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The Folin–Ciocalteu assay revisited: improvement of its

specificity for total phenolic content determination
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Cite this: Anal. Methods, 2013, 5, 5990

Juan Carlos Sánchez-Rangel,a Jorge Benavides,a J. Basilio Heredia,b Luis Cisneros-

Zevallosc and Daniel A. Jacobo-Velázquez*a

Received 8th July 2013
Accepted 8th August 2013
DOI: 10.1039/c3ay41125g
www.rsc.org/methods
This study presents a review of the Folin–Ciocalteu (F–C) assay for total phenolic content (TPC)
determinations and describes different approaches to improve its specificity. Phenolics are regarded as
the molecules with the highest potential to neutralize free radicals. Therefore, their quantification is a
common practice in different areas of food research. However, when determining TPC in plant food
extracts, the presence of reducing interferants [ascorbic acid (AA)] produces inaccurate estimations of
TPC values. Different methodologies have been proposed to improve the specificity of the F–C assay.
These methodologies include: (i) the use of solid phase extraction (SPE) cartridges to separate
interferants from phenolics; (ii) the calculation of a corrected TPC value based on the AA reducing
activity present in the extract; and (iii) the pre-treatment of extracts with oxidative agents prior to TPC
quantification. These methods are described in detail in the present study. Likewise, their advantages
and disadvantages are discussed based on new experimental data. A simple modification of the F–C
assay procedure is proposed to quantify both the TPC value and the AA reducing activity in plant food
extracts. Values obtained by the modified F–C assay can be used to estimate a corrected TPC value.

1 Introduction most commonly used procedure to determine TPC of food

Scientic interest in phenolic compounds (PC) as chemo-
preventive and therapeutic agents against several chronic
diseases was stimulated in the late 1990s, when the
French paradox (dened as the low occurrence of coronary heart
diseases despite diets rich in cholesterol and saturated fat) was
attributed to the high intake of red wine polyphenols by the
French population, specically to resveratrol which is present in
red grape skins.1–4 According to the Scopus database, since 1995
more than 49 000 articles that included the word phenolics in
their title, abstract or keywords have been published, indicating
the high impact of PC on different aspects of research.
PC are the major contributors to the antioxidant capacity of
fruits, vegetables, and grains.5,6 Therefore, the quantication of
PC is a common practice when selecting genotypes, maturity
stages, storage and processing conditions that allow the
production of fresh and processed food products with high
potential to protect against free radicals.7–10 The F–C assay is the
a
Centro de Biotecnologı́a-FEMSA, Department of Biotechnology and Food Engineering,
School of Biotechnology and Food, Tecnológico de Monterrey-Campus Monterrey, E.
Garza Sada 2501 Sur, C.P. 64849, Monterrey, N.L., México. E-mail: djacobov@
itesm.mx; Fax: +52-818-328-4136; Tel: +52-818-358-20-00 ext. 4820
b
Centro de Investigación en Alimentación y Desarrollo, A.C. Unidad Culiacán,
Postharvest Physiology and Quality Laboratory, Carretera a El Dorado Km. 5.5,
Apartado Postal 32-A, Culiacán, Sinaloa, 80129 México
c
Department of Horticultural Sciences, Texas A&M University, Vegetable & Fruit
Improvement Center, College Station, Texas 77843-2133, USA
extracts. The F–C assay is a colorimetric method based on elec-
tron transfer reactions between the F–C reagent and PC. However,
the F–C assay is not specic for TPC determinations. It is known
that other types of compounds that may be present in high
abundance in plant food extracts (i.e. reducing sugars and AA) can
also reduce the F–C reagent, skewing the results of TPC.11,12
Therefore, different methodological approaches to improve
the specicity of the F–C assay have been proposed. Those
methodological approaches include: (i) the partial purication of
phenolic extracts using SPE columns before the F–C assay is
performed;13 (ii) the calculation of a corrected TPC value by sub-
tracting the AA reducing activity from the TPC quantied in the
plant food extract14 and (iii) the treatment of phenolic extracts
with oxidative agents such as hydrogen peroxide (H2O2) at levels
that oxidize the interfering compounds and do not affect PC.15
In this article different methodological approaches to
improve the specicity of the F–C assay for total PC determi-
nations are reviewed. Advantages and disadvantages of each
method are discussed and supported with new experimental
data obtained from plant food extracts and from model systems
prepared with phenolic and interfering compounds.
2 Folin–Ciocalteu (F–C) assay background
and theory
The F–C assay16 was generated in order to improve the Folin and
Denis (F–D) assay,17 which was designed to indirectly determine

