

Wine industry and the market sector are particular examples inwhich the development of sophisticated chemometric techniquesare essential for the improvement of the analytical informationabout wine composition and for assessing wine authenticity(Arvanitoyannis, Katsota, Psarra, Soufleros, & Kallithraka, 1999;Horwitz, 1995). Several chemometric procedures have been usedas the basis for discrimination of wines according to vinificationtechnology and classification according to region, type and variety.Different pattern recognition techniques (PRT), such as PrincipalComponent Analysis (PCA) (Boselli, Giomo, Minardi, & Frega,2008; Recamales, Sayago, Gonzlez-Miret, & Hernanz, 2006), Linearand Canonical Discriminant Analysis (LDA and CDA) (Hernndez,Estrella, Dueas, Fernandez de Simn, & Cadaha, 2007; Makris,Kallithraka, & Mamalos, 2006; Villiers et al., 2005), ProbabilisticNeural Network (PNN) (Daz, Conde, Estvez, Prez-Olivero, &Prez Trujillo, 2003), K-Nearest Neighbours (KNN) (Beltrn et al.,2006), Cluster Analysis (CA) (Villiers et al., 2005), MultiregressionAnalysis (MRA), Partial Least Squares (PLS) (Capron, Smeyers-Verbeke, & Massart, 2007; Le Moigne et al., 2008), and CAIMAN

0308-8146/$ - see front matter ! 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodchem.2010.08.014

Corresponding author. Tel.: +34 957218645; fax: +34 957218644.E-mail address: [email protected] (A. Gmez-Hens).

Food Chemistry 124 (2011) 17531759

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Food Chemistry

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Research Article

Luminescent determination of flavonoids inorange juices by LC with post-columnderivatization with aluminum and terbium

A new post-column derivatization system is described and applied to the determination offlavonoids in citric beverages after their separation by LC using a monolithic column. Thederivatization involves the formation of the chelates of the analytes with aluminum (III)and terbium (III) in the presence of the surfactant SDS and the measurement of theterbium sensitized luminescence at lex 360 and lem 545 nm. Naringin, hesperidin,quercetin, naringenin, and kaempferol have been chosen as analyte models. The largeStokes shift and the relatively long wavelength emission of terbium(III) can minimizeinterferences from background sample matrix, which usually emit at shorter wavelengths.Calibration graphs were constructed in the intervals 6.01700 ng/mL naringin,9.81700 ng/mL hesperidin, 2.12000 ng/mL quercetin, 5.21500 ng/mL naringenin and2.52000 ng/mL kaempferol, with regression coefficients higher than 0.9935 in allinstances. The precision of the method, expressed as RSD%, was established at twoconcentration levels, with values of 1.3 and 4.7%, which corresponded to the minimal andmaximal error zones of the calibration graphs. The practical usefulness of the method isdemonstrated by the analysis of orange juices, which were diluted and directly injectedinto the chromatographic system, obtaining recoveries between 86.9 and 108.2%.

Keywords: Aluminum and terbium complexes / Flavonoid compounds / Orangejuices / Post-column derivatizationDOI 10.1002/jssc.200900696

1 Introduction

Flavonoids are a group of polyphenolic compounds widelydistributed in vegetables and fruits [1]. The determination ofthese compounds is of great interest owing to their multiplebiological effects, including antioxidant activity, antitumor,antimutagenic, antibacterial and angioprotective properties[2, 3]. They also contribute to different plant properties suchas colour, flavour, fragrance, nutrition, stability andtherapeutic properties. Flavonoids such as 2-phenyl-benzo-a-pyrones are classified according to the multitude ofsubstitution patterns in the two benzene rings of theirbasic structure. Variation in their heterocyclic rings givesrise to flavonols, flavones, catechins, flavanones, anthocya-nidins and isoflavones [4]. The flavonoid content in orangejuices has special interest as it contributes to the quality ofthese samples [5].

