2275

Review

Direct Electrochemical Sensing and Detection of Natural

Antioxidants and Antioxidant Capacity in Vitro Systems
Antonio Javier Blasco, Agustn González Crevillén, Mara Cristina González, Alberto Escarpa*
Departamento de Qu mica Anal tica e Ingenier a Qu mica, Facultad de Qu mica, Universidad de Alcalá, 28871 Alcalá de Henares,
Madrid, Spain
*e-mail: alberto.escarpa@uah.es

Received: June 8, 2007

Accepted: August 9, 2007

Abstract
This review highlights the role of electrochemical approaches in the sensing of antioxidants and their antioxidant
capacity with especial attention to the analytical possibilities of electrochemistry in the direct evaluation of
antioxidant capacity exhibited by food and biological samples due to the termed dietary, natural or biological
antioxidants (mainly polyphenols, and vitamins C and E). The analytical potency of the electrochemistry is
comprehensively stated and the selected results found in the literature are summarized and discussed critically. The
main electrochemical approaches used have been cyclic voltammetry (CV) and flow injection analysis with
amperometric detection (FIA-ED). In addition, miniaturization is going to break new frontiers in the evaluation of
antioxidant activity.

Keywords: Electrochemical sensing, Natural antioxidants, Antioxidant activity, Food and Biological samples

DOI: 10.1002/elan.200704004

1. Introduction from being formed in the first place. Another interesting

Clinical and epidemiological studies have established an
inverse correlation between the intake of fruits and
vegetables and the occurrence of diseases such as inflam-
mation, cardiovascular disease, cancer and other related
disorders [1]. Dietary or natural antioxidants, including
polyphenolic compounds, vitamins E and C are believed to
be effective compounds in the prevention of these oxidative
stress related diseases [2]. Antioxidants have thus become a
topic of increasing interest, recently involving interdiscipli-
nary faces with a clear increase in number of publications
[3].
The word antioxidant is increasingly popular in modern
society as it gains publicity through the mass media coverage
of its health benefits. But what is an antioxidant? Depending
on the scientific discipline, the scope and protection targets
are significantly different. The term antioxidant has a
multifaceted nature; however, from a general point of
view, a suitable definition is synthetic or natural substances
added to products to prevent or delay their deterioration by
action of oxygen in air [4]. In food environments, antiox-
idants have a broad scope in that they include components
that prevent fats from becoming rancid as well as dietary
antioxidants as substance in foods that significantly decrease
the adverse effects of reactive species, such as reactive
oxygen (ROS) and nitrogen species (RNS), on normal
physiological function in humans [5]. Therefore, a dietary
antioxidant can sacrificially scavenge ROS/RNS to stop
radical chain reactions, or it can inhibit the reactive oxidants
definition is given by Halliwell [6] who defined biological
antioxidants as molecules which, when present in small
concentrations compared to the biomolecules they are
supposed to protect, can prevent or reduce the extent of
oxidative destruction of biomolecules.
In consequence, it is important to underline that when we
are talking about antioxidants we are referring to dietary
and/or natural and/or biological antioxidants which con-
stitute part of our intake. Therefore, food and biological
samples become target samples involved in the scope of
natural antioxidant analysis.
But the definition of antioxidant does not conceptually
involve the mechanism about how dietary antioxidants are
working in vitro systems covering radical chain reaction
inhibitors, metal chelators, oxidative enzyme inhibitors, and
antioxidant enzyme cofactors. In addition, many terms are
used by different researchers to describe antioxidant
capacity such as efficiency, power or activity. Antioxidant
activity measured by an individual assay reflects only the
chemical reactivity under specific conditions applied in the
assay while antioxidant capacity, efficiency or power are
terms more independent of specific reactions and have
similar chemical meanings. In consequence, in this review
we are going to use antioxidant capacity with independence
of the term used by the authors in their original contribu-
tions.
On the other hand, on the basis of the chemical reactions
involved, major antioxidant capacity assays can be roughly
divided into two categories: (i) hydrogen atom transfer

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2276 A. J. Blasco et al.

