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The Folin-Ciocalteu assay revisited:

Improvement of its specificity for total
phenolic content determination

Article in Analytical methods November 2013

DOI: 10.1039/c3ay41125g

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The FolinCiocalteu assay revisited: improvement of its

specicity for total phenolic content determination
Published on 09 August 2013. Downloaded by University of Edinburgh on 16/10/2013 20:28:06.

Cite this: Anal. Methods, 2013, 5, 5990

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

Zevallosc and Daniel A. Jacobo-Velazquez*a

Received 8th July 2013
Accepted 8th August 2013
DOI: 10.1039/c3ay41125g
www.rsc.org/methods
This study presents a review of the FolinCiocalteu (FC) assay for total phenolic content (TPC)
determinations and describes dierent approaches to improve its specicity. Phenolics are regarded as
the molecules with the highest potential to neutralize free radicals. Therefore, their quantication is a
common practice in dierent 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. Dierent methodologies have been proposed to improve the specicity of the FC 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
quantication. These methods are described in detail in the present study. Likewise, their advantages
and disadvantages are discussed based on new experimental data. A simple modication of the FC
assay procedure is proposed to quantify both the TPC value and the AA reducing activity in plant food
extracts. Values obtained by the modied FC assay can be used to estimate a corrected TPC value.

1 Introduction most commonly used procedure to determine TPC of food

Scientic 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 (dened 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, specically to resveratrol which is present in
red grape skins.14 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 dierent aspects of research.
PC are the major contributors to the antioxidant capacity of
fruits, vegetables, and grains.5,6 Therefore, the quantication 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.710 The FC assay is the
a
Centro de Biotecnologa-FEMSA, Department of Biotechnology and Food Engineering,
School of Biotechnology and Food, Tecnologico de Monterrey-Campus Monterrey, E.
Garza Sada 2501 Sur, C.P. 64849, Monterrey, N.L., Mexico. E-mail: djacobov@
itesm.mx; Fax: +52-818-328-4136; Tel: +52-818-358-20-00 ext. 4820
b
Centro de Investigacion en Alimentacion y Desarrollo, A.C. Unidad Culiacan,
Postharvest Physiology and Quality Laboratory, Carretera a El Dorado Km. 5.5,
Apartado Postal 32-A, Culiacan, Sinaloa, 80129 Mexico
c
Department of Horticultural Sciences, Texas A&M University, Vegetable & Fruit
Improvement Center, College Station, Texas 77843-2133, USA
extracts. The FC assay is a colorimetric method based on elec-
tron transfer reactions between the FC reagent and PC. However,
the FC assay is not specic 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 FC reagent, skewing the results of TPC.11,12
Therefore, dierent methodological approaches to improve
the specicity of the FC assay have been proposed. Those
methodological approaches include: (i) the partial purication of
phenolic extracts using SPE columns before the FC assay is
performed;13 (ii) the calculation of a corrected TPC value by sub-
tracting the AA reducing activity from the TPC quantied 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 aect PC.15
In this article dierent methodological approaches to
improve the specicity of the FC 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 FolinCiocalteu (FC) assay background
and theory
The FC assay16 was generated in order to improve the Folin and
Denis (FD) 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 dierence between the FC
and FD 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 FD assay.17 The FC assay is more sensitive and repro-
ducible than the FD 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 FD assay to determine the total PC content in
Prunus domestica, whereas Singleton and Rossi18 adapted the
FC assay to determine total PC in wine. Both assays are widely
used by the scientic community. Indeed, the procedures
developed by Swain and Hillis19 and Singleton and Rossi18 have
been cited in scientic papers more than 1900 and 5500 times,
respectively. Furthermore, the method has been recently adap-
ted to measure lipophilic antioxidants.20
The FC 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 acidied with 50 mL of concentrated HCl and
50 mL of 85% phosphoric acid. The acidied solution is boiled
for 10 h, cooled, and 150 g of Li2SO4$4H2O is added. The
resultant intense yellow solution is the FC reagent.11,21
Although the chemical nature of the FC reagent has not been
elucidated, it is believed to be composed of heteropoly-phos-
photungstates/molybdates.11 Likewise, the exact chemical
nature of the FC 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 FC reaction
involves sequences of reversible one- or two-electron reduction
reactions.11,21,22 From the components of the FC 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 FC assay, the reaction
between PC and the FC 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
FC reagent.11,21
2.1 Interference during the analysis of total phenolic
content (TPC) by the FolinCiocalteu (FC) assay
As mentioned earlier, crude plant extracts may contain inter-
fering substances (other reducing compounds) that can react
with the FC reagent, skewing the results for TPC
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Analytical Methods
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 signicant concentrations
of vitamin C.23 Under acidic conditions of the FC reagent
(pH " 3), AA and DHA (both enediols) rapidly react with poly-
phosphotungstate, giving a blue color right aer 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 FC reagent. Like-
wise, it has been suggested that AA could have an augmenting
eect on the amount of FC reagent reacting with PC, because it
may reduce the quinones formed during the assay. However,
this augmenting eect 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 FC 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
specicity of the FolinCiocalteu (FC) assay
Dierent procedures have been proposed to reduce the
response of interfering compounds when performing the FC
assay for TPC determinations in plant extracts. The methods
include: (i) the partial purication of PC by using SPE
cartridges; (ii) the calculation of a corrected TPC by subtracting
the AA reducing activity from the TPC quantied; and (iii) the
treatment of phenolic extracts with oxidative agents prior to FC
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 purication of phenolic extracts by solid phase
extraction (SPE)
Biological extracts usually contain a wide variety of dierent
solutes, aside PC, which may generate a positive colorimetric
response in the FC assay. This generates an overestimation of
the TPC in the sample. Therefore, the partial purication 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 purication 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).13,3235
With the aim to obtain a more accurate estimation of TPC
using the FC assay, George 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 FC assay. Thereaer, 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 FC
assay. Based on the hypothesis of the authors, the dierence
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 dicult (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
5992 | Anal. Methods, 2013, 5, 59905999
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Paper
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 FolinCiocalteu (FC) 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 eectiveness of this SPE-aided TPC determina-
tion strategy would depend on factors related to the prole 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 nonspecic
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 FC assay is conducted since most of them are not
compatible with the FC 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 eective 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 FC 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 glucosefructose (1 : 1) at
dierent 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
FC assay. Unfortunately, using correction factors to eliminate
the response given by sugars when performing the FC assay
would be quite impractical because it would be necessary to
determine the exact concentration of the dierent 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 quantication
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 FC 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 specic spectrophotometric method for AA
determinations would be utilized for AA quantication 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 FC assay
before the addition of the alkali, AA rapidly reacts with poly-
phosphotungstate from the FC reagent. Therefore, the blue
color formation observed under the initial acidic conditions of
the FC 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 FC 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 quantied by comparing the
absorbance obtained under acidic conditions of the FC assay
with the absorbance of AA standard solutions under the same
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Analytical Methods
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 quantica-
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-
icity of the FC assay for TPC determinations is the treatment
of plant food extracts or fruit juices with oxidative agents before
performing the FC assay. By treating plant extracts with
oxidative agents, the interfering compounds would be oxidized,
decreasing their response to the FC 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 aected by "10% the total PC content of juice and
non-juice PC model systems, losses that are not signicant
compared with the large errors commonly exerted by AA
("100% of error) or DHA (2040% 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 FC assay is a suit-
able approach to eliminate interferants from diverse plant food
extracts.
4 Comparison between methodological
approaches to improve the specicity of the
FC assay for total phenolic content (TPC)
determinations
Dierent approaches to eliminate interferants during the
quantication of TPC in plant food extracts by the FC 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 FC assay
in order to oxidize interfering compounds and (ii) the simul-
taneous quantication of vitamin C and TPC using the FC
assay to obtain a corrected TPC value. Both methodological
approaches proposed to eliminate the contribution of inter-
fering compounds to the FC assay as well as the methodologies
described by George 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), FC phenol
Anal. Methods, 2013, 5, 59905999 | 5993

