Food Anal.
Methods (2009) 2:41–60
DOI 10.1007/s12161-008-9067-7
Review of Methods to Determine Antioxidant Capacities
Ayse Karadag & Beraat Ozcelik & Samim Saner
Received: 2 September 2008 / Accepted: 25 November 2008 / Published online: 13 January 2009
# Springer Science + Business Media, LLC 2009
Abstract Antioxidant capacity is related with compounds
capable of protecting a biological system against the
potentially harmful effect of processes or reactions involving
reactive oxygen and nitrogen species (ROS and RNS). These
protective effects of antioxidants have received increasing
attention within biological, medical, nutritional, and agro-
chemical fields and resulted in the requirement of simple,
convenient, and reliable antioxidant capacity determination
methods. Many methods which differ from each other in
terms of reaction mechanisms, oxidant and target/probe
species, reaction conditions, and expression of results have
been developed and tested in the literature. In this review, the
methods most widely used for the determination of antiox-
idant capacity are evaluated, presenting the general princi-
pals, recent applications, and their strengths and limitations.
Analysis conditions, substrate, and antioxidant concentra-
tion should simulate real food or biological systems as
much as possible when selecting the antioxidant capacity
method. The total antioxidant capacity value should
include methods applicable to both lipophilic and hydro-
philic antioxidants, with regards the similarity and differ-
ences of both hydrogen atom transfer and electron transfer
mechanism. The methods including various ROS/RNS
also have to be designed to comprehensively evaluate the
antioxidant capacity of a sample.
A. Karadag : B. Ozcelik
Department of Food Engineering,
Faculty of Chemical and Metallurgical,
Istanbul Technical University,
34469 Maslak, Istanbul, Turkey
S. Saner (*)
Kalite Sistem Laboratories Group,
Ar Plaza B Blok Degirmen Sk. No:16 Kozyatagi,
34742 Kadikoy, Istanbul, Turkey
e-mail: saners@kalitesistem.com
Keywords Antioxidant Capacity Methods . Review .
Reactive Oxygen Species
Introduction
After several studies on the importance of antioxidants in
biological systems by counteracting of oxidative stress that
causes several human diseases such as atherosclerosis,
diabetes mellitus, chronic inflammation, neurodegenerative
disorders, and certain types of cancer have been conducted,
there is a great interest of quantification of antioxidants and
determination of antioxidant capacities of a number of
specific food compounds.
In food science, the antioxidant is defined as a substance in
foods that when present at low concentrations compared to
those of an oxidizable substrate significantly decreases or
prevents the adverse effects of reactive species, such as
reactive oxygen and nitrogen species (ROS/RNS), on normal
physiological function in humans (Halliwell et al. 1995;
Huang et al. 2005). According to this definition, not all
reductants involved in a chemical reaction are antioxidants;
only those compounds which are capable of protecting the
biological target from oxidation meet this criterion.
Mechanisms of antioxidant action include serving as (1)
physical barriers to prevent ROS generation or ROS access to
important biological sites, e.g., UV filters, cell membranes; (2)
chemical traps/sinks that “absorb” energy and electrons,
quenching ROS, e.g., carotenoids, anthocyanidins; (3) cata-
lytic systems that neutralize or divert ROS, e.g., the
antioxidant enzymes SOD (superoxide dismutase), catalase,
and glutathione peroxidase (Chaudiere and Ferrari-Iliou
1999); (4) binding/inactivation of metal ions to prevent
generation of ROS, e.g., ferritin, ceruloplasmin, catechins;
and (5) chain-breaking antioxidants which scavenge and
42
destroy ROS, e.g., ascorbic acid (vitamin C), tocopherols
(vitamin E), uric acid, glutathione, flavonoids (Benzie 2003).
Many methods have been developed and tested in the
literature, advantages and limitations of these methods have
still been discussed. It does not seem to have a consensus
for concluding the most convenient method as a standard
method for claiming total antioxidant capacity, for example,
the limitations for determination of hydrophilic antioxi-
dants, the problems occurring in determination of reaction
end point, the concern on light sensitivity of initiators or
probes, carrying out the analysis in the physiological
irrelevance pH, possible interference from certain food
components, the use of different standards for expressing
results that causes the difficulties in comparison. The scope
of this review is to summarize the principals of the most
commonly used in vitro antioxidant capacity assays by
evaluating their strengths and limitations.
Definitions
Different researchers used different terms to express
antioxidant capacity including total antioxidant efficiency,
effectiveness, action, power, parameter, potential, potency,
and activity. Antioxidant activity and antioxidant capacity
are terms that are often used interchangeably; they have
different meanings though (MacDonald-Wicks et al.
2006). The “activity” of a chemical would be pointless
without specific reaction conditions such as pressure and
temperature. The antioxidant capacity gives the informa-
tion about the duration while the activity describes the
starting dynamics of antioxidant action (Roginsky and
Lissi 2005). The antioxidant capacity in complex hetero-
geneous foods and biological systems is affected by many
factors including the partitioning properties of the anti-
oxidants between lipid and aqueous phases, the oxidation
conditions and the physical state of the oxidizable
substrate (Frankel and Meyer 2000). For example, antiox-
idant protection significantly changes according to the
substrate used to conduct evaluation. When brain homog-
enate and linoleic acid emulsion are used as substrates, α-
tocopherol showed a much better protection than Trolox
(Castro et al. 2006).
A dietary antioxidant can sacrificially scavenge ROS/
RNS to stop radical chain reactions, considered as primary
chain-breaking antioxidants or free radical scavengers
(FRS), or it can inhibit the reactive oxidants from being
formed in the first place, considered as secondary or
preventive antioxidants. Primary antioxidants, when present
in trace amounts, may either delay or inhibit the initiation
step by inactivating or scavenging free radicals, thus
inhibiting initiation and propagation reactions by reacting
with peroxyl or alkoxyl radicals.
Food Anal. Methods (2009) 2:41–60
FRS or chain-breaking antioxidants are capable of
accepting a radical from oxidizing lipids species such as


peroxyl LOO
) and alkoxyl LO ) radicals by the following
reactions.



LOO or LO þ FRS ! LOOH or LOH þ FRS

Antioxidant efficiency is dependent on the ability of the
FRS to donate hydrogen to the free radical. As the
hydrogen-bond energy of the FRS decreases, the transfer
of the hydrogen to the free radical is more energetically
promising and rapid. The ability of an FRS to donate
hydrogen to a free radical can be predicted from standard
one-electron reduction potentials. The reduction of an FRS
should be lower than 600 mV, which is a reduction
potential of polyunsaturated fatty acid to work as antioxi-
dant (Lee et al. 2003). Efficient FRS also produces radicals
(FRS
) that do not react rapidly with oxygen to form
peroxides. In foods, the efficiency of phenolic FRS also
depends on additional factors such as volatility, pH
sensitivity, and polarity (Akoh and Min 1998).
Preventive antioxidants, such as superoxide dismutase,
catalase, and peroxidase, are described either as preventing
introduction of initiating radicals or as inhibiting the rate at
which new chains are set up. There are many different
“preventive” antioxidation pathways because of the distinct
range of available oxidation initiators. These pathways
include chelation of transition metals, singlet-oxygen
deactivation, enzymatic ROS detoxification, UV filtration,
inhibition of prooxidant enzymes, antioxidant enzyme
cofactors, etc. (Laguerre et al. 2007). Metal chelators are
preventive antioxidants by complexing with transition
metal ions, thereby delaying metal-catalyzed initiation
reactions and decomposition of lipid hydroperoxides. Other
antioxidant mechanisms include singlet-oxygen quenching,
oxygen scavenging, and blocking the prooxidant effects by
binding certain proteins containing catalytic metal sites
(Frankel and Meyer 2000). Dietary antioxidants often
broadly include radical chain reaction inhibitors, metal
chelators, oxidative enzyme inhibitors, and antioxidant
enzyme cofactors (Huang et al. 2005).
Antioxidant Mechanisms
Due to the confusion in the literature for the reaction
mechanisms, the need to provide a protocol involving
measurement of more than one property due to multiple
activities of polyphenols is outlined and the dominant
activity depends on the medium and type of antioxidants.
The response of antioxidants to different radical or oxidant
sources may be different. For example, carotenoids are not
particularly good quenchers of peroxyl radicals relative to
phenolics but are exceptional singlet-oxygen scavengers.
Food Anal. Methods (2009) 2:41–60
Therefore, no single assay accurately reflects the mecha-
nism of action of all radical sources or all antioxidants in a
complex system (Prior et al. 2005).
On the basis of the inactivation mechanism involved,
major antioxidant capacity methods have been generally
divided into two categories though: (1) hydrogen atom
transfer (HAT) reaction and (2) electron transfer (ET)
reaction-based methods. Bond dissociation energy and
ionization potential are two major factors that determine
the mechanism and the efficiency of antioxidants. ET and
HAT mechanisms almost always occur together in all
samples and their difference can be made difficult. In
Huang et al. (2005) and MacDonald-Wicks et al. (2006),
total phenol assay by using the Folin-Ciocalteu Reagent
(FCR), Trolox equivalent antioxidant capacity (TEAC), and
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
capacity assays was considered under the methods utilizing
ET mechanism, whereas they were classified under the
methods utilizing both ET and HAT mechanisms in Prior et
al. (2005) due to difficulty in the interpretation of inhibition
mechanisms of these radicals.
HAT-based methods measure the classical ability of an
antioxidant to scavenge free radicals by hydrogen donation
to form stable compounds (Prior et al. 2005). HAT-based
methods are more relevant to the radical chain-breaking
antioxidant capacity.