(HAT) reaction based assays and (ii) single electron transfer

(ET) reaction based assays. HAT-based assays monitor
competitive reaction kinetics (i.e., oxygen radical absorb-
ance capacity assay (ORAC), the total radical-trapping
antioxidant parameter assay (TRAP) and inhibited oxygen
uptake assay (IOU). The ET-based assays involve one redox
reaction with the oxidant (i.e., Trolox equivalent antioxidant
capacity assay (TEAC), ferric ion reducing antioxidant
parameter assay (FRAP), DPPH-based assay, copper (II)
reduction capacity, and total phenolics assay by Folin-
Ciocalteu (FC)).
Although, the critical discussion of the advantages/
disadvantages of all antioxidant capacity assays are outside
the scope of this review, in order to write a comprehensive
review dealing with this matter, it is also important to point
out two aspects: (i) the biggest problem is the lack of a
validated assay in vitro that can give a reliable measure of
the antioxidant capacity in food and biological samples and
(ii) the misunderstood Folin – Ciocalteu assay. From the first
one, a convenient method for estimation of antioxidant
content and evaluation of their antioxidant capacity is
desirable. With respect to the last, the Folin – Ciocalteu
method (FC assay) has gained popularity to determine total
phenolics and it is commonly known as the total phenols
(or phenolic) assay. However, FC assay measures the
sampleOs reducing capacity, but this is not reflected in the
name total phenolic assay. In essence, it is believed that the
Mo (VI) (yellow color in the complex Na2MoO4 · 2H2O) is
easier to reduce to Mo (V) (blue complex PMoW11O4)4
.
The redox reaction occurs between Mo (VI) and the
reductants (antioxidants). In addition, we can not forget Fig. 1. Chemical structures of the main antioxidants.
one weak point of the FC assay: although, this assay is
generally accepted as a method to determine total phenolics
ii) Electrochemical approaches evaluate the overall
and a huge volume of data based on this assay are avail-
reducing power of antioxidant compounds present
able, it overestimates the phenolic content in high extent
in food and biological samples, without the use of
[7].
reactive species, thus can be considered as a direct
Further reading about the chemistry behind antioxidant
test of the total antioxidant capacity since it is based
capacity assays, can be found in the excellent review of
only on the chemical-physical properties of the
Huang et al. [8].
antioxidant compounds simplifying notably the over-
all process.
iii) Electrochemistry is the conceptual base of several
2. Why Use Electrochemical Approaches in the
antioxidant capacity assays. Traditionally, the trans-
Sensing of Natural Antioxidant and Antioxidant
ference of charge involved in ET-based capacity
Activity?
assays has been based on redox reactions performed
in natural extracts. However, interestingly their
In the context of the main aspects of natural antioxidants
activity could be evaluated towards their electro-
described in the introduction section, and from a philo-
chemical reactions in suitable electrode materials as
sophical point of view, we are going to describe a series of
shown in this scheme.
keys for the understanding of the relevant role of electro-
chemical techniques in the evaluation of antioxidant
Redox: Oxidant þ e
(from antioxidant) >
capacity.
Reduced oxidant þ Oxidized antioxidant
i) All termed natural, dietary, and biological antiox-
idants (mainly including polyphenolic compounds, Electrochemical: Antioxidant
e
(electrode) !
and E and C vitamins; see Fig. 1) are believed to be Oxidant
effective dietary compounds which act as antiox-
iv) In the particular case of controlled-potential tech-
idants and all of them exhibit a moderate-marked
niques, on the one hand, oxidation potential is
native electroactivity.
conceptually correlated with the antioxidant capaci-