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total protein concentration by measuring the content of tyro-
sine and tryptophan. The principle of both methods is based on
the reaction between the oxidant reagent and tyrosine/trypto-
phan, resulting in blue color formation proportional to the
concentration of protein. The main difference between the F–C
and F–D assays is the proportion of molybdate (Mo) used to
prepare the reagent. Folin and Ciocalteu increased the Mo
content to prevent the formation of a white precipitate observed
in the F–D assay.17 The F–C assay is more sensitive and repro-
ducible than the F–D assay.18
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The application of both assays has been extended to deter-
mine total PC in plant food extracts. Swain and Hillis19 adapted
the F–D assay to determine the total PC content in
Prunus domestica, whereas Singleton and Rossi18 adapted the
F–C assay to determine total PC in wine. Both assays are widely
used by the scientic community. Indeed, the procedures
developed by Swain and Hillis19 and Singleton and Rossi18 have
been cited in scientic papers more than 1900 and 5500 times,
respectively. Furthermore, the method has been recently adap-
ted to measure lipophilic antioxidants.20
The F–C reagent is prepared by rst dissolving 100 g of
sodium tungstate (Na2WO4$2H2O) and 25 g of sodium Mo
(Na2MoO4$2H2O) in 700 mL of distilled water. Then, the
solution is acidied with 50 mL of concentrated HCl and
50 mL of 85% phosphoric acid. The acidied solution is boiled
for 10 h, cooled, and 150 g of Li2SO4$4H2O is added. The
resultant intense yellow solution is the F–C reagent.11,21
Although the chemical nature of the F–C reagent has not been
elucidated, it is believed to be composed of heteropoly-phos-
photungstates/molybdates.11 Likewise, the exact chemical
nature of the F–C reaction that leads to a blue species [possibly
(PMoW11O40)4
] is unknown and likely to remain so due to its
complexity.21 However, it is assumed that the F–C reaction
involves sequences of reversible one- or two-electron reduction
reactions.11,21,22 From the components of the F–C reagent,
molybdates are more easily reduced than tungstates, and thus
it is suggested that most of the electron-transfer reactions in
the assay are between the reductants and the molybdates as
shown in eqn (1).11,21 During the F–C assay, the reaction
between PC and the F–C reagent takes place at a pH of 10,
which is reached by adding sodium carbonate. Under those
basic conditions, dissociation of a phenolic proton leads to the
formation of a phenolate ion, which is capable of reducing the
F–C reagent.11,21
2.1 Interference during the analysis of total phenolic
content (TPC) by the Folin–Ciocalteu (F–C) assay
As mentioned earlier, crude plant extracts may contain inter-
fering substances (other reducing compounds) that can react
with the F–C reagent, skewing the results for TPC
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determinations. Among those reducing compounds, AA, dehy-
droascorbic acid (DHA), and reducing sugars (i.e. glucose and
fructose) have the highest impact on hampering the accuracy of
the assay.12,21
The presence of AA is generally a problem when determining
the total PC of extracts obtained from fruits such as orange,
kiwifruit and strawberry, which have signicant concentrations
of vitamin C.23 Under acidic conditions of the F–C reagent
(pH  3), AA and DHA (both enediols) rapidly react with poly-
phosphotungstate, giving a blue color right aer mixing the
plant extract with the reagent (eqn (2)). Indeed, the observation
of blue color before the addition of the alkali indicates the
presence of AA, DHA or other reducing compounds not
requiring the phenolate form to reduce the F–C reagent. Like-
wise, it has been suggested that AA could have an augmenting
effect on the amount of F–C reagent reacting with PC, because it
may reduce the quinones formed during the assay. However,
this augmenting effect is not generally accepted, because during
the assay, AA is rapidly oxidized before the addition of the alkali
and subsequent oxidation of phenolics takes place.21 DHA is the
rst oxidation product of AA and it is naturally present in fruits
and vegetables, especially in those where the activity of poly-
phenol oxidase (PPO) is high and AA is used as a substrate to
reduce quinones.24 DHA is an enediol and thus it also produces
blue color under acidic conditions during the F–C reaction.21
The presence of reducing sugars is a problem when deter-
mining the TPC of extracts obtained from fruits where the TPC
is low. In these cases most of the response (blue color forma-
tion) may be generated by the sugars present in the sample. The
interference produced by reducing sugars comes from the
enediol reductones formed under the alkali conditions of the
assay (eqn (3)).21
3 Methodological approaches to improve
specificity of the Folin–Ciocalteu (F–C) assay
Different procedures have been proposed to reduce the
response of interfering compounds when performing the F–C
assay for TPC determinations in plant extracts. The methods
include: (i) the partial purication of PC by using SPE
cartridges; (ii) the calculation of a corrected TPC by subtracting
the AA reducing activity from the TPC quantied; and (iii) the
treatment of phenolic extracts with oxidative agents prior to F–C
assay performance, in order to oxidize interferants. In the
following sections each of these methodological approaches are
described.
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3.1 Partial purication of phenolic extracts by solid phase
extraction (SPE)
Biological extracts usually contain a wide variety of different
solutes, aside PC, which may generate a positive colorimetric
response in the F–C assay. This generates an overestimation of
the TPC in the sample. Therefore, the partial purication of
such extracts represents a strategy for eliminating the inter-
ference of such reducing solutes during TPC determinations.
SPE is a commonly used technique for obtaining fractions
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enriched in PC while eliminating interferants. This fraction-
ation technique involves the interaction and adsorption of
analytes (solubilized in a liquid phase) in a solid matrix. This
interaction occurs based on the physicochemical and
biochemical properties (hydrophobicity, electrochemical
charge, size, etc.) of the solutes and the matrix.25 Therefore, the
selection of the solid phase to be used is related to the char-
acteristics of the analytes based on the expected fractionation
behavior. In this regard, the partial purication of phenolic
extracts utilizing SPE has been conducted mainly using reverse
phase or other hydrophobicity-based matrixes.26,27 Reverse
phase SPE has been used for the fractionation and recovery of
PC in wine,28,29 plant tissues,30 fruits and vegetables,31 and
juices,32 among others. The use of SPE-based strategies allows
in most cases the elimination of reducing interferants (i.e.
reducing sugars, organic acids) in food samples resulting in a
more accurate estimation of the content of PC (as well as other
analytes of interest).13,32–35
With the aim to obtain a more accurate estimation of TPC
using the F–C assay, Georgé et al.13 developed a method to
eliminate water-soluble interferants in plant food crude extracts
using an Oasis HLB (hydrophilic-lipophilic balance) cartridge.
The procedure consisted in determining TPC in the crude
extract by the F–C assay. Thereaer, the crude extract (con-