AH þ X ! XH þ A
ð1Þ
Relative reactivity in HAT methods is determined by the
bond dissociation energy of the H-donating group in the
potential antioxidant and ionization potential. HAT reactions
are solvent and pH dependent and are generally quite rapid,
typically completed in seconds to minutes. Most HAT-based
methods monitor competitive reaction kinetics, and the
quantitation is derived from the kinetic curves. HAT-based
methods are generally composed of a synthetic free radical
generator (AAPH, 2,2′-azobis(2amidinopropane) dihydro-
chloride (ABAP), 2,2′-azobis(2,4dimethylvaleronitrile
(AMVN), 2,2′-azinobis(3-ethylbenzothiazolline-6-sulfonic
acid (ABTS; Valkonen and Kuusi 1997; Wolfe and Liu
2007)), an oxidizable molecular probe (dichlorofluorescein
(DCFH; Adom and Lui 2005)), fluorescein (FL; Moore et al.
2006), and an antioxidant.
The HAT-based assay using fluorescent probes has a
mechanistic similarity to lipid peroxidation, but, under the
assay conditions, the substrate (probe) concentration is
often smaller than the antioxidant concentration. This is in
conflict with real situations. In food systems, the antioxi-
dant concentration is much smaller than substrate (e.g.,
lipid) concentration. It is questionable whether the antiox-
idant capacity measured using the HAT-based assay using a
molecular probe can exhibit the situations in a real food
system (Huang et al. 2005).
43
ET-based methods detect the ability of a potential
antioxidant to transfer one electron to reduce any com-
pound, including metals, carbonyls, and radicals.


MðIIIÞ þ AH ! AH þ MðIIÞ
In ET methods, relative reactivity is based on
deprotonation and ionization potential of the reactive
functional group, so ET reactions are pH dependent. In
general, ionization potential values decrease with in-
creasing pH, reflecting increased electron-donating ca-
pacity with deprotonation (Prior et al. 2005). The pH
values have an important effect on the reducing capacity
of antioxidants. At acidic conditions, the reducing capac-
ity may be restrained due to protonation on antioxidant
compounds, whereas, in basic conditions, proton dissoci-
ation of phenolic compounds would increase sample’s
reducing capacity (Huang et al. 2005). ET reactions can be
relatively slow and need long times to reach completion so
traditionally measure relative percent decrease in product
rather than kinetics or total antioxidant capacity (Ozgen et
al. 2006). Compared to HAT, the ET mechanism is
strongly solvent dependent due to solvent stabilization of
the charged species (Ou et al. 2002a).
ET-based methods involve two components in the
reaction mixture, antioxidants and oxidant (probe). The
probe itself is an oxidant and abstracts an electron from
the antioxidant, resulting in color changes of the probe.
The degree of the color change is proportional to the
antioxidant concentration. The reaction is reached to end
point when color change stops. The change of absor-
bance (ΔA) is plotted against the antioxidant concentra-
tion to give a linear curve. The slope of the curve
indicates the antioxidant’s reducing capacity, which is
expressed as Trolox equivalence or Gallic acid equiva-
lent. To make the correlation, it is accepted that
antioxidant capacity is equal to reducing capacity.
However, it is questionable how the assay results relate
to total antioxidant capacity of a sample because there is
no competitive reaction involved and there is no oxygen
radical in the assays (Huang et al. 2005).
The question raised is which mechanism, ET or HAT,
physiologically reflects the antioxidant preventive action.
As cited in Ou et al. (2002a), Wright and co-workers used a
procedure based on density functional theory to calculate
gas-phase bond dissociation enthalpy and ionization poten-
tial for molecules belonging to the class of phenolic
antioxidants, including tocopherols, catechins, aminophe-
nols, and stilbenes related to resveratrol. Their results
demonstrated that in most cases HAT mechanism is
dominant. It is clear that HAT reaction concurs with ET
reaction and plays a dominant role in biological redox
reactions.
44
Antioxidant Capacity Methods
The features of any antioxidant capacity method are an
oxidation initiator, a suitable substrate, and an appropriate
measure of the end point. Initiators may include increased
temperature (Laguerre et al. 2007) and partial pressure of
oxygen, addition of transition metal catalysts (Ou et al.
2002a), exposure to light to promote photosensitized
oxidation by singlet oxygen (Min and Boff 2002; Lee et al.
2004), and variable shaking to enhance reactant contact and
free radical sources (Pulido et al. 2000). However, it should
be realized that the analytical methods of measurement and
the conditions under which the analysis has occurred can
lead to variable results for the same food types (Antolovich
et al. 2002; Nilsson et al. 2005) For example, photosensitized
acceleration underestimates the effects of chain-breaking
antioxidants (Frankel and Meyer 2000) and temperature raise
can change oxidation mechanism.
Several analytical strategies are possible for the end-
point measurements. These include (1) measurement at a
fixed time point; (2) measurement of reaction rate; (3) lag-
phase measurement where the length of the lag time to end-
point change is measured, samples with higher antioxidant
capacity suppress the change longer; and (4) integrated rate
measurement which involves integration of the end point
versus time curve and is used where the reaction kinetics
have no simple order (Antolovich et al. 2002).
Several colorimetric and fluorometric antioxidant capac-
ity assays based on HAT mechanism apply a radical
reaction without a chain propagation step which is an
essential step in lipid autoxidation. Thus, the relevance of
these approaches to radical chain-breaking antioxidant
capacity is disputable. In general, added antioxidant
competes with probes (substrates) for the radicals and
inhibits or retards the probe oxidation. Assays with this
feature include total radical trapping antioxidant parameter
(TRAP) assay, oxygen radical absorbance capacity (ORAC)
assay, and crocin-bleaching assay (Huang et al. 2005)
These assays have the following components: (a) thermo-
labile azo-radical initiator, which gives radicals (R
) that
react fast with O2 to give a steady flux of ROO
radicals
(the most frequently applied radical generators are the
water-soluble AAPH and lipid-soluble AMVN); (b) oxidiz-
able target, a molecular probe (UV or fluorescence) for
monitoring reaction progress; (c) antioxidant, its presence
inhibits or retards the oxidation of probe. Therefore, in the
beginning of the assay, insignificant spectroscopic changes
of the probe would be observed (induction period or lag
phase). As the reaction proceeds, the antioxidants are
consumed by radicals and the oxidation of the probe would
continue at a slower rate compared to control. Finally, when
the antioxidants are depleted, the reaction rate of the probe
oxidation will be similar to that obtained for the control (d)
Food Anal. Methods (2009) 2:41–60
reaction kinetic parameters (Huang et al. 2005; Magalhaes
et al. 2008).
Antioxidant capacity can be measured by the effects of the
antioxidant in controlling the extent of oxidation. Methods
show extreme diversity. The antioxidant capacity assays can
also be based in the peroxyl radical scavenging such as ORAC
(Ou et al. 2001) and TRAP (Schlesier et al. 2002), metal
reducing power such as ferric reducing/antioxidant power
(FRAP; Benzie et al. 1999) and cupric reducing/antioxidant
power (CUPRAC; Apak et al. 2004), hydroxyl radical
scavenging such as deoxyribose assay (Caillet et al. 2007),
organic radical scavenging such as ABTS (Miller et al. 1993)
and DPPH (Rivero-Perez et al. 2007), or quantification of
products formed during lipid peroxidation such as thiobarbi-
turic acid reactive substances (Chumark et al. 2008; Perez-
Jimenez and Sauro-Calixto 2008) and low-density lipoprotein
(LDL) oxidation (Gheldof and Engeseth 2002). The major
difference among these assays is the quantitation approaches.
For example, the ORAC assay applies the area under the
kinetic curve approach; the TRAP assay depends on lag time,
and the crocin-bleaching assay uses initial reaction rate
(Huang et al. 2005).
Oxygen Radical Absorbance Capacity Assay
ORAC measures antioxidant inhibition of peroxyl-radical-
induced oxidations and reflects classical radical chain-
breaking antioxidant activity by H-atom transfer (Ou et al.
2001). In the basic assay, the peroxyl radicals generated from
thermal decomposition of AAPH in aqueous buffer or
hydroxyl radicals generated from Cu2+-H2O2 (Cao et al.
1997) react with a fluorescent probe, an oxidizable pro-
tein substrate, to form a nonfluorescent product, which
can be quantitated easily by fluorescence. Probe reaction
with peroxyl radicals is followed by loss of the intensity
of fluorescence by time. The first version of the ORAC
assay employed B-phycoerythrin (B-PE, a fluorescent
protein) as the probe. B-PE was chosen because of its
excitation wavelengths, high fluorescent yield, sensitiv-
ity to ROS, and water solubility (MacDonald-Wicks et
al. 2006).
In addition to evaluation of antioxidant capacity
samples of fruits and vegetables, dietary supplements,
wines, juices, and nutraceuticals, the ORAC assay have
been used in plasma or serum samples. The protein
fraction of plasma or serum contributes significantly to
the antioxidant capacity, which may mask responses,
particularly if the interest is in small-molecular-weight
antioxidants. Therefore, protein removal is important
(Prior et al. 2003).
In general, samples, controls, and standard (Trolox of
four or five different concentrations for structuring the
standard curve) are mixed with fluorescein solution and
Food Anal. Methods (2009) 2:41–60
incubated at constant temperature (37 °C) before AAPH
solution is added to initiate the reaction (MacDonald-
Wicks et al. 2006). Under this reaction conditions, 1 mol
of AAPH loses a dinitrogen to generate 2 mol of AAPH
radical. In air-saturated solution, the generated AAPH
radical reacts with O2 rapidly to give a more stable


peroxyl radical ROO . The loss of fluorescence of the
probe is an indication of the extent of damage from its
reaction with the peroxyl radical. The fluorescence
intensity [485 nm (ex)/525 nm (em)] is measured every
minute for 35 min at ambient conditions (pH 7.4, 37 °C).
In the presence of antioxidant, the fluorescence decay is
prevented (Ou et al. 2002a).
Advantages
Traditional antioxidant analyses followed extension of
the lag phase only, but antioxidant effects often extend
well beyond early stages of oxidation (Niki 2002; Huang
et al. 2005). For example, glutathione shows no clear lag
phase and thus accurate calculations based solely on a lag
phase are difficult (Cao et al. 1997). The protective effect
of an antioxidant in ORAC assay is measured by the net
integrated area under the fluorescence decay curve of the
sample compared to that of blank (AUCsample −AUCblank)
and stands for lag time, initial rate, and total inhibition in a
single value (Prior et al. 2005). To avoid underestimation
of antioxidant capacity and to account for potential
effects of secondary antioxidant products, the ORAC
assay follows the reaction for extended periods, for
example, ≥30 min.
The advantage of AUC approach is that it operates
equally well for both antioxidants that exhibit distinct
lag phases and those that have no lag phases. It is
particularly useful for samples which often contain
multiple ingredients and have complex reaction kinetics.
The FL probe is inexpensive and the use of a fully
automated microplate fluorescence reader makes the
method readily accessible and the efficiency of the
assay is enhanced with at least a tenfold increase in
sample throughput (Huang et al. 2002b).
Another advantage of ORAC assay is the capability of
employing different free radical generators or oxidants. This is
important because the measured antioxidant capacity of a
compound depends on which free radical or oxidant is used in
the assay. For example, the ORAC assay using AAPH (an