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Natural Antioxidants in Vitro Systems
ty. Indeed, low oxidation potentials found in food
and biological samples inform about their high
antioxidant capacity. On the other hand, the am-
perometric current and/or charge measured under
fixed oxidation conditions should give us an idea
about the extension of their capacity as well as the
estimation of their total content. Also, oxidation
potential allows a control of the selectivity to find the
most suitable conditions for measuring antioxidants
and their antioxidant capacity. This versatility is a
unique feature of electrochemistry versus those
exhibited by spectrophotometric ones in these envi-
ronments.
v) Due to the complexity of the composition of food and
biological samples, and taking also into account the
possible synergistic interactions among the antiox-
idant compounds in the samples, separating each
antioxidant compound and studying it individually is
costly and inefficient. This fact, dramatically opens
up the analytical possibilities of electrochemical
techniques which could be used in direct measure-
ment taking into account their inherent advantages
of selectivity and sensitivity.
In the particular case of polyphenols, a lot of
unknown structures could occur in the final extract.
In consequence, since current is an additive magni-
tude; electrochemistry becomes a very useful tool
because this signal should contain the contribution of
all complex structures involved in the extract.
vi) Responses which are not-dependent on the optical
path length or sample turbidity and their few
instrumental requirements, are additional, attractive
advantages of the electrochemical approaches.
In consequence, from the statements critically discussed
before, and in a general sense, controlled-potential tech-
niques, are inherently capable of simultaneously estimating
the total natural antioxidant content and of evaluating the
resulting total antioxidant capacity as the native techniques
group suited for evaluating antioxidant capacity in vitro
systems.
3. State of the Art in Electrochemical Sensing of Natural
Antioxidants and Antioxidant Capacity
This review highlights the role of electrochemical ap-
proaches in the sensing of antioxidants and their antioxidant
capacity with special attention to the analytical possibilities
of electrochemistry in the direct evaluation of antioxidant
capacity exhibited by food and biological samples due to the
termed dietary, natural or biological antioxidants (poly-
phenols, vitamins C and E, and others).
Because total and direct antioxidant determination versus
individual ones is highly desired, the main electrochemical
approaches used have been the controlled-potential tech-
niques: cyclic voltammetry (CV), differential-pulse voltam-
metry (DPV), other voltammetry approaches as well as flow
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2277
injection analysis with amperometric detection (FIA-ED).
Modified electrodes and electrochemical detection coupled
to separation techniques such as HPLC and CE will not have
been covered except when those approaches were explicitly
used in the evaluation of antioxidant capacity. HPLC-ED
has been widely used when accurate individual determina-
tion has been required but not with evaluation purposes of
antioxidant capacity since, as we have already mentioned,
this is due to the synergic effect from the whole antioxidant-
matrix system.
The analytical information obtained from relevant liter-
ature will be critically discussed in the following sections.
3.1. Electrochemical Sensing of Natural Antioxidants and
Antioxidant Capacity Using Batch Approaches
Kilmartin [9] has proposed CV at a GCE in an acid medium
to rapidly estimate the antioxidant capacity due to flavo-
noids and phenolic acids from complex mixtures such as
blood serum and wine, the most effective antioxidants being
those more readily oxidizable (lower oxidation potential
phenolics). In other works, cyclic voltammograms were used
to determine the concentrations of phenolics in red and
white wines. The total phenols content for the easily
oxidizable polyphenols (PP) in the wines was calculated
from the integral area under the peak up to 500 mV (Q500)
and expressed in catechin equivalents [10 – 14]. This method
provided a rapid and reliable approximation of the phenolic
content from red wines yielding results comparable to those
obtained by the standard Folin – Ciocalteu assay; however,
CV measurements observed for white wines were 4 to 5
times lower than the Folin – Ciocalteu value. The discrep-
ancy observed is because a larger proportion of the
phenolics that contribute to the Folin – Ciocalteu measure-
ment become oxidized at potentials greater than 500 mV
and appear in the larger voltammetric peak beyond 800 mV,
which was not included in the Q500 value. A new method
employing also CV at a carbon electrode to provide a rapid
measurement of easily oxidizable polyphenols in beverages
such as green, oolong, and black teas, and in coffee has also
been carried out by Kilmartin et al. [15, 16].The total level of
components that are oxidized at potentials less than 400 mV,
estimated by the Q400 value, provides a measure of its
reducing strength (antioxidant capacity). A poor correlation
between Q400 and antioxidant capacity in tea extracts,
calculated as ability to retard methyl linoleate oxidation,
were observed in all samples due possibly to a relatively high
content of phenolics with low antioxidant capacity, which
evidently do not contribute to Q400.
Zanoni et al. [17] have evaluated the antioxidant capacity
of some phenolic acids according to their voltammetric
behavior. The results were confirmed by DPPH (2,2-
diphenyl-1-picrylhydrazyl) spectrophotometric test being
the electrochemical method applied to the selective deter-
mination of ferulic, p-cumaric, sinapic, caffeic and chloro-
genic acids in orange juice. The selective interaction of
caffeic or chlorogenic acids on activated glassy carbon
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2278
electrode offers a good method for the detection of these
antioxidants in orange juice with a high degree of sensitivity
without any preextraction step or interference of ascorbic, p-
cumaric, ferulic, sinapic or cinnamic acids since they are not
deposited on activated electrode surface. This interesting
approach using orange juice as real sample containing both
types of antioxidants, ascorbic and polyphenols shows the
power of electrochemistry in their direct analysis. However,
any information about the contribution to the overall
antioxidant capacity from typical citrus flavonoids (flava-
nones) which are largely present in these samples was given.
Likewise, Saso et al. [18] have observed a good agreement
between both CV and FRAP (ferric reducing antioxidant
power) assay in the determination of the antioxidant
capacity of flavonoids. However, when radical scavenging
activities of 34 natural antioxidants (especially phenolic
acids and flavonoids) were investigated by Osakai et al. [19]
from an electrochemical viewpoint, the oxidation potentials
measured by CV showed a low correlation with the DPPH
radical scavenging activities. Antioxidant properties of
natural polyphenols (flavonoids and phenolics acids) have
also been investigated by Testa et al. [20] using CV. A good
correlation between those and the antioxidant capacity
calculated from inhibition of lipid peroxidation was ob-
tained. Kusu et al. [21 – 23] have studied the relationship of
electrochemical oxidation of flavonoids on their antioxidant
capacity. The relation between the E1/2 values of the first
oxidation step obtained from the voltammograms and the
antioxidant data (antioxidant concentration to achieve 50%
inhibition, IC50) in biological systems obtained from refer-
ences was examined. It was found that less positive E1/2
values of flavonoids were usually associated with decreasing
IC50 values. These findings suggest that the flavonoids
electrochemical properties contribute to their antioxidant
activity, and thus the E1/2 values of flavonoids can be used as
indexes of their antioxidant activities in biological systems.
The proposed method is expected to be useful for the quick
screening of flavonoid antioxidants and estimating the
antioxidant capacity of flavonoid containing foods and
medicinal plants.
Since DPV is a selective and sensitive technique, their
possibilities have also been explored in the analytical
detection of natural polyphenolic antioxidants in both
complex fruit samples (apples and pears) and matrix (peel
and pulps) containing several phenolic classes such as
cinnamic acids, flava-3-ols and flavonols [24]. DPV was used
as characterization technique to evaluate the antioxidant
capacity of the fruit samples proposing the voltammetric
profiles found as antioxidant capacity profiles. The relation-
ship between its voltammetric behavior and its in vitro
antioxidant activity was established towards oxidation
potential: waves with low values for oxidation potential
which were associated to high antioxidant activity. In
general terms, the high antioxidant power was always
related to predominant flavonoid and phenolic classes
(acids and flavan-3-ols and flavonols) while the low
antioxidant power was related to finger prints phenolics
such as phlorizin and arbutin (typical from apples and pears,
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A. J. Blasco et al.
respectively). In addition, a selective oxidation of flavonoids
versus phenolic acids (at pH 7.5) was strategically proposed
approaching the discrimination of different matrices and
fruits (mainly at pH 2 where the highest peak resolution was
obtained).
Other voltammetric approaches involving cyclic voltam-
metry and chronoamperometry to evaluate the antioxidant
capacity have also been explored [25]. The proposal was
based on the competitive reactions where DPPH . free
radical reacts with an antioxidant (AH) or radical species
(R .) as follows:
DPPH . þ AH(antioxidant) ! DPPH-H þ A .
DPPH . þ R .(radical) ! DPPH-R
In this way, a new method for evaluation of the antioxidant
capacity based on the amperometric determination of no
reacted DPPH free radical has been reported. The resulting
current on a glassy carbon electrode polarized at 140 mV,
was proportional to the concentration of unreacted DPPH
free radical. Apart from a representative group of water
soluble antioxidants involving nonphenolics and phenolic
ones, eight samples of tea, wine and other beverages were