Analytical Methods
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, Mexico).
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, Mexico). 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 dierent
approaches described herein to improve the specicity of the
FC assay for TPC quantication.
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 FC 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
(0.252.00 M) were prepared in methanol. The TPC of samples
treated with H2O2 was determined by the FC assay. Likewise, to
evaluate the eect of H2O2 treatments on the stability of
phenolics, the compounds were detected and quantied by
HPLC-photodiode array (PDA) aer treating the samples with
H2O2. This methodology is summarized in Fig. 2.
4.1.5 Partial purication of phenolic compounds (PC) by
using solid phase extraction (SPE). PC in plant food extracts
were partially puried using the SPE procedure reported by
George 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 quantied 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 Quantication of total phenolic content (TPC) by the
FolinCiocalteu (FC) assay. The TPC was determined with the
FC assay18 adapted to a 96-well microplate.43 Briey, 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 FolinCiocalteu (FC) 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 FC 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. Thereaer, the FC 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 quantication of total ascorbic acid
(AA) and total phenolic content (TPC) by the FolinCiocalteu
(FC) assay. As described earlier herein, AA reacts at the
beginning of the FC assay when the plant food extract is mixed
with the FC reagent (eqn (2)). At that point, under the acidic
conditions of the FC assay PC do not react with the FC 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
aer the addition of the FC reagent at the beginning of the FC
assay. For this, the plant food extract (15 mL) was diluted with
distilled water (240 mL) in a 96-well microplate well and the FC
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
(0.13.0 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 FC 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 Quantication of total ascorbic acid (AA) by the a, a0 -
bipyridyl method. Total AA in plant food extracts was quantied
by the a, a0 -bipyridyl method41 adapted to the 96-well micro-
plate format.44 Briey, 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. Thereaer, 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 (0.1510 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) quantication. As
suggested by Isabelle et al.14 a corrected TPC value can be
obtained if AA is quantied in the plant food and based on its
AA content its contribution to the FC 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 quantied (in mg mL!1) by either the a, a0 -
bipyridyl method or the FC 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 FC 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 FolinCiocalteu (FC) assay procedure for the simultaneous
quantication 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 FC reagent at the beginning of the FC 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 Identication and quantication of phenolic
compounds (PC) by HPLC-PDA. In order to determine the eect
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 soware V3.1 (Waters Corp, Mildford, MA). The
identication of individual phenolics was based on their PDA
spectra characteristics as compared with authentic standards of
the compounds. For the quantication of individual PC, stan-
dard curves of CHA, RES, Q 3-O-G, and FA were prepared at a
range of 0.5100 mM.
5 Results and discussion
5.1 Treatment of model systems and plant food extracts with
hydrogen peroxide (H2O2)
The eect 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 FC 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 FC 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 eect of H2O2 pre-treatments on
the development of blue color response for model systems of
individual PC prior to the FC assay. Results indicate that H2O2
pre-treatments may increase the sensitivity of certain PC to
donate electrons to the FC 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 dierent concentrations of H2O2 was
determined by HPLC-PDA (Table 2). H2O2 pre-treatment did not
aect 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 FC assay cannot be
attributed to degradation. Regarding the interferants, H2O2
resulted in an eective approach to decrease their response
during the FC assay (Table 1). Reducing sugars (FRU and GLU)
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Analytical Methods Paper