ROO generator) measures all traditional antioxidants
including ascorbic acid, α-tocopherol, β-carotene, glutathi-
one, bilirubin, uric acid, and melatonin while the ORAC


assay using Cu2+-H2O2 (mainly an OH generator) measures
compounds like mannitol, glucose, uric acid, and transition
metal chelators but not ascorbic acid and α-tocopherol (Cao
et al. 1997). ORAC assay measures only the antioxidant
45
capacity against peroxyl and hydroxyl radicals not that
against all reactive oxygen species (e.g., superoxides and
singlet oxygen; Apak et al. 2004).
Disadvantages
The use of B-PE in antioxidant assays has some limitations
such having large interbatch differences, photobleaching of
B-PE after exposure to the excitation light, and interaction
with polyphenols by nonspecific protein binding. These
factors cause inconsistency in assay results and false low
values. To solve these disadvantages, Ou et al. (2001)
replaced B-PE with FL. FL is a synthetic nonprotein probe
and overcomes the shortcomings of B-PE. It is more stable
and less reactive. However, FL is pH sensitive and this
must be carefully monitored (MacDonald-Wicks et al.
2006). In addition, the reaction products of FL with peroxyl
radical have been characterized, and the product pattern
was consistent with a classic HAT reaction mechanism
(Prior et al. 2005).
As originally designed, the ORAC assay is limited to
measurement of hydrophilic chain-breaking antioxidant
capacity against only peroxyl radicals. However, many
antioxidants are lipophilic and are particularly important
against lipid oxidation in all systems as well as other
radicals that are very active physiologically. It is also
known that the antioxidant capacity of a compound is
dependent on reaction media; therefore, an organic
solvent-based ORAC assay has been adapted to measure
lipophilic samples, too. To solve these disadvantages,
Huang et al. (2002a) developed an assay for lipophilic
components using randomly methylated β-cyclodextrin as
a solubility enhancer, which allows for the measurement
of the antioxidant capacity of both hydrophilic and
lipophilic components in a given sample separately using
the same radical source. To obtain an accurate total ORAC
value of a given sample, both hydrophilic and lipophilic
fractions need to be measured (Wu et al. 2004).
However, FL is not sufficiently lipid soluble, and its
fluorescence intensity in nonpolar organic solvent is
rather low. To overcome this problem, 4,4-difluoro-3,5bis
(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene
(BODIPY 665/676) is employed as a fluorescent probe
and AMVN as a peroxyl radical generator (Naguib 2000).
AMVN has the advantage of being highly soluble in
toluene, methanol, ethanol, and hexane (Laguerre et al.
2007). By applying this assay, the antioxidant capacity of
various carotenoids was quantified. However, this assay is
100 times less sensitive than the classical ORAC assay,
probably due to the low efficiency of the radical generator,
AMVN. In addition, the fluorescent quenching mechanism
of BODIPY by peroxy radical remains to be investigated
(Huang et al. 2005).
46
Total TRAP Assay
Total TRAP assay monitors the ability of antioxidant
compounds to interfere with the reaction between
peroxyl radicals generated by AAPH or ABAP and a
target probe (Schlesier et al. 2002). The TRAP assay
uses R-phycoerythrin (R-PE) as a fluorescent probe and
the reaction progress of R-PE with AAPH was monitored
fluorometrically [(ex) 495 nm and (em) 575 nm)].
Valkonen and Kuusi (1997) applied dichlorofluores-
cein-diacetate (DCFH-DA) as the fluorescent oxidizable
substrate (probe). The oxidation of DCFH-DA by peroxyl
radicals yields the formation of highly fluorescent
dichlorofluorescein (DCF) product (ex 480 nm, em
526 nm) and also has absorbance at 504 nm. This enabled
the determination of serum TRAP by simple spectropho-
tometry. The presence of antioxidant compounds compet-
itively inhibits the increase of fluorescence signal. Adom
and Liu (2005) also developed a rapid and sensitive
hydrophilic peroxyl radical capacity (hydro-PSC) assay
incorporating DCHF-DA as a fluorescent probe and
modified the hydro-PSC assay as a lipophilic peroxyl
radical scavenging capacity (lipo-PSC) assay, using
randomly methylated β-cyclodextrin to increase the
solubility of lipophilic compounds.
Wolfe and Liu (2007) developed a method to measure
antioxidant capacity in cell culture. Cellular antioxidant
activity (CAA) assay is a valuable new method and has
advantages over the traditional chemistry antioxidant assays
because it is a more biologically relevant model. The assay
utilizes HepG2 human hepatocarcinoma cells because they
give consistent results with lower coefficient of variation.
Cells are pretreated with antioxidant compounds and
DCFH-DA. The antioxidants bound to the cell membrane
and/or passed through the membrane to enter the cell.
DCFH-DA diffused into the cell where cellular esterases
cleaved the diacetate moiety to form more polar DCFH,
which was trapped within the cell. Cells were also treated
with ABAP or AAPH which was able to diffuse into cells.
They spontaneously decompose to form peroxyl radicals.
These radicals attack the cell membrane to yield more
radicals and oxidized the intracellular DCFH to the
fluorescent DCF and the level of fluorescence measured
upon excitation is proportional to the level of oxidation. In
addition to peroxyl radicals and hydrogen peroxides,
various other species (e.g., peroxynitrite, nitric oxide,
superoxide radicals) have been found to oxidize DCFH to
DCF in cell culture. In a study, where the CAA values of 25
fruits were determined, it has been shown that the CAA
values for fruits were significantly positively related to total
phenolic content and ORAC values (Wolfe et al. 2008).
However, this is in contrast to a study of Eberhardt et al.
(2005) involving broccoli extracts which showed that no
Food Anal. Methods (2009) 2:41–60
correlation was found between ORAC and cellular antiox-
idant capacity methods.
Advantages
TRAP assay involves the initiation of lipid peroxidation by
generating water-soluble peroxyl radicals and is sensitive to
all known chain-breaking antioxidants; the concept has
been very useful for quantifying and comparing antioxidant
capacity.
Disadvantages
The method’s greatest problem is perhaps its greatest
strength; too many different end points have been used,
so comparisons between laboratories are difficult. For
example, the method suffered from the drawback of
oxygen electrode end point in that the electrode would
not maintain its stability over the required time period
(up to 2 h per sample; Apak et al. 2007). TRAP assay is
relatively complex and time-consuming to perform,
requiring a high degree of expertise and experience
However, the TRAP assay has been criticized as employ-
ing water-soluble peroxyl radicals (Frankel and Meyer
2000), but the method can be adapted to use lipid-soluble
initiators (Prior et al. 2005).
Another shortcoming of TRAP assay is the use of the
lag time that corresponds to the inhibition of accumu-
lation of colored radical reagents in the presence of
antioxidants (e.g., the time period required for the
colored radical to emerge in the reaction medium; Apak
et al. 2007) for quantifying antioxidant capacity since not
every antioxidant has an obvious lag phase (Magalhaes et
al. 2008).
Moreover, the value obtained from the lag phase alone
often underestimates antioxidant capacity considerably
because the antioxidant value obtained after the lag phase
is totally ignored (Prior et al. 2005). Another important
limitation of this assay and also of the ORAC assay is that
the oxidative deterioration and antioxidant protection of
fluorescent probe does not mimic a critical biological
substrate (Frankel and Meyer 2000).
In TRAP assay, the probe must be reactive with
ROO
at low concentrations and to maximize the sensi-
tivity there must be a dramatic spectroscopic change
between the native and oxidized state of probe, and no
radical chain reaction beyond probe oxidation should
occur (Prior et al. 2005).
β-Carotene or Crocin-Bleaching Assay
Carotenoids bleach via autoxidation induced by light or
heat or peroxyl radicals (e.g., AAPH or oxidizing lipids).
Food Anal. Methods (2009) 2:41–60
The addition of an antioxidant-containing sample, individ-
ual antioxidants, or plant extracts causes the inhibition of
β-carotene bleaching (Laguerre et al. 2007). The assay
measures the decrease in the rate of β-carotene or crocin
decay provided by antioxidants. Color loss followed
optically at 443 nm in phosphate buffer (pH 7.0), so no
special instrumentation is required. The bleaching rate
becomes linear at ∼1 min after the addition of AAPH and
is monitored for 10 min (Huang et al. 2005).
Advantages
The kinetic approach in the measurement allows the
determination of total inhibiting effects and provides a
more precise evaluation of the efficiency of antioxidant
defense, but two independent parameters, the reactivity
and antioxidant capacity, cannot be determined sepa-
rately. Later, a modified microplate-based version suit-
able for routine determinations was suggested (Roginsky
and Lissi 2005).
Disadvantages
Although β-carotene is often used as the target (probe), its
discoloration at 470 nm can occur by multiple pathways, so
interpretation of results can be difficult. To overcome this
problem, carotenoid derivative crocin, a natural compound
with extremely strong absorbance in visible range, has
become the reagent choice. Crocin has straightforward
reactions and undergoes bleaching only under the attack of
peroxyl radical (Prior et al. 2005).
It is not clear in the presence of some antioxidants,
such as Trolox, ascorbic acid, or uric acid whether a lag
phase, or a period of maximal protection of crocin from
oxidation by peroxyl radicals, can be seen. These
antioxidants usually show a lag phase in the assay
systems that use lipids or proteins as oxidizable
substrate for peroxyl radicals. The antioxidant capacity
of ascorbic acid analyzed using this kinetic method was
reported to be 7.7 Trolox equivalents (Tubaro et al.
1998), much higher than those obtained with any other
methods (Prior and Cao 1999).
This assay has found limited applications in food
samples so far because crocin is not available commer-
cially and must be extracted from saffron which may
result to a low reproducibility between assays. More-
over, crocin absorbs at a rather short wavelength
(450 nm), and many food pigments, such as carote-
noids, absorb at the same wavelength. To eliminate
possible interferences from the sample itself, a blank (a
mixture containing only AAPH and food sample) must
be tested at the same time under the same wavelength
(MacDonald-Wicks et al. 2006). Ordoudi and Tsimidou
47
(2006) have examined the crocin-bleaching assay perfor-
mance and validation procedures.
Total Phenol Assay by Using the Folin-Ciocalteu Reagent
The exact chemical nature of the Folin-Ciocalteu reagent
is not known, but it is accepted that it contains
phosphomolybdic/phosphotungstic acid complexes. The
chemistry behind the FCR assay counts on the transfer of
electrons in alkaline medium from phenolic compounds
and other reducing species to molybdenum, forming blue
complexes that can be monitored spectrophotometrically
at 750–765 nm (Magalhaes et al. 2008). The phenolic
compounds react with FCR only under basic conditions
(adjusted by a sodium carbonate solution to pH∼10;
MacDonald-Wicks et al. 2006). Since Singleton and Rossi
extended this assay to the analysis of total phenols in
wine, it has found many applications (Huang et al. 2005).
They also specified the conditions to minimize the
variability and eliminate inconsistent results: (1) proper
volume ratio of alkali and FCR, (2) optimal reaction time
and temperature for color development, (3) monitoring of
optical density at 765 nm that allows us to minimize
interference from the sample matrix, which is often
colored, (4) use of reference standards such as gallic acid
(Prior et al. 2005).
Advantages
Despite the undefined chemical nature of FCR, the total
phenol assay by FCR is convenient, simple, and reproduc-
ible (Huang et al. 2005). Several publications applied the
total phenol assay by FCR and antioxidant capacity assay
(e.g., DPPH, FRAP, TEAC, ORAC etc.) and often found
excellent linear correlations between the “total phenolic
profiles” and “the antioxidant activity” (Gheldof and
Engeseth 2002; De Beer et al. 2003; Madhujith et al.
2006; Shahidi et al. 2006; Stratil et al. 2006).
Disadvantages
Instead of recommended gallic acid reference standard
catechin equivalents (Vinson et al. 2001; Katsube et al.
2003), tannic acid equivalents (Nakamura et al. 2003),
chlorogenic acid equivalents (Wang et al. 2003b), caffeic
acid equivalents (Maranz et al. 2003), and ferulic acid
equivalents (Velioglu et al. 1998; Stratil et al. 2006) have
also been used. Lack of standardization of methods can lead
to several orders of magnitude difference. The final
absorbance values are usually proportional to the number
of reacting phenolic hydroxyl groups and also depend on
the molecule structure. If the standard used for calibration is
highly reactive and gives a high absorbance, then the
48
measured values of samples will be low. For example, the
absorbance value for caffeic acid (two reacting OH) is
approximately twice and for catechin (three reacting OH)
three times higher than that for phenol (one reacting OH;
Stratil et al. 2006). The choice of standard as a “critical
control point” of the antioxidant capacity assays is
examined in Nenadis et al. (2007) in detail.
The FCR-based assay has been known as the total
phenol assay. The FCR reagent is not specific for phenolic
compounds as it can also be reduced by many nonphenolic
compounds (MacDonald-Wicks et al. 2006). The total
phenol assay by FCR is carried out in water, an aqueous
phase. For lipophilic antioxidants, this assay in its current
form is not applicable. The FCR actually measures a
sample’s reducing capacity, but this is not reflected in the
name “total phenolic assay.” There is always the disagree-
ment on what is being detected in total antioxidant capacity
assays, whether only phenols or phenols plus reducing
agents plus possibly metal chelators.
TEAC Assay
TEAC assay was first reported by Miller et al. (1993) and
has been improved and widely used in testing antioxidant
capacity in food samples. ABTS is a peroxidase substrate,
which when oxidized by peroxyl radicals or other oxidants
in the presence of H2O2 generates a metastable radical
cation ABTS
þ ) which is intensely colored and can be
monitored spectrophotometrically in the range of 600–
750 nm. The antioxidant capacity is measured as the ability
of test compounds to decrease the color reacting directly
with ABTS
þ radical and expressed relative to Trolox
(Roginsky and Lissi 2005).
The order of addition of reagents and sample was
modified in the improved version. The sample to be tested
was added after generation of a certain amount of radical
cation and the remaining radical cation concentration after
reaction with antioxidant compound/sample was then
quantified to minimize the interference of compounds with
oxidants during radical formation and prevent the possible
overestimation (Magalhaes et al. 2008).
In further modified methods that utilize ABTS radical in
common, ABTS was generated by enzymatic (e.g., perox-
idase, myoglobin) or chemical reactions (manganese diox-
ide, potassium persulfate, ABAP; Schlesier et al. 2002).
Generally, chemical reactions require a long time (e.g., up
to 16 h for potassium persulfate generation) or high
temperatures (e.g., 60 °C for ABAP generation), whereas
enzyme generation is faster and the reaction conditions are
milder (Prior et al. 2005).
The TEAC assay measures the antioxidant capacity of
the parent compound plus that of reaction products. These
reaction products may have considerable contribution to the
Food Anal. Methods (2009) 2:41–60
TEAC. For example, the relatively high TEAC of chrysin
compared to its moderate antioxidant activity is due to the
formation of reaction products that also scavenge ABTS
þ .
The TEAC assay is often used to rank antioxidants and for
the construction of structure activity relationships (Arts et
al. 2004). There is no relation between the TEAC value and
the number of electrons that an antioxidant can donate
(MacDonald-Wicks et al. 2006).
Advantages