also tested using this procedure to determine their antiox-
idant activity. The amperometric method for the evaluation
of antioxidant capacity had the same advantages and
disadvantages as the compared spectrophotometric method
(spectrophotometric control of no reacted DPPH free
radical) since both methods are based on the same chemical
reaction and kinetics. However, the spectrophotometric
ones can be easily affected by the color and turbidity of a
sample, demanding pretreatment, but the electrochemical
response showed a good stability, and was not affected by
turbid or nontransparent samples. This interesting advant-
age of electrochemistry underlined by the authors of this
work was also included in the introduction of the conceptual
keys to understand the relevant role of electrochemical
approaches in these environments (see Sec. 2)
Korotkova et al. [26] have applied an effective and
convenient voltammetric approach for the determination
of antioxidant capacity by recording the current of the
electrochemical oxygen reduction at a mercury film elec-
trode (MFE). In these experiments the results obtained with
the MFE were compared with those found using a glassy
carbon electrode and the main conclusion was that MFE
proved to be more effective and sensitive. The investigation
was performed in diverse synthetic substances, green tea,
apple vinegar, and pharmaceuticals products. The results
showed the greater antioxidant capacity in ascorbic acid and
glucose.
3.2. Electrochemical Sensing of Natural Antioxidants and
Antioxidant Capacity Using Flow Approaches
Flow injection analysis with electrochemical detection has
also had a predominant role in the evaluation of antioxidant
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Natural Antioxidants in Vitro Systems
capacity since it allows to work at selective target potential
giving an easy and direct way for both, evaluation of
antioxidant capacity and the quantification of the total
antioxidants involved.
Mannino et al. [27] developed a simple method in 1998,
based on a FIA system with amperometric detection to
evaluate the antioxidant capacity of red and white wines
using a GCE operating at a potential of þ 0.4 V, the
antioxidant capacity of white wines being well correlated
with total phenols content obtained by the traditional Folin-
Ciocalteu assay. However, no such good correlation exists
for red wines due to the fact that some red wines show a high
antioxidant capacity even though they have low concen-
trations of total phenols. Later, the same research team
reported a similar procedure for the evaluation of the
antioxidant power of olive oils [28] operating, in this case, at
detection potential of þ 0.5 V. A comparison of the pro-
posed procedure with two other methods used to carry out
the measurement of the olive oil antioxidant activity
(Rancimat method, the most used for the evaluation of the
olive oils oxidative stability, and ABTS .þ radical cation
decoloration assay) was performed. A good agreement
between the results of the proposed electrochemical method
and those of the ABTS .þ decoloration assay was obtained.
This agreement is due to the fact that both methods supply a
measurement of the total amount of natural antioxidants
(phenols and tocopherols). However, on comparing the
proposed procedure and the Rancimat method no good
correlation between both methods was found observing that
the value of the antioxidant capacity from Rancimat method
tends towards a plateau as the induction time increases. This
fact is probably due to the high temperature used in
Rancimat method which can cause an improvement in the
efficiency of some antioxidants and/or synergistic effect
among natural antioxidants, when they are present at high
concentration and phospholipids are also present in the
matrix. Recently, Mannino et al. [29] have also successfully
employed their electrochemical method for monitoring of
antioxidant capacity in herb extracts, with results in good
agreement with those obtained by the more frequently used
DPPH test. The electrochemical method proposed by
Mannino et al. has also been used by different research
groups: Brenna et al. [30] to determine the antioxidant
capacity of wines obtaining a good correlation with the
Folin – Ciocalteu assay, Del Carlo et al. [31] to evaluate the
antioxidant capacity of the phenolic fraction of extra virgin
olive oil and Cerretani et al. [32] to determine the antiox-
idant capacity of several phenolic compounds isolated from
extra virgin olive oil.
Escarpa et al. have also carried out several works dealing
with the relevant role of electrochemistry as an important
alternative to the spectrophotometric ones in the analysis of
natural antioxidants and their antioxidant capacity [33 – 35].
Firstly, they introduced Electrochemical Index (EI) concept
defined as the total polyphenolic content obtained by non
selective oxidation of all polyphenols at the target potential
þ 0.8 V [33]. To demonstrate the suitability of the concept, a
wide and heterogeneous sample of food involving wines,
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2279
fruits, and vegetables was examined and the total polyphe-
nolic content was measured using both electrochemical (EI)
and traditional (Total phenolics spectrophotometric) pro-
tocols. Total phenolics was always higher than EI, a fact that
probably demonstrated the inherent selectivity of EI in the
determination of their total content. In addition, the fraction
of polyphenols with expected high antioxidant capacity was
also obtained by just changing the oxidation potential at
þ 0.5 V (high antioxidant polyphenolic fraction, HA). EI
and HA were well correlated with the total phenolics
obtained using the spectrophotometric assay (r ¼ 0.95, r ¼
0.94) indicating their suitability to determine both total
polyphenolic fractions. The accuracy of the EI concept has
been recently demonstrated adjusting the previously pro-
posed protocol towards the determination of total isofla-
vones in soy samples as a particular case of EI [34]. The
accuracy was evaluated using a secondary standard from
Drug Master File (SW/1211/03) as metrological reference.
The error obtained when the total isoflavonoid content was
compared with the certified value was less than 11% while a
high overestimation was obtained when spectrophotometric
assay was used. The accuracy obtained, allowed the
introduction of the term Isoflavonoid Index defined as the
total amount of isoflavones obtained in soy samples when
they are amperometrically monitorized at þ 1.0 V. In
addition, this work clearly demonstrates that the spectro-
photometric assay is not suitable to determine total
phenolics while electrochemical protocol is a good alter-
native, at least in samples where only one flavonoid class is
present.
In another contribution, Total phenolics (TP) (using
Folin – Ciocalteu assay), Antioxidant capacity (AC) (using
DPPH method), Electrochemical Index (EI) (total poly-
phenolics at þ 0.8 V) and High Antioxidant polyphenolic
fraction (HA) (total polyphenolics at þ 0.5 V) have been
evaluated in honey samples by the same group [35].
Correlation studies and Principal Component Analysis
(PCA) was performed in depth. The most relevant infor-
mation obtained could be summarized as follows: (i) EI was
highly correlated with total phenolics which indicates the
suitability of the electrochemical approach as an alternative
to measure total phenolics. (ii) EI and, mainly HA, were
highly correlated with AC, which indicates that the electro-
chemical protocol become a new tool for estimating the
antioxidant capacity of the target honeys. In this way, PCA
confirmed that HA fraction was clearly associated with AC
revealing this fraction as the fraction of polyphenols with
high antioxidant activity. These results revealed the suit-
ability of the use of electrochemical reactions to estimate
antioxidant capacity and antioxidant content in complex
samples such as honeys since good correlation between HA
and AC, as well as between EI and FC were obtained,
respectively. The relative antioxidant capacity was finally
demonstrated to be correlated with the honey color dark >
intermediate > clear.
Very recently, Buratti et al. [36] have also evaluated the
antioxidant capacity of honeybee products by measuring the
current resulting from the oxidation of electroactive com-
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2280
pounds present in the samples at the established potential of
þ 0.5 V. This potential appears to be selective to discrim-
inate only the compounds having effective antioxidant
capacity and at this potential the reducing power decreases
in order: quercetin > isorhamnetin > kampferol > caffeic
acid > galangin > ferulic acid > p-cumaric acid. Hespertin,
naringenin, chysin, and pinocembrin are not revealed and
presumably they have very little or no antioxidant effect.
The samples of honey, propolis and royal jelly were also
analyzed by the DPPH assay and their total phenolic
content was determined by the Folin – Ciocalteu assay. The
data obtained by the proposed method were substantially
confirmed by the DPPH assay since a good correlation was
observed between the two methods. On the other hand, the
correlation analysis between the proposed method and the
Folin – Ciocalteu assay also gave good results suggesting
that the antioxidant capacity of propolis and honey samples
are due to their phenolic contents. Cosio et al. [37] have also
estimated the antioxidant capacity of selected herbs at
þ 0.5 V using a conventional FIA system. The data obtained
by this electrochemical method were substantially con-
firmed by the DPPH method. The two methods were highly
correlated. Among analyzed herbs, rosemary and sage
exhibit the highest antioxidant capacity. This is probably
due to high content of rosmarinic acid, carnosic acid and
their derivatives. The antioxidant capacity of the other herbs
decreases in the following order: oregano > thyme >
peppermint > laurel > basil. The results showed that the
antioxidant capacity was not necessarily correlated with the
phenolic content. In fact, rosemary and sage have a high
total phenolic content and a high antioxidant capacity, while
other herbs, such as laurel and basil, have a high phenolic
content and a low antioxidant capacity. However, the
relationship between the total phenolic and the antioxidant
capacity evaluated by the proposed method shows a good
correlation.
Electron-donating ability as a criterion to assay the