taining phenolics and interferants) was passed through the HLB
matrix. The interferants were eluted from the cartridge with
water, and this eluted fraction was analyzed also by the F–C
assay. Based on the hypothesis of the authors, the difference
between the response of the crude extract and the eluted polar
fraction represented the TPC in the original sample (Fig. 1).
This subtraction approach aims at achieving a more accurate
estimation of TPC from biological samples.
Certainly SPE is a well-characterized technique with
advantages such as selectivity,26,32 reproducibility,36 and a wide
diversity of solid matrixes (i.e. C18, HLB, MCX, etc.) to separate
compounds based on their physicochemical characteristics.27
However, this method also has some disadvantages that need
to be considered. PC are very diverse in nature and vary within
a wide range of hydrophobicity (from very polar to fairly
hydrophobic), while in most cases the reducing interferants
usually found in biological samples are primordially hydro-
philic. Therefore, it is difficult (or even impossible in some
cases) to select a solid matrix capable of fractionating polar
phenolic compounds from the polar interferants. For instance,
low retention yields are usually achieved for phenolic acids
(hydroxycinnamic and hydroxybenzoic acids as well as their
derivatives) when using SPE.27 This is due to the weak
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Fig. 1 Estimation of total phenolic content (TPC) by subtraction of reducing
interferants using solid phase extraction. The crude extract is introduced into the
SPE column where phenolics are retained due to interactions with the solid
matrix. The reducing interferants are eluted in the most polar fraction, which is
analyzed using the Folin–Ciocalteu (F–C) assay. The response from this fraction is
subtracted from the response of the crude extract, resulting in an estimation of
the TPC.
interactions between these compounds and the solid
matrix.37,38 This unintended elution of phenolic acids along
with the interferants may cause an underestimation of TPC.13
Therefore, the effectiveness of this SPE-aided TPC determina-
tion strategy would depend on factors related to the prole of
PC and interfering compounds, creating the necessity for a
case-to-case optimization process. Furthermore, in order to
prevent saturation of the solid matrix it is necessary to have a
priori information regarding the range of concentration of both
the phenolics and interferants. It is always possible to use
unnecessarily high solid matrix volumes in order to prevent
saturation. However, this usually generates lower recovery
yields of the product of interest while promoting nonspecic
interactions between undesirable solutes and the surface of the
matrix. Additionally, depending on the solid phase selected
and the solutes being fractioned, the use of SPE may require
using a strong organic solvent during the elution stage.
Organic solvents (such as diethyl ether and ethyl acetate) need
to be removed using reduced pressure and/or high tempera-
ture before F–C assay is conducted since most of them are not
compatible with the F–C assay.33 Although a vacuum drying
step may be used for solvent removal, this process induces
losses in the products of interest due to hydrolysis, isomeri-
zation, and polymerization at temperatures above 40  C.33
Once the organic solvent is removed, the sample may be
reconstituted with methanol before analysis. Likewise, the
reuse of the cartridge can result in decreased reproducibility
because some polymeric polyphenols can be retained in the
matrix, resulting in weaker interactions between PC and the
cartridge.27,39 Based on this, the cartridge may be used for a
limited number of times before being replaced,13 generating
further drawbacks from the economical and methodological
point-of-view. These disadvantages imply that although the use
of SPE-aided strategies may be effective in some cases, the
amount of work involved in their optimization and standard-
ization based on the characteristics of the sample being
analyzed is not trivial.
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3.2 Calculation of a corrected total phenolic content (TPC)
value by subtracting the reducing activity of interferants
present in the plant crude extract
The subtraction of reducing activity of interferants from the
TPC value obtained in plant food extracts has been proposed as
an approach to obtain a better estimation of TPC. This approach
has been reported to deduct the contribution of sugars21,40 and
vitamin C13 during TPC determinations, which are the most
important interfering compounds present in fruits and vegeta-
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bles that react in the F–C assay.13,21,33
As suggested by Slinkard and Singleton,40 the corrected TPC
values in sweet wines can be obtained by adding sugar to the
standard solutions (i.e. gallic acid solution) equivalent to the
level in the samples. Corrections to be subtracted from the TPC
found in sweet wine were determined by preparing standard
curves of gallic acid (GA) added with glucose–fructose (1 : 1) at
different levels. In addition, those sugars were added to dry
wines prior to analysis. The authors observed that fructose was
the sugar that produced more blue color formation during the
F–C assay. Unfortunately, using correction factors to eliminate
the response given by sugars when performing the F–C assay
would be quite impractical because it would be necessary to
determine the exact concentration of the different sugars in
each biological sample. In order to determine the individual
sugars content it is usually necessary to utilize sophisticated
laboratory equipment such as an HPLC coupled to a refractive
index detector. Once they are determined, standard solutions
containing the exact concentrations of sugars present in the
extract must be prepared.
Isabelle et al.14 proposed the subtraction of AA contribution
to TPC results in order to obtain a more accurate quantication
of TPC in common vegetables. In this approach, AA was rst
determined by a HPLC method. In addition, an AA standard was
tested for TPC using the F–C assay and it was found that AA
possesses a reducing activity of 0.872 mg of GA equivalents per g
AA. To obtain the corrected TPC of the vegetables analyzed, the
authors multiplied the AA content of the sample by 0.872 and
the result was subtracted from the TPC obtained. This strategy
could be simpler if a specic spectrophotometric method for AA
determinations would be utilized for AA quantication instead
of an HPLC method. Okamura41 described a spectrophoto-
metric method that would be suitable for this purpose.
As mentioned earlier herein, when performing the F–C assay
before the addition of the alkali, AA rapidly reacts with poly-
phosphotungstate from the F–C reagent. Therefore, the blue
color formation observed under the initial acidic conditions of
the F–C assay may be attributed to the AA content in the plant
food extract (eqn (2)). It would be interesting to explore the
feasibility of subtracting the absorbance value at 765 nm
obtained under the acidic conditions of the F–C assay (blue
color developed by AA) from that obtained under alkaline
conditions (blue color developed by PC) to obtain a corrected
TPC value. By using such an approach, the AA content of plant
food extracts would be also quantied by comparing the
absorbance obtained under acidic conditions of the F–C assay
with the absorbance of AA standard solutions under the same