ABTS þ can be solubilized in both aqueous and in organic
media and is not affected by ionic strength, so the
antioxidant capacity can be measured due to the hydrophilic
and lipophilic nature of the compounds (Arnao 2000).
TEAC method which is based on the oxidation of ABTS in
the presence of H2O2 and metmyoglobin only measures
hydrophilic antioxidants. Preparation of ABTS radical by
filtering ABTS solution through manganese dioxide pow-
der allows the assay to measure lipophilic antioxidants like
carotenoids and tocopherols. By changing the solvent from
water to ethanol, TEAC method also enables the measure-
ment of both kinds of antioxidants (Schlesier et al. 2002).
TEAC assay is operationally simple and has been
used in many research laboratories. Absorption maxima
were shown to be at wavelengths of 414, 752, and
842 nm in aqueous media and 414, 730, and 873 nm in
ethanolic media (Arnao 2000). Another advantage of
TEAC assay is that it permits study over a wide pH range.
Although frequently used at pH 7.4, the stability of ABTS
radical at this pH has been reported to be problematic. For
standard antioxidants such as Trolox or ascorbic acid,
ABTS radical at pH 7.4 provided reliable end-point values
after 10 min. However, with standard phenolics, the
results at 10 min are estimates only and do not represent
equilibrium end-point values based on oxidation. Also,
with ABTS radical at pH 7.4, values for the antioxidant
capacity of the standard phenolics were 5–20% greater
than the values determined at pH 4.5 (Ozgen et al. 2006).
Disadvantages
As for the limitations of this method, TEAC value actually
characterizes the capability of the tested sample to react with