antioxidant properties, in connection with three electro-
chemical methods, including cyclic voltammetry, hydro-
dynamic linear sweep voltammetry (using a rotating disk
electrode) and flow injection amperometry, have also been
explored [38]. Real samples used included bovine milk,
whey and low-molecular weight (LMW) fractions of whey.
The main antioxidants found were ascorbate and, mainly
ureate.
In addition, two contributions using separation tech-
niques coupled to electrochemical detection where explic-
itly antioxidant capacity was evaluated have been found.
A HPLC-ED (coulometric detection) work dealing with
antioxidant capacity has been reported. In this work, Peyrat-
Maillard et al. [39] have established a correlation between
the potential corresponding to maximal detector response
(MDR, obtained from the HDV profile) of phenolic acids
and flavonoids and their antioxidant capacity. The potential
corresponding to MDR of phenolic acids was shown to be
inversely proportional to its antioxidant capacity as deter-
mined by the DPPH test. However, such a relationship was
not observed for flavonoids. Although this paper showed an
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A. J. Blasco et al.
interesting and systematic study, the findings using stand-
ards were not confirmed using any real sample.
Yuhua et al. [40] have provided a simple method to screen
out high efficient antioxidant capacity from Chinese herbs.
They have carried out studies on OH . scavenging activities
of rutin as a typical flavonoid, widely distributed in Chinese
herbs using capillary electrophoresis with electrochemical
detection (CE-ED) at a carbon disk electrode. The hydroxyl
radicals (OH .) were produced by Fenton reaction. It reacts
with salicylic acid to produce 2.3-dihydroxy benzoic acid
(2.3-DHBA) and 2.5-dihidroxy benzoic acid (2.5-DHBA) as
probe of OH ., and they can be analyzed by CE-ED. When
pH is lower than 7.4 and the detection potential applied was
þ 0.6 V, 2.3 DHBA and 2.3-DHBA elute simultaneously
from the capillary. As a result, only one peak appears in the
electropherogram. Since peak currents of the two analytes
are additive, the overall peak current of two compounds can
be used for the determination of hydroxyl radical. Scaveng-
ing OH . activities at different scavenger concentrations
extracted from herbs clearly prove that OH . scavenging
activity is obviously correlated to the concentration of
scavenger. In this work, the CE-ED analytical system was
employed just to evaluate the antioxidant activity, previ-
ously carried out.
3.3. Electrochemical Sensing of Non-(poly-)phenolic
Antioxidants and Antioxidant Capacity
CV has also been employed [41, 42] as an instrumental tool
to measure the plasma antioxidant capacity which stems
from the low molecular weight antioxidants such as ascorbic
acid. Two anodic waves were identified in the CV tracings of
human plasma at E1/2 ¼ 420  25 and 920  25 mV, two
specific components of the first anodic wave being identified
confirming by HPLC-ED: ascorbate and urate [41]. Param-
eters that indicate the antioxidant capacity of samples such
as the peak potential Ep, the peak current Ip and the charge
Q below voltammetric waves were also calculated for the
waves obtained. The plasma antioxidant capacity values
have also been compared with the results of the DPPH test
being a good correlation between both methods, reflecting
the antioxidant capacity of the two prominent water-soluble
low molecular weight antioxidants in plasma: ascorbic and
uric acid [42].
Mannino et al. have also reported [43] a method based on
a FIA system with amperometry detection using a GCE for
the evaluation of the antioxidant capacity of lipophilic
compounds present in vegetables such as tocopherols and
others. This method is similar to that published for wine and
olive oil antioxidant capacity [27, 28].
Finally, Ziyatdinova et al. [44] developed a method to
evaluate the total antioxidant capacity (TAC) in animal fats
and vegetable oils and juices by coulometric titration using
electro-generated bromine. The bromine was used as
reagent for estimation of the integrated antioxidant proper-
ties of these samples caused by the presence of a-tocopher-
ol, vitamins A and D. TAC of blood and plasma was also
G 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Natural Antioxidants in Vitro Systems
evaluated by this method showing good correlation with
catalase activity and low density lipoprotein concentration
[45]. In another work, this group developed a potentiomet-
ric method for the determination of ascorbic acid using
iodine modified platinum electrode and, furthermore,
evaluated the contribution of this compound on the total
antioxidant capacity (TAC) of some phyto preparations and
juices [46].
3.4. Electrochemical Sensing of Natural Antioxidants and
Antioxidant Capacity Using Miniaturized
Approaches
One of the trends in analytical chemistry is the miniatur-
ization of all the analytical operations termed lab-on-a-chip
where electrochemical detection has demonstrated a rele-
vant role due to inherent miniaturization without loss of
performance and high compatibility with microfabrication
techniques [47]. However, microfluidic systems in real food
analysis have been employed in minor extension because of
the complexity of this type of samples, being CE microchips
the main miniaturized system used [48].
For this reason, only a few contributions have been found.
Three works appeared close to each other in the literature
dealing with detection of phenolic acids in wine [49],
catechins in tea extracts [50] and natural antioxidants
involving both flavonoids and ascorbic acid in pear samples
[51]. Although all of them offered an ultra fast separation of
the main natural antioxidants involved and the detection of
some of them in real food samples; however, no study
explicitly confirms the antioxidant capacity.
Recently, an interesting characterization of antioxidants
involving the evaluation of their antioxidant capacity using a
fluidic chip has been reported [52]. The antioxidant in vitro
model was based on simultaneous detection of superoxide
and hydrogen peroxide (ROS species) and their possible
scavengers. The fluidic chip developed consists of an ROS-
generation chamber, a mixing section and a compartment
for the biosensor chip with two sensors for each species.
Antioxidants of enzymatic and nonenzymatic nature as well
as of a model cosmetic cream doped with green tea extract
(polyphenol antioxidants) were tested with promising
results. In fact, it was found that the sample could react
with superoxide as well as with hydrogen peroxide indicat-
ing the role of polyphenols as radical scavengers.
Finally, an interesting contribution regarding the evalua-
tion of antioxidant capacity using gold nanoparticles has
been proposed [53]. The antioxidant capacity of several
phenolic acids and related food samples was estimated from
the generation and growth of gold nanoparticles. The
intensity of the resulting particle plasmon absorption bands
correlated well with the redox characteristics of these
phenolic acids using CV. The highest capacity of reducing
Au(III) to Au(0) nanoparticles corresponds to the highest
antioxidant activity, consistent with the tendency of phe-
nolic acids to donate electrons obtained directly and easily
by CV. This work reconfirms the premier role of electro-
Electroanalysis 19, 2007, No. 22, 2275 – 2286 www.electroanalysis.wiley-vch.de
2281
analytical approaches as suitable tools in antioxidant
capacity evaluation.
4. Conclusions
Electrochemistry is now among the most important ap-
proaches in the analytical evaluation of antioxidant capacity
because it has the same conceptual base as those exhibited
by antioxidants in vitro systems. In addition, since the
synergistic interactions among the antioxidant compounds
in a food mixture can be present, direct, selective and
sensitive determination of both, total antioxidants and
antioxidant activity, in the food and biological samples is
highly desirable. Electrochemical techniques allow both the
simultaneous estimation of the total antioxidants in the real
sample as well as the evaluation of their antioxidant activity.
Controlled-potential techniques in both, batch and flow
approaches, have been commonly used to evaluate antiox-
idant capacity and estimate antioxidant content. On the
contrary, other electrochemical approaches such as modi-
fied electrodes or detectors coupled to HPLC and CE have
not been explored to this end because these approaches are
focused in the accurate determination of each compound,
therefore sometimes being irrelevant in these environments.
Scanning controlled-potential techniques give rapid in-
formation about the relative position of the oxidation waves
which inform potentially about antioxidant capacity while
FIA approaches allow the fixing of the selectivity by
strategically decreasing potential to evaluate the high
antioxidant capacity of the food and biological samples.
Electrochemical approaches are demonstrating their ana-
lytical power in terms of selectivity giving more realistic
antioxidant contents than those found in spectrophotomet-
ric ones. The unique versatility of electrochemistry is of
particular interest because a decrease of oxidation potential
gives direct information about the antioxidants with high
antioxidant activity.
The suitability of the electroanalytical approaches has
been studied for correlation studies. In general, correlation
studies between electrochemical approaches and commonly
used antioxidant capacity assays to demonstrate their
possibilities as new tools in the evaluation of the antioxidant
capacity have been performed. Since polyphenols are the
main antioxidants involved in the antioxidant capacity
exhibited by the samples, correlation studies between
electrochemical approaches with Folin – Ciocalteu assay to
attribute the antioxidant capacity to the polyphenols, have
also been carried out. These studies are demonstrating the
antioxidant properties of food and biological samples due to
the presence of polyphenolic compounds and vitamins C
and E.
In addition, following the lab-on-a-chip concept, some
initial works emulating model ROS-systems and ultra fast
separation of the main antioxidants are emerging with
promising results. Also, relevant nanoelectrochemistry
strategies based on the redox reactions between antioxi-
dants and oxidants to generate gold nanoparticles are
G 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2282 A. J. Blasco et al.