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conditions. This methodology has not been explored yet by
other research groups to the best of our knowledge. In this
regard, preliminary data evaluating the performance of the
proposed two-stage approach for the simultaneous quantica-
tion of AA and TPC are presented in the following sections.
3.3 Treatment of phenolic extracts with hydrogen peroxide
(H2O2) to oxidize interferants
An additional approach that may be used to improve the spec-
icity of the F–C assay for TPC determinations is the treatment
of plant food extracts or fruit juices with oxidative agents before
performing the F–C assay. By treating plant extracts with
oxidative agents, the interfering compounds would be oxidized,
decreasing their response to the F–C reagent. This approach has
been recently proposed by Ford et al.15 and is still under
investigation. The authors suggested that AA interferants could
be eliminated by treating fruit juices with ascorbate oxidase
(AO) followed by the addition of H2O2. In this method, AA is
transformed to DHA by AO, and the remaining DHA is oxidized
into non-reducing compounds by H2O2 (600 ppm). H2O2
treatments affected by 10% the total PC content of juice and
non-juice PC model systems, losses that are not signicant
compared with the large errors commonly exerted by AA
(100% of error) or DHA (20–40% error) present in orange juice
samples. In addition, H2O2 can oxidize reducing sugars.42
Further experiments are required to evaluate if the application
of oxidative agents prior to performing the F–C assay is a suit-
able approach to eliminate interferants from diverse plant food
extracts.
4 Comparison between methodological
approaches to improve the specificity of the
F–C assay for total phenolic content (TPC)
determinations
Different approaches to eliminate interferants during the
quantication of TPC in plant food extracts by the F–C assay
have been discussed earlier herein. In this section of the paper,
the feasibility of applying two simple methodologies to elimi-
nate the contribution of interferants to TPC is evaluated and
compared with those previously proposed based on the SPE
strategy13 and based on calculation of a corrected TPC value.14
The methodologies evaluated in the present paper consist of: (i)
the application of H2O2 to plant food extracts prior to F–C assay
in order to oxidize interfering compounds and (ii) the simul-
taneous quantication of vitamin C and TPC using the F–C
assay to obtain a corrected TPC value. Both methodological
approaches proposed to eliminate the contribution of inter-
fering compounds to the F–C assay as well as the methodologies
described by Georgé et al.13 and Isabelle et al.14 are explained in
detail in the following sections of the paper.
4.1 Experimental approach
4.1.1 Chemicals. AA, DHA, a-D-glucose (GLU), D-(
)-fruc-
tose (FRU), GA, chlorogenic acid (CHA), resveratrol (RES),
quercetin 3-O-glucoside (Q 3-O-G), ferulic acid (FA), F–C phenol
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reagent (2 N), sodium carbonate (Na2CO3), a,a0 -bipyridyl,
dithiothreitol (DTT), N-ethylmaleimide (NEM), trichloroacetic
acid (TCA), ferric chloride (FeCl3), methanol, formic acid, and
water (HPLC grade) were purchased from Sigma Chemical Co.
(St. Louis, MO, USA). Orthophosphoric acid (H3PO4) and H2O2
were obtained from DEQ (Monterrey, NL, México).
4.1.2 Plant material and preparation of plant food extracts.
Strawberries (Fragaria  ananassa), kiwifruit (Actinidia deli-
ciosa), and carrots (Daucus carota) were obtained from a local
market (HEB, Monterrey, NL, México). Plant tissues (5 g) were
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homogenized with methanol (20 mL) and centrifuged (10 000 
g, 15 min, 4  C). The clear supernatant (further referred to as
plant food extract) was used to evaluate the different
approaches described herein to improve the specicity of the
F–C assay for TPC quantication.
4.1.3 Preparation of model systems containing phenolic
and interfering compounds. Model systems containing AA,
DHA, GLU, FRU, GA, CHA, RES, Q 3-O-G, and FA alone and in
combination were prepared in order to determine if H2O2 pre-
treatments could reduce interferants (AA, DHA, GLU, FRU)
during the F–C assay. Model systems were prepared in meth-
anol. The concentrations of the interferants used in the model
systems were established based on their concentration expected
in methanol extracts of fruits rich in AA, DHA (i.e. strawberry,
kiwifruit),23 and reducing sugars (banana, apple, pineapple) as
reported in the USDA nutritional database. The nal concen-
tration of individual PC, AA, and DHA (commercial standards)
in the model system treated with H2O2 was 312.5 mM, whereas
the nal concentration of GLU and FRU was 625 mM and
468 mM, respectively.
4.1.4 Treatment of model systems and plant food extracts
with hydrogen peroxide (H2O2). The H2O2 treatments were
performed by mixing either the model system or plant food
extracts (500 mL) with H2O2 solution (300 mL). H2O2 solutions
(0.25–2.00 M) were prepared in methanol. The TPC of samples
treated with H2O2 was determined by the F–C assay. Likewise, to
evaluate the effect of H2O2 treatments on the stability of
phenolics, the compounds were detected and quantied by
HPLC-photodiode array (PDA) aer treating the samples with
H2O2. This methodology is summarized in Fig. 2.
4.1.5 Partial purication of phenolic compounds (PC) by
using solid phase extraction (SPE). PC in plant food extracts
were partially puried using the SPE procedure reported by
Georgé et al.13 The SPE cartridge used in this procedure was an
Oasis HLB (Waters, Milford, MA, USA). Before passing the plant
food extract through the cartridge, it was conditioned with 4 mL
of pure methanol and rinsed with 2  4 mL of water. Then, the
extract (3 mL) was passed through the cartridge and the water-
soluble compounds were recovered with 2  2 mL of distilled
water. The nal volume of the water-soluble compounds was
adjusted to 10 mL. The TPC was quantied in the crude extract
and in the water-soluble extract eluted from the column. To
obtain the corrected TPC calculations were performed as shown
in Fig. 1.
4.1.6 Quantication of total phenolic content (TPC) by the
Folin–Ciocalteu (F–C) assay. The TPC was determined with the
F–C assay18 adapted to a 96-well microplate.43 Briey, plant
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Fig. 2 Procedure to oxidize interfering compounds in crude extracts by treating
extracts with hydrogen peroxide (H2O2) prior to total phenolic content determi-
nations by the Folin–Ciocalteu (F–C) assay. H2O2 pre-treatments are performed by
mixing the plant food extracts (500 mL) with 1.5 M H2O2 solutions (300 mL). The
mixture is vortexed and subjected to the F–C assay. Absorbance values are
compared against a standard curve of chlorogenic acid treated with H2O2.
food extracts or model systems where TPCs were to be deter-
mined (15 mL) were diluted with distilled water (240 mL) in a
96-well microplate well. Thereaer, the F–C reagent (0.25 N, 15
mL) was added. The mixture was incubated for 3 min, and
Na2CO3 (1 N, 30 mL) was added. The nal mixture was incu-
bated for 2 h at room temperature in the dark. Spectrophoto-
metric readings at 765 nm were collected using a plate reader
(Epoch, BioTek Instruments, Inc. Winooski, VT). Absorbance
values were compared against a standard curve of CHA and
results were reported in mg of CHA equivalents per 100 g fresh
weight (FW).
4.1.7 Simultaneous quantication of total ascorbic acid