ABTS þ rather than to inhibit the oxidative process. The
TEAC reaction with many phenolics and samples of natural
products may take a long time to reach an end point. Thus,
by taking this fixed end point of short duration (4 or 6 min),
false low TEAC values can be read before the reaction is
finished (Huang et al. 2005). As studied by Perez-Jimenez
and Saura-Calixto (2008), comparison between the antioxi-
dant capacity obtained at a fixed end point and the kinetics
behavior of the samples, when both the concentration and the
Food Anal. Methods (2009) 2:41–60
time necessary to deplete the radical are considered, shows
some differences. For example, BHA shows higher antiox-
idant capacity than ferulic acid when results are obtained at a
fixed end point, but it has low value indicating slower
kinetics than ferulic acid. The fact that the ABTS radical
used in TEAC assays is not found in biological systems and
is not similar to radicals found in those systems is also
another problem (MacDonald-Wicks et al. 2006).
DPPH Radical Scavenging Capacity Assay
The DPPH radical is a long-lived organic nitrogen radical
and has a deep purple color. It is commercially available
and does not have to be generated before assay. In this
assay, the purple chromogen radical is reduced by antiox-
idant/reducing compounds to the corresponding pale yellow
hydrazine. The reducing ability of antioxidants towards
DPPH can be evaluated by electron spin resonance or by
monitoring the absorbance decrease at 515–528 nm until
the absorbance remains stable in organic media. This
widely used method was first reported by Brand-Williams
et al. (1995). The percentage of the DPPH (%DPPHrem)
remaining is calculated as:
 antioxidants that may react quickly with peroxyl
%DPPHrem ¼ 100  ½DPPHrem ½DPPHT ¼0
%DPPHrem is proportional to the antioxidant concentra-
tion, and the concentration that causes a decrease in the
initial DPPH concentration by 50% is defined as EC50. The
time needed to reach the steady state with EC50 is defined
as TEC50.
Based on TEC50 values, the kinetic behavior of the
antioxidant is classified as follows: <5 min (rapid), 5–
30 min (intermediate), and >30 min (slow; Sanchez-
Moreno et al. 1998). Sanchez-Moreno et al. (1998) further
introduced another parameter to express antioxidant capac-
ity, called “antiradical efficiency” [AE=(1/EC50) TEC50]
which is more discriminative than TEC50 and more useful
because it takes into account the reaction time.
A similar and new parameter designed as “radical
scavenging efficiency (RSE), combining scavenging activ-
ity in terms of the amount of radicals scavenged and initial
scavenging rate, was suggested by De Beer et al. (2003).
RSE is calculated as the ratio of the initial scavenging rate
(obtained during the first minute) and the EC50 value. The
main limitation of determination is that the percentage of
scavenged radical is dependent on of the initial DPPH
radical concentration.
Advantages
The DPPH assay is technically simple and rapid and needs
only a UV-Vis spectrophotometer that might explain its
49
widespread use in antioxidant screening. Analyses of a
large number of samples could be made by using micro-
plates (Fukumoto and Mazza 2000).
Disadvantages
There are some drawbacks which limit its application. DPPH
can only be dissolved in organic media (especially in alcoholic
media), not in aqueous media, which is an important limitation
when interpreting the role of hydrophilic antioxidants (Arnao
2000). Although widely used for measuring and comparing
the antioxidant status of phenolic compounds and foodstuffs,
the evaluation of antioxidant capacity by the changes in
DPPH absorbance should be carefully inferred since absor-
bance of DPPH radical at 517 nm after reaction with an
antioxidant is decreased by light (Ozcelik et al. 2003),
oxygen, and type of solvent (Apak et al. 2007). It was
concluded that above a certain limit of water content of
solvent, the antioxidant capacity decreased, since a part of
the DPPH coagulates and it is not easily accessible to the
reaction with antioxidants (Magalhaes et al. 2008).
DPPH molecule has little similarity to the highly
reactive and transient peroxyl radicals. Also, many
radicals in vivo may react slowly or may even be inert
to DPPH. The reaction kinetic between DPPH and
antioxidants is not linear to DPPH concentration;
therefore, to express antioxidant capacity using EC50 is
problematic. Moreover, interpretation of the results is
complicated if the test compounds have spectra that
overlap DPPH at 515 nm (e.g., in particular, carotenoids;
Prior et al. 2005). It was also reported that the reaction of
DPPH with eugenol was reversible (Huang et al. 2005).
This would result in falsely low readings for antioxidant
capacity of samples containing eugenol and other phenols
bearing a similar structure.
Originally, it was believed that the DPPH assay was a
hydrogen transfer reaction but recent studies suggested
otherwise (MacDonald-Wicks et al. 2006). The rate-
determining step for this reaction occurs very quickly as
initial electron transfer and subsequent hydrogen transfer
occurs very slowly and depends on the neutral hydrogen-
bond accepting solvent such as methanol and ethanol
(Huang et al. 2005) In addition, basic and acidic impurities
in the solvent may influence the ionization equilibrium of
phenols and therefore cause a reduction or enhancement of
the measured rate constants (MacDonald-Wicks et al.
2006).
FRAP Assay
FRAP assay is based on the ability of phenolics to reduce
yellow ferric tripyridyltriazine complex (Fe(III)-TPTZ) to
50
blue ferrous complex (Fe(II)-TPTZ) by the action of
electron-donating antioxidants (Benzie et al. 1999). The
resulting blue color measured spectrophotometrically at
593 nm is taken as linearly related to the total reducing
capacity of electron-donating antioxidants. Ferric salt is
used as an oxidant and its redox potential (<0.70 V) is
comparable to that of ABTS (0.68 V). Therefore, essential-
ly, there is not much difference between TEAC assay and
the FRAP assay except TEAC is carried out at neutral pH
whereas FRAP assay needs acidic (nonphysiologically low
pH value=3.6) conditions to maintain the iron solubility.
One FRAP unit is defined as the reduction of 1 mol of Fe
(III) to Fe(II) (Huang et al. 2005).
Advantages
FRAP assay is simple, rapid, inexpensive, and robust and
does not require specialized equipment. The FRAP assay
can be performed using automated, semiautomatic, or
manual methods (Prior et al. 2005).
Disadvantages
Fe(II) is a well-known “prooxidant.” It can react with H2O2
to produce hydroxyl radical (OH
), the most harmful free
radical found in vivo. The ability of a compound to produce
Fe(II) from Fe(III) defined as “antioxidant power” in the
FRAP assay because it is probable that some antioxidants,
such as ascorbic acid and uric acid, can reduce both reactive
species and Fe(III) and their ability in reducing Fe(III) may
reflect their ability in reducing reactive species. But not all
reductants that are able to reduce Fe(III) are antioxidants
(Prior and Cao 1999). Any electron-donating substance
even without antioxidant properties with redox potential
lower than that of the redox pair Fe(III)/Fe(II) can
contribute to the FRAP value and indicate falsely high
values (Nilsson et al. 2005). In addition, an antioxidant that
can effectively reduce prooxidants may not be able to
efficiently reduce Fe(III). For example, the FRAP assay
does not measure GSH (glutathione), an important antiox-
idant in vivo (Prior and Cao 1999).
Potential problems take place as the mixture contains
other Fe(III) species, which can bind to chelators in the
food extract and these complexes are capable of reacting
with the antioxidants. Results show that, similar to
TEAC, there is no relation between the FRAP value and
the number of electrons that an antioxidant can donate
(MacDonald-Wicks et al. 2006). The FRAP values for
ascorbic acid, α-tocopherol, and uric acid are identical
(2.0). The FRAP value of bilirubin is onefold higher than
that of ascorbic acid. These results suggest that 1 mol of
ascorbic acid can reduce 2 mol of Fe(III) and 1 mol of
bilirubin can reduce 4 mol of Fe(III). This is in conflict
Food Anal. Methods (2009) 2:41–60
with the fact that both ascorbic acid and bilirubin are both
two-electron reductants (Huang et al. 2005).
FRAP cannot detect compounds that act by radical
quenching (H transfer), particularly thiols and proteins (Ou
et al. 2002a). This causes a serious underestimation in
serum. Both the FRAP and TEAC assays rely on the
hypothesis that the redox reactions proceed so rapidly that
all reactions are complete within 4 and 6 min, respectively,
but in fact this is not always true. Pulido et al. (2000)
measured the FRAP values of several polyphenols in water
and methanol. However, for polyphenols with such behav-
iors including caffeic acid, tannic acid, ferulic acid, ascorbic
acid, and quercetin, the absorption (A593) does not stop at
4 min; instead, it slowly increased even after several hours
of reaction time. Thus, determination of antioxidant
capacity by utilizing a fixed end point may not represent
a completed reaction.
Total Antioxidant Potential Assay Using Cu(II)
as an Oxidant
The method is based on reduction of Cu(II) to Cu(I) by
reductants (antioxidants) present in a sample. It has
been introduced as Bioxytech AOP-490 and CUPRAC
developed by Apak et al. (2004). In the Bioxytech AOP-
490 assay, bathocuproine (2,9-dimethyl-4,7-diphenyl-
1,10-phenanthroline) forms a 2:1 complex with Cu(I),
producing a chromophore with maximum absorbance at
490 nm. Reaction rate and concentration of products are
followed by bathocuproine complexation of the Cu(I)
produced (Prior et al. 2005).
The CUPRAC assay uses the copper(II)-neocuproine
reagent as the chromogenic oxidant. It involves mixing
the antioxidant solution (directly or after acid hydroly-
sis) with solutions of CuCl2, neocuproine, and ammoni-
um acetate at pH 7, and measuring the absorbance at
450 nm after 30 min. Flavonoid glycosides needed to be
hydrolyzed to their corresponding aglycons for fully
exhibiting their antioxidant capacity. Slow-reacting anti-
oxidants needed increased temperature incubation to
complete their oxidation with the CUPRAC reagent (Apak
et al. 2008).
Advantages
CUPRAC values are comparable to TEAC values for
polyphenols, whereas FRAP values are usually consider-
ably lower (Apak et al. 2004). Due to the lower redox
potential of the CUPRAC reagent, reducing sugars and
citric acid—which are not true antioxidants but oxidizable
substrates in other similar assays, e.g., FRAP—are not
oxidized with the CUPRAC reagent (Apak et al. 2008). At
the same time, the low redox potential enhances redox
Food Anal. Methods (2009) 2:41–60
cycling, so copper reduction may be and even more
sensitive indicator of potential prooxidant activity of
antioxidants (Prior et al. 2005).
Other advantages of the CUPRAC method over other
similar assays are: (a) the CUPRAC reagent is fast enough to
oxidize thiol-type antioxidants, whereas the FRAP method
does not measure certain thiol-type antioxidants like glutathi-
one; (b) the reagent is more stable and accessible than other
chromogenic reagents (e.g., ABTS, DPPH); (c) easily and
diversely applicable in rutin laboratories; (d) the absorbance
versus concentration curves are linear over a wide range,
unlike those of other methods yielding polynomial curves; (d)
the redox reaction yielding colored species is carried out at
pH 7 buffer as opposed to the acidic conditions of FRAP
(pH 3.6) or basic conditions of the Folin-Ciocalteu assay
(pH 10); (e) the method can concurrently measure hydrophilic
and lipophilic antioxidants (unlike FCR and DPPH; Apak et
al. 2008). CUPRAC assay is complete in minutes for
ascorbic acid, uric acid, gallic acid, and quercetin but
requires 30–60 min for more complex molecules.
Disadvantages
The copper reduction assays still have similar problems
with a complex mixture of antioxidants in terms of
selecting an appropriate reaction time (Prior et al. 2005).
Methods that Measure the Inhibition of Induced Lipid
Autoxidation
This method artificially induces autoxidation of linoleic acid
or LDL particles or serum proteins without prior isolation of
LDL particles (Gheldof and Engeseth 2002), by either a
transition element such as Cu(II) or thermal decomposition
of AAPH, a water-soluble diazo ROO
initiator, and
peroxidation of the lipid components determined through
the formation of conjugated dienes, followed at 234 nm
spectrophotometrically (Hendelman et al. 1999). The use of
AAPH as peroxyl radical generator is preferred over Cu(II)
since it bears a strong resemblance to oxidative reactions in
biological systems (Magalhaes et al. 2008). Basically, three
mechanisms are involved in LDL antioxidant activity; free
radical scavenging activity, binding to critical sites on LDL,
and metal chelation (Katsube et al. 2004). In general, in vitro
LDL oxidation has been used effectively in several
researches to characterize antioxidant capacity of a number
of different phytochemicals (Hu et al. 2000; Katsube et al.
2004; Shahidi et al. 2006; Chumark et al. 2008).
Advantages
The reaction can be carried out in micelles or in organic
solvents. In micelles, reaction progress cannot be followed
51
directly by a UV spectrophotometer and sample workup is
necessary; this limits the efficiency of the method (Huang
et al. 2005). On a limited group of samples, a good
relationship was observed between LDL oxidation using
AAPH and the ORAC value (Hendelman et al. 1999);
however, the relationship was not present when Cu(II) was
used as the oxidant.
Disadvantages
Problems arise because it is difficult to measure the small lag
times that may occur, and many substances in foods also
absorb at 234 nm. Additionally, linoleic acid will form
micelles in the presence of water that can be overcome by
using methyl esters, although products of methyl esters can
show interbatch differences which are not desired in a
chemical quantitation method and further complicate the
assay because the antioxidant distribution between two phases
is critical (MacDonald-Wicks et al. 2006).
Cu(II) alone does not induce the autoxidation of lipids.
Instead, the reaction was initiated by antioxidants (e.g.,
tocopherols) present in the LDL. Flavonoids might present
antioxidant or prooxidant activities depending on the radical-
generating system and concentration (Hassimotto et al. 2005).
The method has a major drawback that LDL must be
isolated on a regular basis, and obtaining blood samples
from different individuals is necessary. Thus, it is not
possible to get consistent preparations. According to Prior
et al. (2005), this method is not much suitable for the
development of a consistent reproducible high-throughput
assimilable organic carbon (AOC) assay.
Chemiluminescence Assay
The general principle of these methods is based on the
ability of luminol and related compounds to lumines-
cence under the attack of free radicals (chemilumines-
cence (CL); Roginsky and Lissi 2005). This reaction
produces low-intensity light emission that may decay
rapidly. The addition of para-iodophenol gives a more
intense, prolonged, and stable light emission. The light
emission is interfered by antioxidants but will be restored
when all the added antioxidants have been depleted (Prior
and Cao 1999). Oxidant sources of peroxyl radicals
include the enzyme horseradish peroxidase (Whitehead et
al. 1992) and H2O2-hemin (Bastos et al. 2003). Luminol is
the most widely used marker compound to trap oxidants
and convert weak emissions into intense, prolonged, and
stable emissions (Whitehead et al. 1992), although
lucigenin and bioluminescent proteins such as Pholasin®
are becoming more popular. Pholasin emits light in the
presence of different systems capable of generating free
radicals such as superoxide and hydroxyl radical (but not
52
peroxyl radical) and certain oxidants (Reichl et al. 2001;
Jaffar et al. 2006).
The addition of the antioxidant/sample brings on a CL-
lag phase (time interval which CL emission was not
monitored). The magnitude of the lag phase was directly
related to the antioxidant concentration or it results to the
decrease of CL emission, expressed as inhibition percent-
age (Magalhaes et al. 2008).
Advantages
By changing the initiator, the reaction can be altered to
differentiate quenching of specific oxidants, for example,