Table 1. In Vitro electrochemical approaches in the evaluation of antioxidant capacity of food and biological samples.
Sample Antioxidants Electrochemical Antioxidant Total pheno- Remarks Ref.
technique1 capacity assay2 lics3 ( TP )

Red wine and blood Ascorbic acid, flavo- Cyclic voltamme- RANDOX kit Not deter- Estimation of the relative [9]
serum noids, phenolic acids try ( ABTS .þ ) mined reducing strength of each
and transresveratrol antioxidant
Red and white wines Flavonoids and phe- Cyclic voltamme- Not performed Folin-Ciocal- Estimation of the relative [10]
nolic acids try teu reducing strength of each
antioxidant. Qualitative
assessment of wine phe-
nolics
Red and white wines No analytes standards Cyclic voltamme- Ability to re- Folin-Ciocal- Direct evaluation of anti- [11]
try tard methyl li- teu (r ¼ 0.81) oxidant capacity in real
noleate oxida- ACA and FC samples.
tion (r ¼ 0.94) High correlation between
different methods.
RSDs < 10% (in wines)
Red and white wines Flavonoids and phe- Cyclic voltamme- Not performed Folin-Ciocal- Better correlation for [12]
nolic acids try teu (r ¼ 0.91 white than for red wines
for white)
Red wine No analytes standards Cyclic voltamme- Ability to re- Folin-Ciocal- Low correlation between [13]
try tard methyl li- teu antioxidant capacity and
noleate oxida- wine age.
tion
Red and white wines ( þ )-Catechin Cyclic voltamme- Not performed Folin-Ciocal- Low correlation [14]
try teu
Teas No analytes standard Cyclic voltamme- Ability to re- Not deter- [15]
try tard methyl li- mined
noleate oxida-
tion (r2 ¼ 0.82
for black and
r2 ¼ 0.44 for
green)
Teas and coffee Flavonoids and galic Cyclic voltamme- Not performed Not deter- Identification of individual [16]
acid try mined antioxidants by HPLC
Orange juice Phenolic acids Cyclic voltamme- DPPH Not deter- Presence of ascorbic acid [17]
try mined and polyphenols
RSDs < 3%
LOD 10
5 M
(caffeic acid)
No real sample stu- Flavonoids, resvera- Cyclic voltamme- FRAP Not deter- Oxidation potential and [18]
died trol, trolox and uric try mined FRAP were inversely cor-
acid related
No real sample stu- Flavonoids phenolic Cyclic voltamme- DPPH Not deter- Low correlation [19]
died acids, ascorbic acid try (r ¼ 0.73) mined
and cysteine
No real sample stu- Flavonoids and phe- Cyclic voltamme- Inhibition of Not deter- Compounds with its low [20]
died nolic acids try lipid peroxida- mined oxidation potential yet
tion poor antioxidant capacity.
No real sample stu- Flavonoids Cyclic voltamme- Inhibition of Not deter- Hight correlation [21,
died try, flow-trough lipid peroxida- mined 22,
column electrolysis tion 23]
(r ¼ 0.907)
Apple and pears Phenolic acids & fla- Differential pulse Not performed Not deter- Predominant flavonids as- [24]
peels and pulps & vonoids volammetry mined sociated with high-antiox-
comercial juices idant capacity
RSDs < 9%
Recovery 100%
LODs 0.1–4.2 mM