(AA) and total phenolic content (TPC) by the Folin–Ciocalteu
(F–C) assay. As described earlier herein, AA reacts at the
beginning of the F–C assay when the plant food extract is mixed
with the F–C reagent (eqn (2)). At that point, under the acidic
conditions of the F–C assay PC do not react with the F–C reagent
because they need to be in the form of phenolate ions to be able
to donate electrons (eqn (1)), which occurs only under alkaline
conditions. Therefore, to quantify AA in the plant food extract,
blue color formation was spectrophotometrically determined
aer the addition of the F–C reagent at the beginning of the F–C
assay. For this, the plant food extract (15 mL) was diluted with
distilled water (240 mL) in a 96-well microplate well and the F–C
reagent (0.25 N, 15 mL) was added. The mixture was incubated
for 3 min and absorbance values were determined at 765 nm
using a plate reader. The absorbance values obtained were
attributed to the presence of AA in the extract and thus were
compared against a standard curve prepared with AA solutions
(0.1–3.0 mM) in order to quantify total AA in the extract.
To obtain the TPC value in the sample, the assay was
continued by adding Na2CO3 (1 N, 30 mL) to the mixture used to
determine AA (with extract, water and F–C reagent) and it was
incubated for 2 h at room temperature in the dark. Spectro-
photometric readings at 765 nm were collected using a plate
reader and absorbance was compared against a CHA standard
curve.
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4.1.8 Quantication of total ascorbic acid (AA) by the a, a0 -
bipyridyl method. Total AA in plant food extracts was quantied
by the a, a0 -bipyridyl method41 adapted to the 96-well micro-
plate format.44 Briey, the extract (100 mL) was placed in a 2 mL
tube and mixed with DTT solution (20 mM, 100 mL). The
mixture was incubated for 10 min at room temperature and in
the dark. Thereaer, NEM solution (0.5%, 100 mL) was added to
the mixture and incubated for 30 s. Finally, TCA (10%, 500 mL),
H3PO4 (43%, 400 mL), a, a0 -bipyridyl (4%, 400 mL) and FeCl3 (3%,
200 mL) solutions were added to the assay tubes. The assay tubes
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were incubated at 37  C for 1 h. Then, 200 mL of the reaction
solutions from the assay tubes were placed in a well of a clear
96-well microplate and absorbance readings were collected at
525 nm. Absorbance values were compared against an AA
standard curve (0.15–10 mM) prepared in methanol. AA calcu-
lated by this method was used to obtain corrected values of TPC
as described in the following section.
4.1.9 Calculation of a corrected total phenolic content
(TPC) value based on total ascorbic acid (AA) quantication. As
suggested by Isabelle et al.14 a corrected TPC value can be
obtained if AA is quantied in the plant food and based on its
AA content its contribution to the F–C assay can be subtracted
from the TPC of the extract to obtain a corrected value.
Following this approach, to obtain the corrected TPC value, the
total AA content quantied (in mg mL
1) by either the a, a0 -
bipyridyl method or the F–C assay (as described earlier) was
used to estimate the AA reducing activity in the plant food
extract. For this, a standard solution of AA was evaluated
following the F–C assay for TPC determination, and it was
found that 1 mg of AA had a reducing activity equivalent to
1.43 mg of CHA. Therefore, the total AA content in the plant
food extract was multiplied by 1.43. The value obtained repre-
sented the AA reducing activity in the extract and was subtracted
Fig. 3 Proposed Folin–Ciocalteu (F–C) assay procedure for the simultaneous
quantification of ascorbic acid (AA) and total phenolic content (TPC) in plant
crude extracts, and for the calculation of a corrected TPC value. To quantify AA,
blue color formation is spectrophotometrically determined after the addition of
the F–C reagent at the beginning of the F–C assay. To obtain the corrected TPC,
the AA reducing activity is determined by multiplying the AA content (mg mL
1)
with 1.43 and the value obtained is subtracted from the TPC. Results calculated
are expressed in mg of chlorogenic acid (CHA) equivalents per mL.
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Analytical Methods
from the TPC obtained in the plant food extract to calculate the
corrected TPC. This methodology is summarized in Fig. 3.
4.1.10 Identication and quantication of phenolic
compounds (PC) by HPLC-PDA. In order to determine the effect
of H2O2 treatments on individual PC, the qualitative and
quantitative analysis of PC was performed by high performance
liquid chromatography with photodiode array detection (HPLC-
PDA) as previously described.45 The HPLC system used was
composed of two 515 binary pumps, a 717-plus autosampler,
and a 996-photodiode array detector (Waters Corp, Mildford,
MA). The PC were separated on a 4.6 mm  250 mm, 5 mm, C18
reverse phase column (Luna, Phenomenex, Torrance, CA, USA).
The mobile phases consisted of water (phase A) and meth-
anol:water (60 : 40, v/v, phase B) adjusted to pH 2.4 with formic
acid. The gradient solvent system was 0/100, 3/70, 8/50, 35/30,
40/20, 45/0, 50/0, and 60/100 (min/% phase A) at a constant ow
rate of 1 mL min
1. Chromatographic data were processed with
the Millennium soware V3.1 (Waters Corp, Mildford, MA). The
identication of individual phenolics was based on their PDA
spectra characteristics as compared with authentic standards of
the compounds. For the quantication of individual PC, stan-
dard curves of CHA, RES, Q 3-O-G, and FA were prepared at a
range of 0.5–100 mM.
5 Results and discussion
5.1 Treatment of model systems and plant food extracts with
hydrogen peroxide (H2O2)
The effect of H2O2 pre-treatments on the development of blue
color response (absorbance @765 nm) during the determina-
tion of total PC in model systems containing pure individual PC
and interferants is shown in Table 1. The methodological
approach followed to apply the H2O2 pre-treatments is
summarized in Fig. 2. Interestingly, for CHA, FA and Q 3-O-G
solutions, H2O2 pre-treatments induced a higher response
during the F–C assay as compared with the controls. In contrast,
H2O2 pre-treatments induced slightly lower absorbance values
in GA and RES model systems evaluated by the F–C assay. The
model system containing mixtures of the 5 individual PC pre-
treated with H2O2 showed higher absorbance values as
compared with the controls. There are no previous reports in
the literature evaluating the effect of H2O2 pre-treatments on
the development of blue color response for model systems of
individual PC prior to the F–C assay. Results indicate that H2O2
pre-treatments may increase the sensitivity of certain PC to
donate electrons to the F–C reagent during the assay, and thus a
higher absorbance is observed.
To determine the stability of each PC when treated with
H2O2, the concentration of phenolic compounds in model
systems treated with different concentrations of H2O2 was
determined by HPLC-PDA (Table 2). H2O2 pre-treatment did not
affect the concentration of individual PC. Therefore, the higher
or lower blue color formation shown by individual PC when pre-
treated with H2O2 and subjected to the F–C assay cannot be
attributed to degradation. Regarding the interferants, H2O2
resulted in an effective approach to decrease their response
during the F–C assay (Table 1). Reducing sugars (FRU and GLU)
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Analytical Methods Paper