The main separation technique applied to the determi-nation of flavonoids in orange juices is LC [611], usingreversed-phase columns, although gas chromatography [12],

involving the formation of trimethylsilyl derivatives, hasbeen also described for this purpose. The usefulness of amonolithic column, which can operate at higher flow rateswith lower backpressures than conventional columns, hasbeen previously described for the LC separation of someflavonoids in wine samples [13]. However, to the best of ourknowledge, this type of column has not been used up to datefor the determination of these compounds in orange juices.

Photometric [610], coulometric [10] and MS [6, 11]detection systems have been reported in the LC methodsdescribed for the analysis of these samples. Some flavo-noids, such as quercetin and kaempferol, have been fluor-imetrically determined in body fluids using aluminum(III)as post-column derivatization reagent to obtain fluorescentchelates [14]. Another post-column derivatization systempreviously described for the determination of thesecompounds in wine samples involves the formation of thecorresponding terbium(III) chelates and the measurementof the sensitized luminescence [13]. This phenomenon is anintermolecular energy transfer process, in which the ligandacts as donor and terbium(III) acts as acceptor and emitsluminescence, that allows very sensitive and selectivedeterminations, although its use as detection system in LChas been relatively limited [15].

This article reports for the first time the use of amonolithic column for the direct separation of flavonoids inorange juice samples and a new post-column derivatization

Alvaro Andreu-NavarroJuan Manuel Fernandez-RomeroAgustina Gomez-Hens

Department of AnalyticalChemistry, Faculty of Sciences,University of Cordoba, Cordoba,Spain

Received October 23, 2009Revised December 18, 2009Accepted December 21, 2009

Correspondence: Professor A. Gomez-Hens, Departmentof Analytical Chemistry, Faculty of Sciences, University ofCordoba, Marie Curie Annex Building, Campus of Rabanales,E-14071 Cordoba, SpainE-mail: [email protected]:134-957-218644

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2010, 33, 509515 509

Long-Wavelength Fluorescence Detectionof Flavonoids in Orange Juices by LC

Alvaro Andreu-Navarro, Juan Manuel Fernandez-Romero, Agustina Gomez-Hens&

Department of Analytical Chemistry, Faculty of Sciences, University of Cordoba, Marie Curie Annex Building,Campus of Rabanales, 14071 Cordoba, Spain; E-Mail: [email protected]

Received: 16 April 2010 / Revised: 7 September 2010/ Accepted: 7 September 2010

Abstract

A new post-column liquid chromatographic reaction system for the determination of flavo-noids in orange juices is based on the use of the long wavelength fluorophor cresyl violet andcerium(IV) in a cetyl trimethylammonium bromide micellar medium. Two flavone aglycones(quercetin and kaempferol), a flavonone aglycone (naringenin), one flavone-O-glycoside(rutin) and two flavanone-O-glycosides (hesperidin and naringin) have been used as analytemodels. The reaction process involves the interaction between the analyte, cerium(IV) andcresyl violet giving rise to a decrease in the fluorescence, measured at kex 585, kem 625 nm,which is proportional to the analyte concentration. Dynamic ranges of the calibration graphsand detection limits, obtained with standard solutions of the analytes are (ng mL-1): quer-cetin (12.24,000, 3.7), kaempferol (3.51,000, 1.0), naringenin (6.71,000, 2.0), rutin(5.0800, 1.5), hesperidin (10.11,000, 3.0), and naringin (17.8800, 1.8). The deter-mination coefficients were higher than 0.993 in all instances. The precision of the method,expressed as RSD%, was established at two concentration levels, with values rangingbetween 2.8 and 6.2%. The practical usefulness of the developed method is demonstrated bythe analysis of natural and commercial orange juices, which were filtered, diluted anddirectly injected into the chromatographic system, with apparent recoveries between 86.9and 107.0%.