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Natural Antioxidants in Vitro Systems 2283

Table 1. (cont.)

Sample Antioxidants Electrochemical Antioxidant Total pheno- Remarks Ref.

technique1 capacity assay2 lics3 ( TP )

Tea, wine and other Water soluble (non- Cyclic voltamme- DPPH (as Not deter- Independence of turbidity [25]
beverages pheolic and phenolics) try, chronoam- HAT mechan- mined in amperometry vs. spec-
perometry (at ism) trophotometry
140 mV )
Green tea, apple Glucose, ascorbic acid Electrochemical Not performed Not deter- Mercury film electrode for [26]
vinegar oxygen reduction mined evaluation of antioxidant
at mercury film capacity
electrode
Red and white wines ( þ )-Catechin FIA-ED Not performed Folin-Ciocal- Better correlation for [27]
Amperometry teu (r ¼ 0.95 white than for red wines.
( E ¼ þ 0.4 V ) for white and
r ¼ 0.79 for red
wines)
Olive oil Phenolic acids & Fla- FIA-ED ABTS .þ Not deter- High correlation. Direct [28]
vonoids Amperometry (r ¼ 0.988) mined evaluation of AOA in real
( E ¼ þ 0.5 V ) samples.
RSDs < 4%
LOD ¼ 0.4 mg/L
Herb extracts (La- Phenolic acids & Fla- FIA-ED DPPH Not deter- High correlation. [29]
biatae family) vonoids Amperometry (r ¼ 0.986) mined RSDs < 4%
( E ¼ þ 0.5 V ) Recovery 100%
Red wine Phenolic acids & Fla- FIA-ED Not performed Folin-Ciocal- [30]
vonoids Amperometry teu (r ¼ 0.83)
( E ¼ þ 0.4 V )
Extra virgin olive oil No analytics standard FIA-ED DPPH Folin-Ciocal- Direct evaluation of anti- [31]
Amperometry (r ¼ 0.884) teu (r ¼ 0.896) oxidant capacity in real
samples
Virgin olive oil No analytes standard Amperometric de- DPPH Not deter- Direct evaluation of anti- [32]
tection ( HPLC ) mined oxidant capacity in real
samples
Apple peels Total polyphenolics FIA-ED Not performed Folin-Ciocal- Estimation of total poly- [33]
Apple pulps Amperometry teu (r ¼ 0.95) phenolics (introduction of
Pear peels ( E ¼ þ 0.8 V ) Electrochemical Index
Pear pulps ( E ¼ þ 0.5 V ) concept, EI )
Red, rose and white Estimation of total poly-
wines phenolics with high anti-
Fresh & processed oxidant capacity ( HA )
green beans Good correlation in both
cases
RSDs < 8%v
LODs 0.03–0.88 mg/L
Secondary standard Total isoflavones FIA-ED Not performed Folin-Ciocal- Evaluation of accuracy of [34]
soy samples Amperometry teu EI.
( E ¼ þ 1.0 V ) Introduction of the term
isoflavonoid index.
RSDs < 3%
LODs 2 mg/mL
11 representative Total polyphenolics FIA-ED DPPH Folin-Ciocal- EI & HA in honeys [35]
honeys (clear, inter- Amperometry (r ¼ 0.94) teu (r ¼ 0.93) HA exhibited high antiox-
mediate and dark) ( EI: E ¼ þ 0.8 V ) idant capacity
( HA: E ¼ þ
0.5 V )
Honeybee Flavonoids (flavonols FIA-ED DPPH Not deter- Flavonols exhibited the [36]
mainly) Amperometry (r2 ¼ 0.96) mined highest antioxidant activ-
( E ¼ þ 0.5 V ) ity
Herbs Phenolics rosmaric & FIA-ED DPPH Not deter- Antioxidant capacity was [37]
carnosic acids Amperometry (r2 ¼ 0.96) mined established: oregano >
( E ¼ þ 0.5 V ) peppermint > laurel