O2 , HO , HOCl, ROO , OONO, and 1O2. CL can be
quite sensitive in detecting low-level reactions because it
provides a detectable response below the detection limit of
most chemical assays (Prior et al. 2005). The promising
characteristic of CL methods is their productivity; com-
monly, one run takes a few minutes only; in addition, the
assay can be easily automated and run in microwell plates
(Roginsky and Lissi 2005).
Disadvantages
CL detection needs special equipment that both set samples
close to the detector and detects light at single-photon levels
and, in addition, provides temperature control. The mecha-
nism for chemical processes resulting in CL is not known in
detail which may create problems with interpreting data
obtained (Roginsky and Lissi 2005). The choice of emitter is
also a critical consideration. The use of luminol is acceptable
when single oxidants are being measured. However, because
the intensity of emissions changes considerably with the
oxidant, use of luminol in systems with mixed oxidants is
not straightforward (Prior et al. 2005).
Photochemiluminescence Assay
This assay was described by Popov and Lewin, was
commercialized by Analytik Jena AG (Jena, Germany), and
is commercialized as a complete system under the name
PHOTOCHEM® (Prior et al. 2005). This assay involves the
photochemical generation of superoxide O
 2 free radicals
combined with CL detection. The optical excitation of a
photosensitizer results in the generation of the superoxide
radical. The free radicals are monitored with a CL reagent.
Luminol acts as a photosensitizer as well as an oxygen
radical detection reagent.
Advantages
PCL-based methods principally differ from other antioxi-
dant evaluation procedures because they do not require
Food Anal. Methods (2009) 2:41–60
oxidizing reagents for the generation of radical species
(Vertuani et al. 2004). In contrast to other commonly used
AOC assays, the PHOTOCHEM method is not limited to a
specific pH value or temperature range (Prior et al. 2005).
PCL assay has been used effectively in several investiga-
tions to characterize antioxidant activity (Madhujith et al.
2006; Sclesier et al. 2002). Other advantages of PCL are: it
does not require high temperatures to generate radicals and
it is more sensitive (nanomolar range) in measuring, in few
minutes (Vertuani et al. 2004).
Disadvantages
The weak correlation between ORAC and PCL and FRAP
and PCL is not unexpected because different radical
sources are being evaluated (Sclesier et al. 2002). The
complete reaction mechanism is not known which makes
the interpretation of results difficult.
Total Oxidant Scavenging Capacity Assay
Total oxidant scavenging capacity (TOSC) assay is based on
oxidation of α-keto-γ-methiolbutyric acid (KMBA) to ethyl-
ene by peroxyl radicals produced from AAPH. The antioxi-
dant capacity of a molecule is quantified from its ability to
inhibit ethylene formation relative to a control. The time
duration of ethylene formation is followed by gas chromatog-
raphy (GC) headspace (Regoli and Winston 1999).
The approach for quantification of antioxidant capacity is
based in the area under the curve that defines the inhibition of
ethylene formation as a function of time, which can be up to
300 min (Huang et al. 2005). The antioxidant or TOSC value
is from 0 to 100 (Prior and Cao 1999).
This method points out an important issue in terms of being
able to evaluate different antioxidants with different biolog-
ically relevant radical sources, peroxyl radicals, hydroxyl
radicals, generated by the reaction of iron and ascorbate, and
peroxynitrite generated from 3-morpholinosydnonimine N-
ethylcarbamide (Regoli and Winston 1999; Prior et al. 2005).
For example, reduced glutathione was an efficient scavenger
of peroxyl radicals but a relatively poor scavenger of
peroxynitrite and hydroxyl radicals. Uric acid, Trolox, and
ascorbic acid were comparable scavengers of peroxynitrite
and peroxyl radicals. TOSC assay is useful and robust in
distinguishing the reactivity of various oxidants and the
relative capacities of antioxidants to scavenge these oxidants
(Regoli and Winston 1999).
Advantages
In addition to reproducibility, the main advantages of the
TOSC assay are its effectiveness in detecting the scaveng-
ing capacity of both hydrophilic and lipophilic antioxidants
Food Anal. Methods (2009) 2:41–60
and its ability to distinguish between fast- and slow-acting
antioxidants (Regoli and Winston 1999).
Disadvantages
The long reaction time (>100 min), the instability of the
assay solutions, and the necessity of multiple manual
injections from the same sample into a gas chromatograph
to monitor the ethylene formation make this assay not
adaptable to routine analysis (Huang et al. 2005).
Measurement of Other Reactive Oxygen/Nitrogen
Species
Although HAT- and ET-based methods, designed to measure
a sample’s capacity to react with peroxyl radical, are most
frequently employed in the determination of antioxidant
capacity, the methods including other ROS have to be
designed to comprehensively evaluate the antioxidant capac-
ity of a sample. This fact is implied in several studies
(Kumaran and Karunakaran 2006; Madhujith et al. 2006;
Mathew and Abraham 2006; Rivero-Perez et al. 2007)
The main ROS and RNS causing an oxidative damage in


biological tissues are superoxide anion (O2 ), hydroxyl


radical (HO ), hydrogen peroxide (H2O2), peroxyl radicals


(ROO ), singlet oxygen (1O2), peroxynitrite (ONOO−),



nitric oxide (NO ), and nitric dioxide (NO2 ). Living cells
have a biological defense system consisting of enzymatic
antioxidants that convert ROS to harmless species. For


example, O2 is converted to oxygen and hydrogen
peroxide by superoxide dismutase (SOD) or reacts with


nitric oxide (NO ) to form peroxynitrite. H2O2 can be
converted to water and oxygen by catalase enzyme.


Superoxide anion (O2 ) is a reduced form of molecular
oxygen by receiving one electron. Superoxide is a relatively
weak oxidant; therefore, the reason for the discovery of


compounds that scavenge O2 is that this radical can
decompose to form more potent and reactive ROS such as


the hydroxyl radical ( OH) and peroxynitrite (ONOO−),
singlet oxygen which initiate the lipid oxidation. Flavonoids,
including epicatechin, myricetin, rutin, catechin, epigalloca-
techin, quercetin, galangin, and morin can scavenge O2



(Hort et al. 2008), but the scavenging of O2 is not the only
mechanism for inhibition of lipid oxidation in either
biological or lipid food systems (Frankel and Meyer 2000).
The analytical methods for determination of O2

scavenging capacity employ enzymatic or nonenzymatic


O2 generation systems. Enzymatic system uses xanthine
oxidase (XOD)/hypoxanthine or xanthine to generate O2

radicals at pH 7.4 (Madhujith et al. 2006). In normal tissue,
XOD is a dehydrogenase enzyme that transfers electrons to
nicotinamide adenine dinucleotide. During times of stress,
53
this enzyme is converted to an oxidase enzyme and
produces superoxide and hydrogen peroxide. Nonenzymat-
ic system utilizes a nonenzymatic reaction of phenazine
methosulfate in the presence of nicotinamide adenine
dinucleotide (Rivero-Perez et al. 2007).
Detection methods may include spectrophotometric
measurements using nitroblue tetrazolium (NBT), cyto-
chrome c, hydroethidine (HE) as fluorescent probes in
redox reactions; CL-based determinations; GC and electron
spin resonance (ESR) spectroscopy.
In spectrophotometric measurements that uses NBT as


probe, O2 may reduce NBT into formazan and be
measured at 560 nm. Antioxidant compounds compete


with NBT for O2 and decrease the reaction rate.
Another spectrophotometric detection method competi-



tion utilizes kinetics of O2 reduction of cytochrome c


(probe) and O2 scavenger (sample). The kinetic analysis of
reduction of ferricytochrome c to ferrocytochrome c was
monitored at 550 nm (Aruoma et al. 1993). Cytochrome c
can be reduced directly by antioxidants, which can also
inhibit the xanthine oxidase. This is an important issue as
far as ferricytochrome c is concerned, as it is easily reduced
by ascorbic acid. Furthermore, the reduced antioxidant


formed by attack of O2 could also reduce NBT or
ferricytochrome c. Therefore, this method is not suitable
for quantifying nonenzymatic antioxidant (Huang et al.
2005; Magalhaes et al. 2008).
Another spectrophotometric detection method uses HE
as a redox sensitive probe. Nonfluorescent HE is oxidized


by O2 generated from XOD/xanthine system to form a
fluorescent marker product that has signal at 586 nm. The
oxidation mechanism still remains unclear (Benov et al.
1998; Zhao et al. 2003). This approach can avoid the
problem of direct reduction of the probe by antioxidants,
but possible inhibition of xanthine oxidase by antioxidants
(sample) still exists (Magalhaes et al. 2008).