Keywords

Column liquid chromatographyLong wavelength fluorescence detectionFlavonoid compoundsOrange juice samples

Introduction

The use of long-wavelength fluorophores(LWF) as analytical reagents is anattractive option to improve the selectiv-ity of fluorimetric analysis and avoid orminimize the sample treatment step of theanalytical process. These dyes allowingmeasurements are obtained in a region ofthe electromagnetic spectrum (>600 nm)in which the potential absorption oremission associated to sample matrix isminimized. Also, Raman interference isgreatly diminished and the probability ofnon-radiative quenching processes isdecreased due to the usually short fluo-rescence lifetime of these fluorophores.LWFs have been described as enzymaticsubstrates, immunoassay labels, sensingsystems and derivatizing reagents inliquid chromatography (LC) and capil-lary electrophoresis [1]. Although the lackof sufficient reactive groups for targetingof analytes is a limitation of these fluo-rophores, their analytical usefulness hasbeen expanded using electrostatic andredox interactions [26].

This article describes for the first timethe use of the oxazine LWF cresyl violet(CV) in a post-column LC reactionsystem for the indirect determination of

DOI: 10.1365/s10337-010-1774-8! 2010 Vieweg+Teubner Verlag | Springer Fachmedien Wiesbaden GmbH

Original

Author's personal copy

Analytica Chimica Acta 695 (2011) 1117

Contents lists available at ScienceDirect

Analytica Chimica Acta

journa l homepage: www.e lsev ier .com/ locate /aca

Determination of antioxidant additives in foodstuffs by direct measurement ofgold nanoparticle formation using resonance light scattering detection


Department of Analytical Chemistry, Faculty of Sciences, University of Crdoba, Marie Curie Annex Building, Campus of Rabanales, E-14071 Crdoba, Spain

a r t i c l e i n f o

Article history:Received 21 January 2011Received in revised form 14 March 2011Accepted 24 March 2011Available online 1 April 2011

Keywords:Antioxidant additivesGold nanoparticlesStopped-flow mixing techniqueResonance light scattering detectionFoodstuff samples

a b s t r a c t

The capability of antioxidant compounds to reduce gold(III) to gold nanoparticles has been kineticallystudied in the presence of cetyltrimethylammonium bromide using stopped-flow mixing technique andresonance light scattering as detection system. This study has given rise to a simple and rapid method forthe determination of several synthetic and natural antioxidants used as additives in foodstuff samples.The formation of AuNPs was monitored by measuring the initial reaction-rate of the system in about5 s, using an integration time of 0.1 s. Dynamic ranges of the calibration graphs and detection limits,obtained with standard solutions of the analytes, were (!mol L1): gallic acid (0.040.59, 0.01), propylgallate (0.041.41, 0.01), octyl gallate (0.030.35, 0.08), dodecyl gallate (0.020.30, 0.007), butylatedhydroxyanisol (0.070.39, 0.009), butylated hydroxytoluene (0.040.32, 0.01), ascorbic acid (0.111.72,0.03) and sodium citrate (0.071.29, 0.02). The regression coefficients were higher than 0.994 in allinstances. The precision of the method, expressed as RSD%, was established at two concentration levelsof each analyte, with values ranging between 0.6 and 4.8%. The practical usefulness of the developedmethod was demonstrated by the determination of several antioxidant additives in foodstuff samples,which were extracted, appropriately diluted and assayed, obtaining recoveries between 95.4 and 99.5%.The results obtained were validated using two reference methods.

2011 Elsevier B.V. All rights reserved.

1. Introduction

Natural and synthetic antioxidants are a group of compoundsused as additives to prevent or retard oxidation reactions in foodproducts. There is a trend to limit the use of synthetic antioxidants,owing to their potential toxic effects, but they are still found in a rel-atively wide range of foodstuffs, although their use is subject to verystrict safety regulations [16]. Phenolic compounds such as propyl(PG), octyl (OG) and dodecyl (DG) esters of gallic acid (GA), buty-lated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)are the main synthetic antioxidants still authorized, although theyare being replaced by natural antioxidants, such as ascorbic (AA)and citric (CA) acids. CA is also used as a food additive for otherpurposes, such as acidifier and flavouring agent.