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2284 A. J. Blasco et al.

Table 1. (cont.)

Sample Antioxidants Electrochemical Antioxidant Total pheno- Remarks Ref.

technique1 capacity assay2 lics3 ( TP )

Bovine milk Ascorbate CV Not performed Not deter- Quantitative analysis of [38]
Hydrodynamic mined antioxidants using FIA-
voltammetry ED
FIA-ED
No real sample stu- Flavonoids and phe- Coulometric DPPH Not deter- High correlation for acids [39]
died nolic acids ( HPLC ) (r2 ¼ 0.96) mined but not for flavonoids.
Chinese herbs Flavonoids CE-ED Generation of Not deter- CE is used as method to [40]
(amperometry at OH by Fenton mined monitories the antioxidant
þ 0.6 V ) reaction and capacity
scavenging by
antioxiants
Human plasma Low molecular weight Cyclic voltamme- Not performed Not deter- Two anodic waves; the [41]
antioxidants (ascorbic try mined first wave ( E1/2[ A] ¼
acid and uric acid) 420 mV ) corresponding to
uric and ascorbic acid.
Plasma Low molecular weight Cyclic voltamme- DPPH . Not deter- Two anodic waves; the [42]
antioxidatns (ascorbic try (r ¼ 0.991) mined first wave high correlation
acid and uric acid) ABTS .þ with DPPH and low with
(no data) ABTS.
ABTS includes the two
anodic waves.
Fruits and vegetables Carotenoids, chloro- FIA-amperometry ABTS .þ Not deter- High correlation. [43]
phylls, tocopherol, ( E ¼ þ 0.5 V ) (r ¼ 0.9) mined Selectivity for lipophilic
capsaicin (lipophilic compounds; high speed of
extract) analysis (60 samples/h)
RSDs < 4% (n ¼ 12)
LOD 0.2 mg/mL (b-caro-
teno)
Vegetable oils, fish- Vitamin E Coulometric titra- Not performed Not deter- Determination of indivi- [44]
liver oil, carrot juice Vitamin A tion with electro- mined dual antioxidants and total
genarated bromine antioxidant capacity
( TAC)
Blood and plasma S-containing amino Coulometric titra- Low density li- Not deter- Total antioxidant capacity [45]
acids, vitamin C, Fe- tion with electro- poprotein mined ( TAC) can be used to
containing porphyrins, generated bromine ( LDL ) con- predict antioxidant de-
albumin and uric acid. centration fense system state on pa-
Glutatione and cy- (r ¼ 0.785) tients.
steine Catalase activ-
ity
(r ¼ 0.988)
Phytopreparations Vitamin C Potentiometric Not performed Not deter- Contribution of vitamin C [46]
and juice mined on TAC
RSDs < 10%
Recovery 100%
Wines & fruits & tea Phenolic acids CE-ED microchip Not performed Not deter- Miniaturized approaches [49–
extracts Catechins and ascorbic mined Ultra fast separation of 51]
acid main antioxidants
Cream matrix dop- Polyphenolics Microfluidics sys- Generation of Not deter- Evaluation of antioxidant [52]
ped with tea extract tem coupled two ROS and reac- mined capacity using “lab-on-a-
pairs of electrodes tion with anti- chip” concept.
for independent oxidatns
determination of
superoxide and
hydrogen peroxide
( ROS especies)

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Natural Antioxidants in Vitro Systems
Table 1. (cont.)
Sample Antioxidants Electrochemical
technique1
Beverages Phenolic acids Cyclic voltamme-
try
1
Electrochemical technique used in the evaluation of antioxidant capacity of the samples.
2
Assay used in the evaluation of antioxidant capacity of the samples. Between brackets: correlation obtained between Electrochemical approach and
Antioxidant capacity assay.
3
“Total phenolics” obtained using Folin Ciocalteu assay. Between brackets: correlation obtained between electrochemical approach and/or Antioxidant
capacity with “total pehnolics”.
dramatically opening up the electrochemistry possibilities in
the evaluation of antioxidant capacity of samples with food
and biological significance.
5. Acknowledgements
Financial support from the Spanish Ministry of Science and
Technology CTQ2005-09079-C03-03 is gratefully acknowl-
edged. A.J.B. acknowledges the fellowship received from
Alcalá University. A.G.C. acknowledges the fellowship
received from the Spanish Ministry of Education and
Science.
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