In CL-based determination of O2 scavenging capacity,


a pale yellow solution of O2 in dimethyl sulfoxide
(DMSO), obtained from potassium superoxide and 18-
crown-6, stable at room temperature for at least 1 h, emits
a high CL. This reaction was considered a good method
for examining antioxidative or prooxidative properties of
many biologically important compounds (Aboul-Enein
et al. 2005).
In GC detection method, the reaction product of KMBA

 

and O2 , ethylene, is measured. O2 is generated by using
XOD/xanthine-generating system (Lavelli et al. 1999).



The scavenging capacity of O2 can also be detected by


using ESR spectroscopy. Here, the O2 is trapped by 5,5-
dimethyl-1-pyrroline-N-oxide (DMPO), and the resultant
DMPO superoxide adduct (DMPO-OOH) is detected by
ESR using manganese oxide as the internal standard
(Calliste et al. 2001). A major disadvantage of DMPO is
54
that the DMPO superoxide adduct (DMPO-OOH) sponta-
neously decomposed to the DMPO hydroxyl adduct
(DMPO-OH) that makes spectral interpretation more
confusing. To overcome this potential drawback, Zhang et
al. (2000) employed 5-ethoxycarbonyl-5-methyl-pyrroline-
N-oxide (EMPO) to trap superoxide radical. The superoxide
adduct of EMPO (EMPO-OOH) does not spontaneously
decompose its corresponding hydroxyl adduct; thus, its
electron spectra is less complex and easier to interpret.
Rivero-Perez studied the antioxidant profile of 321
wines by superoxide radical and hydroxyl radical scaveng-
ing assays and compared the results to TEAC, DPPH,
FRAP, and ORAC methods. While superoxide radical
scavenging assay showed positive correlations with ABTS,
DPPH, and FRAP, hydroxyl radical scavenging assay
showed negative correlations with ORAC. These results
confirm the hypothesis that different mechanisms are
involved in capturing both types of radicals or their related
compounds. The negative correlation implies that, in
general, the wines with the greatest ability to transfer
hydrogen atoms are the least effective at stabilizing the
hydroxyl radical (Rivero-Perez et al. 2007).
Hydrogen peroxide (H2O2) can be generated through a
dismutation reaction from superoxide dismutase. Enzymes
such as amino acid oxidase and xanthine oxidase also
produce hydrogen peroxide from superoxide anion. Hydro-
gen peroxide is regarded as poorly reactive because of its
weaker oxidizing and reducing capabilities; it is stable
under physiological pH and temperature in the absence of
metal ions. Biologically, H2O2 is converted to oxygen and
water by catalase. It can generate the hydroxyl radical in the
presence of metal ions and superoxide anion. It also can
produce singlet oxygen through reaction with superoxide
anion or with HOCl or chloramines in living systems.
Hydrogen peroxide can degrade certain heme proteins, such
as hemoglobin, to release Fe ions (Lee et al. 2004).
In the most common methods for detection of H2O2,
scavenging capacity depends on the change of the absor-
bance value at 230 nm when H2O2 concentration is
decreased by scavenger compounds. Nevertheless, there
might be possible interferences from samples that also
absorb at the same wavelength; thus, performance of a
“blank” measurement is measured (Magalhaes et al. 2008).
Other common assay employs horseradish peroxidase
which uses H2O2 to oxidize scopoletin into a nonfluores-
cent product. In the presence of antioxidants, the oxidation
is inhibited and this reaction can be fluorimetrically
monitored (Huang et al. 2005).
Another widely used fluorimetric method is based on
homovanillic acid (Magalhaes et al. 2008). The nature of
the inhibition is ambiguous because there are several
potential inhibition pathways. The antioxidants can inhibit
the reaction by (a) reacting directly with H2O2, (b) reacting
Food Anal. Methods (2009) 2:41–60
with intermediates formed from enzyme and H2O2, or (c)
inhibiting the horseradish peroxidase from binding H2O2.
Therefore, it is difficult to explain the actual chemical
meaning of the data (Martinez-Tome et al. 2001).
Singlet oxygen (1O2) is a nonradical and excited state of
molecular oxygen that has no unpaired electrons and it is
known to be a powerful oxidizing agent, reacting directly
with a wide range of biomolecules. Singlet oxygen is
normally generated in the presence of light and photo-
sensitizers. It was reported that metastable phosphatidyl-
choline hydroperoxides present in the living organism also
produced singlet oxygen during their breakdown in the
presence of Cu2+ in the darkness (Lee et al. 2004).
The singlet-oxygen detection during photosensitized
oxidation of foods is difficult due to the short lifetime.
One of the most common detection methods is ESR
spectroscopy, which is highly sensitive for the detection
of free radicals. A spin-trapping agent such as 2,2,6,6-
tetramethyl-4-piperidone (TMPD) can react with singlet
oxygen to form a stable radical nitroxide radical adduct
2,2,6,6-tetramethyl-4-piperiodine-N-oxyl (TAN) which is
measured by ESR. Although other ROS such as superoxide
and hydroxyl radicals can react with TMPD, they do not
convert TMPD to TAN. This method is highly specific to
singlet oxygen (Min and Boff 2002).
Singlet oxygen can be physically scavenged by transferring
its excitation energy to another molecule. β-carotene is an
excellent physical scavenger of 1O2. The energy difference
between singlet oxygen and ground-state oxygen can be
released as a 1,270 nm photon, which is very specific to
singlet oxygen. The decay rate of the light intensity can be
used to measure the singlet-oxygen scavenging ability of
compounds (MacDonald-Wicks et al. 2006). Nevertheless,
the intensity of luminescence based on the self-emission of
1
O2. is often insufficient to provide reproducible quantitative
information (Magalhaes et al. 2008).
A more sensitive method is reported where the quench-
ing of singlet-oxygen-sensitized delayed fluorescence of
tetra-t-butylphthalocyanine at 703 nm is monitored. Al-
though this method may provide sensitive measurement
where the 1,270-nm luminescence is difficult to detect, it is
not widely applied since it requires specialized equipment
(Huang et al. 2005). The assay was applied to compounds
which are well known as singlet-oxygen quenchers such as
β-carotene, α-tocopherol, and lauric acid (Magalhaes et al.
2008). Cholesterol reacts with singlet oxygen to form
specific oxidative products via the “ene” reaction. This
makes use of cholesterol as an effective indicator of singlet-
oxygen oxidation where the use of other detection methods
is difficult (Min and Boff 2002).


Hydroxyl radical (HO ) is the most reactive free radical
known and can react with everything in living organisms.
These short-lived species can hydroxylate DNA, proteins,
Food Anal. Methods (2009) 2:41–60
and lipids. It can be formed from superoxide anion and
hydrogen peroxide in the presence of metal ions such as
copper or iron. When a hydroxyl radical reacts with
aromatic compounds, it can add on across a double bond,
resulting in hydroxycyclohexadienyl radical. The resulting
radical can undergo further reactions, such as reaction with
oxygen, to give peroxyl radical or decompose to phenoxyl-
type radicals by water elimination (Lee et al. 2004).
The direct scavenging of the hydroxyl radical by dietary
antioxidants in a biological system is not meaningful
because the cellular concentration of dietary antioxidants
is negligible and very high concentrations of scavenger are
required to compete in vivo or in the food matrix for any


HO generated. On the other hand, it is possible to prevent
the formation of hydroxyl radicals by either deactivating
free metal ions [e.g., Fe(II)] through chelation or converting
H2O2 to other harmless compounds (such as water and
oxygen). For example, catalase converts H2O2 to O2 and
H2O, and metal chelators bind metal ions so that they
become inert toward H2O2. Thus, dietary nutrients perform
in this way would act as preventive antioxidants (Huang et al.
2005; Magalhaes et al. 2008).


Moore et al. (2006) reviewed available HO -generating
systems and classified them into five categories including (1)
the classic Fenton reaction including the pH 7.4 buffered
ferric iron–EDTA/ascorbic acid/H2O2 system used in the


“deoxyribose assay.” HO attacks unsaturated membrane
lipids to form malonaldehyde, which may be detected by its
ability to react with thiobarbituric acid (TBA) to form a pink
chromogen. The role of ascorbic acid is to reduce the Fe(III)
to Fe(II) and thus to favor the Fenton reaction (Caillet et al.
2007); (2) the superoxide-driven Fenton reaction using the
hypoxanthine/xanthine oxidase system to generate HO ; (3)

Fenton-like systems including the Co(II)/H2O2 (Aboul-Enein
et al. 2005) and the Cu(II)/H2O2 systems (Cao et al. 1997);
(4) pulse radiolysis of water (Joshi et al. 2005); (5) ultraviolet
excitation of compounds such as pyridine-N-oxides, often
referred to as “photoFenton” reagents. The generation of
hydroxyl radicals from such reagents has been identified by
trapping with coumarin-carboxylic acid and the consequent
formation of the highly fluorescent 7-hydroxycoumarin
carboxylate (Botchway et al. 2007). There are also some
researches in literature that have used γ-irradiation to


produce HO radicals (Yoshioka et al. 2001; Saran and
Summer 1999).
Biologically, the hydroxyl radical is widely believed
to be generated when hydrogen peroxide reacts with Fe
(II) ion (Fenton reaction). However, when the sample is
mixed with Fe(II), it may alter the activity of Fe(II) by
chelation. So, it is impossible to distinguish if the
antioxidants are simply good metal chelators or HO
scavengers. To solve this problem, a chelating agent like
diethylenetriaminepentaacetic acid (DETAPAC) was usu-
55
ally added to the solution. However, antioxidants like
EGCG have strong chelating activity, so the effect of the
addition of DETAPAC is doubtful. In addition, antiox-
idants may consume H2O2. Actually, it is known that
EGCG scavenges H2O2. This action also disturbs the


HO -generating system (Yoshioka et al. 2001).