Table 1 Effect of H2O2 pre-treatments (0.25–2.0 M) on the development of blue color (absorbance @765 nm) in model systems, containing phenolics and inter-
ferants, subjected to the Folin–Ciocalteu (F–C) assay

Percentage (%) of absorbance (@765 nm) in the model systems pre-treated with H2O2 subjected to
the F–C assay as compared with the absorbance shown in the controls (non-H2O2 treated)a

Concentration of H2O2 (M)b

Control 0.25 0.50 1.00 1.50 2.00

Phenolic CHA 100  3.9 cc 179.1  9.0 b 198.0  3.7 a 191.4  5.8 ab 198.0  5.3 a 179.1  4.9 b
compound (PC) FA 100  1.8 c 109.6  1.9 b 110.6  2.4 b 112.0  1.8 b 122.0  5.1 a 114.7  0.8 ab
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GA 100  2.6 a 84.9  1.9 c 88.0  1.4 bc 95.0  4.1 ab 92.6  2.3 abc 99.8  4.9 a
Q 3-O-G 100  2.3 d 126.4  3.0 ab 131.6  2.0 a 115.5  3.2 c 120.8  2.0 bc 125.4  2.4 ab
RES 100  3.0 a 92.2  1.5 b 93.8  2.2 ab 92.2  1.4 b 87.5  2.9 b 89.4  2.1 b
Mixture 100  2.9 c 127.0  2.4 ab 126.3  2.8 b 130.3  3.1 ab 134.0  2.3 a 127.8  2.1 ab
Interferants FRU 100  0.4 a 41.9  2.0 b 23.2  0.2 c 15.6  0.2 d 11.1  0.2 e 10.8  0.5 e
GLU 100  0.9 a 30.4  1.5 b 15.7  0.8 c 10.4  0.9 d 8.2  0.7 de 6.3  0.6 e
AA 100  4.3 a 29.2  1.7 b 26.6  0.9 b 28.3  0.8 b 29.0  1.1 b 30.1  0.5 b
DHA 100  2.1 a 67.7  6.6 b 49.7  5.4 c 45.3  8.2 cd 35.4  4.2 d 50.0  6.9 bcd
Mixture 100  4.1 a 30.5  0.8 b 27.5  0.4 bc 25.0  0.8 c 25.1  0.8 c 22.2  1.0 c
PC + interferants 100  1.2 a 90.3  2.0 b 84.8  1.9 c 83.1  1.1 c 78.4  0.7 d 78.2  0.7 d
a
Values represent the mean of 5 replications  standard error of the mean. b H2O2 pre-treatments were performed by mixing the model system (500
mL) with H2O2 solution (300 mL). c Different letters in the same row indicate statistical difference by the LSD test ( p < 0.05). Abbreviations:
chlorogenic acid (CHA); ferulic acid (FA); gallic acid (GA); quercetin 3-O-glucoside (Q 3-O-G); resveratrol (RES); fructose (FRU); glucose (GLU);
ascorbic acid (AA); dehydroascorbic acid (DHA).