The determination of synthetic antioxidants in food samplesis mainly carried out by using reverse-phase liquid chromatogra-phy (LC) with photometry or mass spectrometry (MS) as detectionsystems [7,8]. Methods based on gas chromatography (GC), with

Corresponding author. Tel.: +34 957 218645; fax: +34 957 218644.E-mail address: [email protected] (A. Gmez-Hens).

flame ionization detector (FID) or MS, and capillary electrophore-sis (CE) with photometric or electrochemical detection have beenalso described [7]. The limits of detection (LODs) reported for mostof these methods are at the level of !mol L1. These methods arevery useful for the identification of the analytes, but they are time-consuming for screening purposes.

The use of gold nanoparticles (AuNPs) as analytical reagentshas allowed the development of very sensitive methods based ontheir special optical and electrochemical properties. For instance,they have been used as labels in immunoassays and hybridiza-tion assays and as nanoscaffolds to develop chemical sensors [9].These methods require the previous synthesis of the NPs, usinggenerally tetrachloroauric acid and a reducing reagent, usually cit-rate [10,11]. Other reagents such as cysteine [12], ascorbic acid[10,13,14], sodium borohydride [15,16] and gallic acid [17,18] havealso shown their usefulness for this purpose. The experimental con-ditions chosen to obtain AuNPs are critical factors that affect thesize, shape and potential aggregation of the NPs and, consequently,their properties. Some of these methods [14,15] involve the use ofcetyltrimethylammonium bromide (CTAB) to favour the synthesisof the NPs. The formation of ion pairs between AuCl4 and cationicsurfactants prior to the formation of AuNPs was described [19,20].

0003-2670/$ see front matter 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2011.03.047

1

Determination of polyphenolic content in beverages using laccase, gold nanoparticles and long wavelength fluorimetry

A. Andreu-Navarro, J.M. Fernndez-Romero, A. Gmez-Hens (*)

Department of Analytical Chemistry, Faculty of Sciences, University of Crdoba, Marie

Curie Annex Building. Campus of Rabanales, E-14071 Crdoba, Spain ABSTRACT An enzymatic fluorimetric method for the determination of polyphenol compounds in beverages is described, which is based on the temporal inhibition caused by these compounds on the oxidation of the long wavelength fluorophor indocyanine green ( ex 764 nm, em 806 nm), in the presence of the enzyme laccase and positive charged gold nanoparticles (AuNPs). The oxidation of the dye gives rise to a fast decrease in its fluorescence, but it is delayed by the polyphenol, obtaining a time period directly proportional to its concentration, which has been used as the analytical parameter. The behaviour of several benzenediols and benzenetriols in the system and the modification of the activity of the enzyme by its interaction with AuNPs have been studied. The system has been optimized using gallic acid as a polyphenol model, but the dynamic ranges of the calibration graphs and the detection limits for several of the polyphenols assayed were obtained ( mol L-1): gallic acid (0.13-5, 0.04), catechol (0.08-5, 0.01), hydroquinone (0.05-2, 0.01), hydroxyhydroquinone (0.09-5, 0.03), pyrogallol (0.17-5, 0.04). Most of the values of the regression coefficients were 0.999 and the precision of the method, expressed as RSD% and checked at two concentration levels of each analyte, ranged between 1.8 and 5.6%. The method has been applied to the determination of polyphenol content in several foodstuff samples and the results compared with those obtained with the standard Folin-Ciocalteu method. KEYWORDS: Polyphenols; laccase; gold nanoparticles; stopped-flow mixing technique; long wavelength fluorimetric detection; beverage samples

* Author to whom correspondence should be addressed (telephone +34 957 218645; fax

+34 957 218644. E-mail address: [email protected] )