The detection methods of HO radicals may include the
separation by high-performance liquid chromatography
(HPLC) combined with electrochemical or UV detectors,
pulse radiolysis, fluorometric methods, ESR, spectrophoto-
metric detection, capillary electrophoresis (CE), and CL-
based determination methods.
The hydroxylated products generated from the reac-
tion of hydroxyl radical with aromatic compounds such
as benzoic acid or salicylic acid are separated by HPLC
and detected with either electrochemical or UV detec-
tion (Saran and Summer 1999). Though this method is
handy and sensitive, the multiple reaction products make
it complicated to quantitative detection of hydroxyl
radical. Hantzsch reaction method and TBA method are
simple and do not need expensive instruments; however,
both of the reactions need high temperature (60–100 °C)
and long reaction time (30–60 min; Zou et al. 2002).
Moreover, the organic mobile phase of the HPLC assay, in


which HO is generally detected by determining the
dihydroxylated benzoic acid products derived from sali-
cylic acid, will possibly cause environmental pollution and
harm to health (Cheng et al. 2003).
Zhu et al. (2000) reported a metal-independent organic


Fenton reaction that production of HO by tetrachlorohy-
droquinone (a major metabolite of the widely used biocide
pentachlorophenol) in the presence of H2O2 was obtained.


Salicylate was used as an HO trap, which forms the
hydroxylation products 2,3- and 2,5-dihydroxybenzoic acid
(DHBA). The DHBA derivatives can be separated by HPLC
and detected with an electrochemical detector. This method
is extremely sensitive and detects DHBAs in the picomolar


range. The process was inhibited by HO scavenging agents
without being affected by iron chelators. This new assay may
provide a more direct way of measuring hydroxyl radical
scavenging capacity of antioxidants.
“Pulse radiolysis” is the most often used technique for


measuring the reaction between HO and antioxidants,
requires specialized equipment, and can be expensive
(Huang et al. 2005). The radiolysis and photosensitization


systems do not measure the HO scavenging capacity
under physiologically relevant conditions. The hypoxan-
thine/xanthine oxidase system generates superoxide in


addition to OH and may have interference from enzyme


inhibitors, which result in overestimation of HO scav-

enging capacity (Moore et al. 2006). The methods using
the generation of hydroxyl radicals from photoFenton
reagents have a potential problem. One-photon excitation
56
of photoFenton reagents such as 2-hydroxypyridine-N-
oxide and 2-mercaptopyridine-N-oxide has their short
wavelength (UV) absorption maxima at ∼310 and
∼330 nm, respectively, at physiological pH. Such wave-
lengths are absorbed by several biochemical chromo-
phores and have limited penetration in tissues. The use
of multiphoton excitation in the near infrared (approxi-
mately 700–900 nm) has the potential to avoid these
problems (Botchway et al. 2007).
Ou et al. (2002a, b) developed a fluorometric method
termed as hydroxyl (HO) radicals averting capacity
(HORAC) for screening the metal chelating capacity of
dietary antioxidants. HO
was generated by a Co(II)-complex-


mediated Fenton-like reaction and the HO formation under
the experimental conditions was indirectly confirmed by the
hydroxylation of p-hydroxybenzoic acid measured by
HPLC–mass spectrometry. FL was used as the probe. The
fluorescence decay curve of FL is monitored in the absence
or presence of antioxidants; the quantitation method is the
same with ORAC assay except gallic acid is used as the
standard. This method has been validated for linearity,
precision, accuracy, and ruggedness. HORAC and ORAC
assays measure two different but equally important aspects of
antioxidant properties; the HORAC primarily reflects metal
chelating radical prevention ability, and the ORAC reflects
peroxyl radical absorption capacity. Therefore, no correlation
found between HORAC and ORAC values is expected.
Another simple and sensitive fluorometric hydroxyl
radical detection method is described. This method


employs the reaction between HO and DMSO to
produce formaldehyde, which then reacts with ammonia
and 1,3-cyclohexanedione at pH 4.5. The product yields
the characteristic fluorescence with its excitation and
emission wavelength at 400.4 and 452.3 nm, respectively.
The quantitative analysis of hydroxyl radical can be done
through the determination of the fluorescence intensity.
The temperature of 95 °C seems to be the major drawback
of this method and make it unsuitable for the analysis of
some unstable biological materials (Tai et al. 2002).
The other novel hydroxyl radical scavenging capacity
named HOSC was described and validated by Moore et
al. (2006). The HOSC assay uses FL as the probe. HO is
employed to monitor peroxynitrite in various systems.
generated under physiological pH using a Fenton-like
reaction with a definite end point. The experimental
conditions were evaluated and confirmed using electron
spin resonance with DMPO spin-trapping measurement.
HOSC assay has acceptable sensitivity, ruggedness,
precision, and accuracy. Compared to the commonly used
deoxyribose method, the HOSC method has better
compatibility with acetone and more efficiently generates
hydroxyl radicals under physiological pH.
ESR spectroscopy measures the electron paramagnetic
resonance spectrum of a spin adduct derivative after trapping.
Food Anal. Methods (2009) 2:41–60
This technique involves the inclusion in the experimental
system of a nitrone or nitroso compound (spin trap) that can


react with a short-lived free radical such as HO to generate a
long-lived nitroxide free radical (spin adduct). The spin
adduct is usually detected by ESR spectroscopy. The two
nitrone spin traps that have found wide use are DMPO
(Cheng et al. 2007) and phenyl-tert-butylnitrone (Ma et al.
1999). In the spin-trapping method with DMPO, the ESR
signal of the DMPO-OH adduct formed by the Fenton
reaction disappeared rapidly in the presence of Fe(II) ions,
and this quenching effect was enhanced by phosphate ions
(Li et al. 2004). Besides, the expensive instruments make it
unsuitable for routine analysis.
The use of high-performance CE to determine HO

generated by Fenton reaction was reported. CE is an
important analytical separation technique due to its speed,
efficiency, reproducibility, ultrasmall sample volume, and
ease of clearing up the contaminants. In addition, with
electrochemical detection (ED), CE-ED (Cao et al. 2003)
and amperometric detection (Wang et al. 2003a) offer
higher sensitivity than CE-UV detection.
The CL-based determination of scavenging capacity


against OH using luminol has also been described. The
methods take advantage of the transient properties of the
Fenton reaction and chemiluminescent reaction between

OH and luminol. However, other ROS, including O2 and

H2O2, are generated at the same time, so it is difficult to


detect OH specifically with this detection process (Cheng
et al. 2003; Chen et al. 2006).
Peroxynitrite (ONOO−), the reaction product of nitric
oxide (NO) and superoxide, is a potent and relatively long-
lived oxidant with its high diffusibility across cell mem-
branes. Its protonated form, peroxynitrous acid (ONOOH),
is a very strong oxidant. The main route of damage is the
nitration or hydroxylation of aromatic compounds, partic-
ularly tyrosine. Under physiological conditions, peroxyni-
trite also forms an adduct with carbon dioxide dissolved in
body fluid. The adduct is believed to be responsible for the
oxidative damage of proteins.
Two fluorogenic probes, DCFH and dihydrorhodamine-
123 (DHR-123), considered to be ideal, have been widely
These methods are based on the oxidation of the reduced
nonfluorescent forms of fluorescent dyes such as fluores-
cein and rhodamine by peroxynitrite to produce the parent
dye molecule, resulting in a dramatic increase in fluores-
cence response. Rhodamine derivatives have the advan-
tages of having great photostability, pH insensitivity over a
broad range (low to neutral pH), and giving high quantum
in aqueous solution and being excitable at long wavelength
(Yang et al. 2002). Although these assays are commonly
used, there are some debates in the literature concerning the
mechanism of peroxynitrite-mediated oxidation of fluores-
Food Anal. Methods (2009) 2:41–60
cent indicators. These fluorogenic indicators are also less
suitable for peroxynitrite formation in vivo (Glebska and
Koppenol 2003) and the synthesis of these probes is rather
difficult and inconvenient. Additionally, the use of organic
dyes is very likely to result in environmental contamination
and usually should be avoided (Liang et al. 2005).
A fluorometric method using folic acid as a fluorescent
probe has been proposed because folic acid can act as a
peroxynitrite scavenger. Folic acid is a low-fluorescent
substance, but the oxidation of folic acid by peroxynitrite
can give high-fluorescent emission product. Compared with
the commonly used probe DHR-123, the fluorescent probe
reported here has some advantages including higher
sensitivity that is desirable for probing of peroxynitrite in
lower concentrations, greater photostability, commercial
availability, and not being toxic to biological system
(Huang et al. 2007).
Another novel spectrofluorimetric method for the deter-
mination of peroxynitrite uses hemoglobin (Hb) as a
catalyst and thiamine (Cao et al. 2005) or l-tyrosine as the
substrate (Liang et al. 2005). Hb is used as the mimetic
enzyme for substituting horseradish peroxidase for two
reasons: (1) for the purpose of the mildness of reaction
conditions and the minimization of cost and (2) Hb is a
kind of nature protein in human body and it is much likely
to coexist with peroxynitrite in cell or organism (Liang et
al. 2005). Other peroxynitrite determination methods are
direct voltammetric detection at a mercury film electrode at
alkaline pH (Zakharova et al. 2007) and luminol-enhanced
chemiluminescence methods (Magalhaes et al. 2008).
Conclusion
In this review, numerous antioxidant capacity methods are
reported; they differ from each other in terms of reaction
mechanisms, oxidant and target/probe species, reaction
conditions, and expression of results. When selecting the
most appropriate method, it should be considered that
analysis conditions, substrate, and concentration of antiox-
idants should simulate real food or biological systems as
much as possible. Antioxidant activity occurs by different
mechanisms which means employing a method depending
on one mechanism may not reflect the true antioxidant
capacity. The total antioxidant capacity value should
include assays applicable to both lipophilic and hydrophilic
antioxidants and regards the similarity and differences of
both HAT and ET. The assays including various ROS/RNS


such as superoxide anion (O2 ), hydroxyl radical (HO ),

hydrogen peroxide (H2O2), singlet oxygen (1O2), peroxyni-


trite (ONOO−), nitric oxide (NO ), nitric dioxide (NO2 )
Calliste CA, Trouillas P, Allais DP, Simon A, Duroux JL (2001) Free
have to be designed to comprehensively evaluate the
antioxidant capacity of a sample.
57
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