Table 2 Effect of H2O2 pre-treatments (0.25–2.0 M) on the concentration of individual phenolic compounds determined by high performance liquid chromatography
with photodiode array detection (HPLC-PDA)

Concentration of individual phenolic compounds (mg L
1)a

CHA FA GA Q 3-O-G RES

Control 13.74  0.78 7.10  0.31 14.92  0.96 18.30  0.92 13.85  0.67
H2O2 0.25 13.79  1.57 7.09  0.38 15.21  0.83 18.16  1.15 14.10  0.79
concentration (M)b 0.5 13.25  1.39 6.98  0.24 14.56  0.75 17.21  0.10 13.23  0.88
1.0 13.67  0.39 6.80  0.38 13.86  1.28 17.10  0.99 13.34  0.74
1.5 13.97  0.73 6.45  0.09 14.04  1.24 18.10  0.19 13.00  0.63
2.0 14.07  0.82 6.89  0.32 13.96  1.18 17.52  0.11 12.94  0.98
a
Values represent the mean of 5 replications  standard error of the mean. b H2O2 treatments were performed by mixing solutions containing pure
commercial standards (500 mL) with H2O2 solution (300 mL). Abbreviations: chlorogenic acid (CHA); ferulic acid (FA); gallic acid (GA); quercetin
3-O-glucoside (Q 3-O-G); resveratrol (RES); fructose (FRU).

showed a higher degree of degradation when compared to AA
and DHA. H2O2 pre-treatments on model systems containing a
mixture of interferants reduced up to 75% the development of
response in the F–C assay. Furthermore, the model system
containing individual PC + interferants showed a lower
response to the F–C assay when pre-treated with H2O2. Mixing
500 mL of this model system (individual PC + interferants) with
1.50 M H2O2 (300 mL) reduced by approximately 20% the
absorbance values obtained in the non-treated model system
when subjected to the F–C assay. Since the mixture of individual
PC pre-treated with H2O2 and subjected to the F–C assay showed
a higher response to the F–C assay, the decrease in response
development observed in the H2O2 treated model system con-
taining individual PC + interferants can be attributed to the
degradation of the interferants.
These results demonstrate that indeed H2O2 pre-treatments
may result in an improvement of the specicity of the F–C assay
for total PC determinations. However, it is important to take
into consideration that H2O2 can increase the sensitivity of
certain PC to donate electrons during the F–C assay. In order to
compensate this effect the standard curve used to calculate total
PC must be performed with the standard compound pre-treated
with H2O2 under the same conditions of the sample. Likewise, it
is important to consider that if the extract contains trace tran-
sition metal ions, they can catalyse the oxidation of PC in the
presence of H2O2, decreasing their response in the F–C assay.46
5.2 Calculation of a corrected total phenolic content (TPC)
value based on the simultaneous quantication of total
ascorbic acid (AA) and phenolic compounds (PC) in plant food
extracts
As mentioned earlier, plant food methanol extracts (strawberry,
kiwifruit, carrot) were prepared to determine their total PC by

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Paper Analytical Methods

Table 3 Ascorbic acid content in strawberry, kiwifruit, and carrot determined by represents a remarkable advantage because with the simple F–C
the Folin–Ciocalteu (F–C) assay and by the a,a0 -bipyridyl method assay it is possible to quantify both the TPC and total AA
without additional requirements of chemicals and equipment.
Ascorbic acid content (mg ascorbic acid/100 g
FW)a F–C assay is a widely used, economic, well-known methodology
for the quantication of TPC. Therefore, all the infrastructure
Sample F–C assayb a,a0 -bipyridyl methodc and reagents needed are already available. In addition, results
obtained using this new proposed approach to the methodology
Strawberry 63.5  7.6 64.4  3.9
Kiwifruit 50.6  1.2 53.2  1.5 can be directly compared with results already in the literature
Carrot 6.0  0.8 3.5  0.4 for the quantication of TPC with the classical F–C assay.
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a
Values represent the mean of 5 replications  standard error of the
mean. b To quantify AA by the F–C assay, blue color formation was
spectrophotometrically determined aer the addition of the F–C
reagent to the plant food extract at the beginning of the F–C assay.
For this, the plant food extract (15 mL) was diluted with distillated
water (240 mL) in a 96-well microplate well and the F–C reagent (0.25
N, 15 mL) was added. The mixture was incubated for 3 min and
absorbance values were taken at 765 nm using a plate reader. The
absorbance values obtained were attributed to the presence of
ascorbic acid in the extract and thus were compared against a
standard curve ( y ¼ 0.4758x + 0.0048; R2 ¼ 0.9992) prepared with AA
solutions in order to quantify total AA in the plant food extract. c The
a,a0 -bipyridyl method41,44 also was used to determine the AA content
in the samples. In this method, the plant extract was compared
against a standard curve of AA ( y ¼ 5.9603x + 0.0111; R2 ¼ 0.9999).
the F–C assay using the traditional procedure and the methods
reviewed and proposed herein to improve the specicity of the
method. One of the strategies evaluated to improve the speci-
city of the assay, consisted of the quantication of total AA to
obtain a corrected TPC value by subtracting from the TPC the AA
reducing activity in the extract. Therefore, the total AA content
in the plant food extract was quantied by the a,a0 -bipyridyl
method. Likewise, the procedure proposed herein involving the
simultaneous quantication of AA and TPC based on the F–C
assay was evaluated (Fig. 3). The results showed no signicant
difference ( p > 0.05) between the total AA values obtained by
either of the two methods utilized (F–C assay or the a,a0 -bipyr-
idyl method) (Table 3).
These results conrm that the F–C assay-based strategy
proposed herein indeed can be applied for the simultaneous
quantication of total AA and TPC in plant food extracts. This
5.3 Comparison between methodological approaches to
improve the specicity of the Folin–Ciocalteu (F–C) assay for
total phenolic compound (TPC) determinations
The TPC values for strawberry, kiwifruit, and carrot obtained in
the methanol extracts and aer the application of the different
approaches to improve the specicity of the F–C assay for TPC
determinations are shown in Table 4. As expected, the different
procedures (SPE, H2O2 pre-treatment, subtraction of AA
reducing activity from TPC) to eliminate the interferants
studied resulted in a lower TPC value as compared with the TPC
quantied in the methanol extract. For strawberry, which has a
high avonoid and vitamin C content23,47 the oxidation of
interferants with H2O2 (Fig. 2) resulted in 54% lower quanti-
cation of TPC, followed by the corrected TPC value (based on
the total AA content, Fig. 3) and by the TPC quantied in the
methanol extract using the SPE approach (Fig. 1).
A similar behaviour was observed for kiwifruit. However,
the application of the different methodologies to eliminate
interferants reduced in a higher percentage the TPC values
obtained in the methanol extracts. For instance, H2O2 treated
methanol extracts of kiwifruit showed 84% lower TPC values.
This can be attributed to the lower TPC and higher reducing
sugar content reported for kiwifruit as compared with straw-
berry.23,48 Interestingly, for carrot the treatment that showed
the lower TPC value when compared with the methanol extract
was the elimination of interferants by SPE. This approach
reduced by 89% the TPC quantied in the crude extract.
Compared with strawberry and kiwifruit, where avonoids are