Crdoba, 14 de enero de 2010

II ENCUENTRO SOBRE

NANOCIENCIA Y NANOTECNOLOGA DE INVESTIGADORES Y TECNLOGOS

DE LA UNIVERSIDAD DE CRDOBA

Universidad de Crdoba

Instituto Andaluz de Qumica Fina y Nanoqumica

Consejera de Innovacin, Ciencia y Empresa

REUNIN DEL GRUPO REGIONAL ANDALUZ DE LA SOCIEDAD ESPAOLA DE QUMICA ANALTICA

Departmento de Qumica Analtica, Facultad de Ciencias. Universidad de Crdoba. Edificio Anexo Marie Curie

- 53 -

P-1

DETERMINATION OF ANTIOXIDANT ADDITIVES IN FOODSTUFFS BY DIRECT MEASUREMENT OF GOLD NANOPARTICLE FORMATION USING

RESONANCE LIGHT SCATTERING DETECTION

A. Andreu-Navarro, J.M. Fernndez-Romero, A. Gmez-Hens Department of Analytical Chemistry

University of Crdoba Campus of Rabanales

Annex to Marie Curie building 14071-Crdoba. Spain

E-mail: [email protected] Web: http://www.uco.es/FQM-303/

The capability of antioxidant compounds to reduce gold(III) to gold nanoparticles (AuNPs) has

been kinetically studied in the presence of cetyltrimethylammonium bromide using stopped-flow mixing technique and resonance light scattering as detection system. This study has given rise to a simple and rapid method for the determination of several synthetic and natural antioxidant used as additives in foodstuff samples. The formation of AuNPs was monitored by measuring the initial reaction-rate of the system in about 5 s, using an integration time of 0.1 s. Dynamic ranges of the calibration graphs and detection limits, obtained with standard solutions of the analytes, were ( mol L-1): gallic acid (0.04 0.59, 0.01), propyl gallate (0.04 1.41, 0.01), octyl gallate (0.03 0.35, 0.08), dodecyl gallate (0.02 0.30, 0.007), butylated hydroxyanisol (0.07 0.39, 0.009), butylated hydroxytoluene (0.04 0.32, 0.01), ascorbic acid (0.11 1.72, 0.03) and sodium citrate (0.07 1.29, 0.02). The regression coefficients were higher than 0.994 in all instances. The precision of the method, expressed as RSD%, was established at two concentration levels of each analyte, with values ranging between 0.6 and 4.8 %. The practical usefulness of the developed method was demonstrated by the determination of several antioxidant additives in foodstuff samples, which were extracted, appropriately diluted and assayed, obtaining recoveries between 95.4 and 99.5 %. The results obtained were validated using two reference methods.

FLUORIMETRIC DETERMINATION OF POLYPHENOLS USING A LONG- WAVELENGTH-FLUOROPHOR AND LACCASE IMMOBILISED ON GOLD NANOPARTICLES

lvaro Andreu-Navarro, Juan Manuel Fernndez-Romero, Agustina Gmez- Hens

Department of Analytical Chemistry. Institute of Fine Chemistry and Nanochemistry (IUQFN- UCO) Campus de Rabanales. Marie Curie Building (Annex) University of Crdoba E-14071- Crdoba, Spain. Phone: 34- 957218645, Fax: 34-957218644, email: [email protected] web: http://www.uco.es/investiga/grupos/FQM-303

A new method for the determination of polyphenols based on their competitive redox interaction with a long-wavelength-fluorophor (LWF) in the presence of the enzyme lacasse immobilised on gold nanoparticles (AuNPs) is presented. The biocatalyst causes a fluorescence inhibition of the LWF, but their effect is delayed in the presence of polyphenols. The competitive redox reaction has been kinetically studied using stopped-flow mixing technique and fluorimetry as detection system. The behaviour of several polyphenols (phenol, gallic acid, catechol, hydroquinone, resorcinol, pyrogallol and phloroglucinol) on the system has been comparatively studied and a simple and rapid method for the determination of these compounds has been developed. Initial-rate and induction time measurements have been used as analytical parameter using Cardio Green ( ex= 764 nm, em= 806 nm) as LWF. Dynamic ranges of the calibration graphs and detection.

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