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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
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
AIM
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
Anal. Chim. Acta 563
Food Control 16
J. Chromatogr., A 1040
J. Chromatogr., A 1052
Food Chem.
Food Chem. 64
Eur. Food Res. Technol. 224
J. Agric. Food Chem. 51
J. Chromatogr., A 912
Anal. Chim. Acta 513
J. Chromatogr., A 1049
Food Chem. 89
J. Chromatogr., A 973
J. Chromatogr., A 1085
J. Sep. Sci. 29
J. Chromatogr., A 1066
Anal. Chem. 55
Trends Anal. Chem. 21
J. Agric. Food Chem. 54
Anal. Chim. Acta 578
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 (*)
92
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
93
Introduction
94
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
95
1. Experimental
1.1. Wine samples
1.2. LC-separation and photometric/fluorimetric detection
96
2. Results and discussion
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
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
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
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
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.
Tabl
e 5.
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
111
Table 5
Figure 1
Figure 1.
112
3. Conclusions
Acknowledgement
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
113
References
114
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
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
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
117
Figure 1
Figure 1.
Captulo 2
118
1. Experimental
1.1. Instrumentation
1.2. Reagents
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
119
1.3. Manifold and Procedure
Figure 2
1.4. Analysis of orange juice samples
Captulo 2
120
Figure 2.
2. Results and discussion
2.1. Study of the luminescence reaction
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
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.
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
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
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
125
Figure 4
Figure 4.A
Figure 4.B
Figure 4.C
2.3. Analytical features
Table 2
Captulo 2
126
Figure 4.
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
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.
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
129
Figure 5.
130
3. Conclusions
Acknowledgements
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
131
References
Chu, Y.H.; Chang, C.L.; Hsu, H.F.; J. Sci. Food Agricul. 2000, 80, 561-566
132
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
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
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
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
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
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
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
139
2. Results and discussion
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)
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
141
Table 2
Table 2. Type of Variables
Variables Range studied Optimum value
Captulo 2
142
Figure 4
Figure 4a
Figure 4b
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
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
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
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.
Innovaciones en la determinacin cromatogrfica de antioxidantes en alimentos
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.
v0
2. Results and discussion
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
A. Andreu-Navarro, J.M. Fernndez-Romero, A. Gmez-Hens
Captulo 3
176
Introduction
Innovaciones en la determinacin no cromatogrfica de antioxidantes en alimentos
177
Figure 1
Figure 1
Captulo 3
178
Fig 1.
Innovaciones en la determinacin no cromatogrfica de antioxidantes en alimentos
179
1. Experimental
Trametes versicolor
Captulo 3
180
Figure 2
Figure 2.
Innovaciones en la determinacin no cromatogrfica de antioxidantes en alimentos
181
Figure 3.
Captulo 3
182
2. Results and discussion
Figure 3
Figure 3
Innovaciones en la determinacin no cromatogrfica de antioxidantes en alimentos
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
Innovaciones en la determinacin no cromatogrfica de antioxidantes en alimentos
185
Figure 4.A
Figure 4.B
Figure 4.C
Captulo 3
186
Table 1.
Table 1
Table 2
Innovaciones en la determinacin no cromatogrfica de antioxidantes en alimentos
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.
Innovaciones en la determinacin no cromatogrfica de antioxidantes en alimentos
189
Table 3
3. Conclusions
Captulo 3
190
References
Singleton, V. L.; Rossi, J. A.; Am. J. Enol. Viticult. 1965, 16, 144-158.
.
Innovaciones en la determinacin no cromatogrfica de antioxidantes en alimentos
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
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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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
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Determination of antioxidant additives in foodstuffs by direct measurement ofgold 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
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
P-16
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
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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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