Table 4 Comparison between different methodological approaches to improve the specificity of the Folin–Ciocalteu assay for total phenolic content (TPC)
determinations

TPC (mg chlorogenic acid equivalents per 100 g FW)a

Oxidation of Corrected TPC by

Elimination of interferants with subtraction of AA
Sample Methanol extractb interferants by SPEc H2O2d reducing activitye

Strawberry 294.4  28.7 af 159.6  25.6 bc 134.1  18.2 c 203.6  18.6 b

Kiwifruit 105.1  4.10 a 31.7  5.40 b 13.2  1.10 c 32.7  3.43 b
Carrot 41.5  1.40 a 4.3  1.60 d 24.8  1.60 c 32.9  1.24 b
a
Values represent the mean of 5 replications  standard error of the mean. b Values shown in the methanol extract column represent the TPC
determined in the plant crude extract before subtracting the contribution of interferants to the F–C assay. c Values shown in this column
represent the TPC determined in the plant extract aer partially purifying phenolic compounds with an Oasis HLB cartridge13 (Fig. 1). d Values
shown in this column represent the TPC determined in plant crude extracts pre-treated with a H2O2 solution (1.5 M) before performing the F–C
assay (Fig. 2). e Ascorbic acid (AA) reducing activity (1.43 mg chlorogenic acid per mg AA) was determined by testing an AA standard for TPC by
the F–C assay; AA contribution to the TPC of each plant food extract was determined by multiplying the AA content in the methanol extract
(obtained by the F–C assay, Table 3) with 1.43 (Fig. 3). f Different letters in the same row indicate statistical difference by the LSD test ( p < 0.05).

This journal is ª The Royal Society of Chemistry 2013 Anal. Methods, 2013, 5, 5990–5999 | 5997

Analytical Methods
the main PC in the plant tissue, the main PC in carrots are
phenolic acids.43,45 Phenolic acids have been reported to have
low retention yields when using SPE cartridges.27 This is due to
the weak interactions between these polar compounds and the
solid matrix.37,38 This unwanted elimination of phenolic acids
along with the interferants resulted in an underestimation of
the TPC when using the SPE strategy (Fig. 1).13 Therefore, the
use of this strategy to eliminate interferants from plant food
extracts is only recommended when most of the PC are
amphipathic in nature, such as avonoids. On the other hand,
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the corrected TPC value obtained (by subtracting AA reducing
activity, Fig. 3) showed 20% lower TPC value and the appli-
cation of H2O2 pre-treatments in the extract reduced by 40%
its TPC value.
Results suggest that the calculation of a corrected TPC value,
based on the calculation of the reducing activity of AA present in
the extract, is the best approach to obtain a better estimation of
TPC values obtained by the F–C assay. Likewise, the H2O2 pre-
treatment strategy could be a feasible approach. However,
further experiments are required to better understand how
H2O2 pre-treatments improve the sensitivity of certain pheno-
lics to react with the F–C reagent. The SPE strategy would be
only suggested when most of the PC of the plant food extracts
are avonoids and not phenolic acids, to avoid underestimation
of TPC values. However, as new and more efficient solid
matrixes are developed the current drawbacks associated with
the SPE strategies may be overcome or at least alleviated to
some extent. The procedure proposed to improve the specicity
of the F–C assay for TPC determinations is summarized in
Fig. 3, where by using the F–C assay the simultaneous quanti-
cation of total AA and TPC can be achieved.
6 Conclusions
In this paper, different methods to improve the specicity of the
F–C assay for TPC determinations were reviewed. These
different approaches were evaluated in model systems (con-
taining phenolics and interferants) and plant food extracts to
determine which method would result in more accurate TPC
value estimations. The methods tested included the use of SPE
to determine the content of interferants in the extract, the use of
H2O2 treatments to oxidize interferants prior to the F–C assay,
and the quantication of AA in the plant food extract to obtain a
corrected TPC calculated by subtracting the AA reducing activity
in the extract. Likewise, we proposed a modication to the F–C
assay procedure that allows the simultaneous quantication of
total AA and TPC in plant food extracts. Results indicate that the
SPE approach would be only recommended when the main PC
in the extracts are avonoids and not phenolic acids, because
they are weakly retained in the SPE matrix and thus under-
estimated TPC values are obtained. On the other hand, H2O2
treatments increase the sensitivity of certain phenolic
compounds to react with the F–C reagent and thus, if the proper
PC to construct the standard curve is not selected, over-
estimation of TPC values would be obtained. The method
proposed in this paper to improve the TPC estimation consists
of obtaining the total AA content to calculate a corrected TPC,
5998 | Anal. Methods, 2013, 5, 5990–5999
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Paper
which can be obtained by subtracting the AA reducing response.
In addition, the method proposed for the simultaneous quan-
tication of AA and TPC using the F–C assay may be used for
this purpose. This approach represents a remarkable advantage
because with the simple F–C assay it is possible to quantify both
the TPC and total AA without additional requirements of
chemicals and equipment.
Acknowledgements
This study was supported by research funds from the Tec-
nológico de Monterrey – Research Chair Initiative (CAT 161) and
Cátedra de Nutrigenómica-FEMSA. Author J.C.S.-R. also
acknowledges the scholarship (169222) from the Consejo
Nacional de Ciencia y Tecnologı́a (CONACYT, México).
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