Table 1 Eect of H2O2 pre-treatments (0.252.0 M) on the development of blue color (absorbance @765 nm) in model systems, containing phenolics and inter-
ferants, subjected to the FolinCiocalteu (FC) assay

Percentage (%) of absorbance (@765 nm) in the model systems pre-treated with H2O2 subjected to
the FC 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 Dierent letters in the same row indicate statistical dierence 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 Eect of H2O2 pre-treatments (0.252.0 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 FC assay. Furthermore, the model system
containing individual PC + interferants showed a lower
response to the FC 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 FC assay. Since the mixture of individual
PC pre-treated with H2O2 and subjected to the FC assay showed
a higher response to the FC 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 specicity of the FC 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 FC assay. In order to
compensate this eect 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 FC assay.46
5.2 Calculation of a corrected total phenolic content (TPC)
value based on the simultaneous quantication 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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Table 3 Ascorbic acid content in strawberry, kiwifruit, and carrot determined by represents a remarkable advantage because with the simple FC
the FolinCiocalteu (FC) 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 FC assay is a widely used, economic, well-known methodology
for the quantication of TPC. Therefore, all the infrastructure
Sample FC 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 quantication of TPC with the classical FC assay.
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a
Values represent the mean of 5 replications % standard error of the
mean. b To quantify AA by the FC assay, blue color formation was
spectrophotometrically determined aer the addition of the FC
reagent to the plant food extract at the beginning of the FC assay.
For this, the plant food extract (15 mL) was diluted with distillated
water (240 mL) in a 96-well microplate well and the FC 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 0.4758x + 0.0048; R2 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 5.9603x + 0.0111; R2 0.9999).
the FC assay using the traditional procedure and the methods
reviewed and proposed herein to improve the specicity of the
method. One of the strategies evaluated to improve the speci-
city of the assay, consisted of the quantication 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 quantied by the a,a0 -bipyridyl
method. Likewise, the procedure proposed herein involving the
simultaneous quantication of AA and TPC based on the FC
assay was evaluated (Fig. 3). The results showed no signicant
dierence ( p > 0.05) between the total AA values obtained by
either of the two methods utilized (FC assay or the a,a0 -bipyr-
idyl method) (Table 3).
These results conrm that the FC assay-based strategy
proposed herein indeed can be applied for the simultaneous
quantication of total AA and TPC in plant food extracts. This
5.3 Comparison between methodological approaches to
improve the specicity of the FolinCiocalteu (FC) assay for
total phenolic compound (TPC) determinations
The TPC values for strawberry, kiwifruit, and carrot obtained in
the methanol extracts and aer the application of the dierent
approaches to improve the specicity of the FC assay for TPC
determinations are shown in Table 4. As expected, the dierent
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
quantied 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 quantied in the
methanol extract using the SPE approach (Fig. 1).
A similar behaviour was observed for kiwifruit. However,
the application of the dierent 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 quantied in the crude extract.
Compared with strawberry and kiwifruit, where avonoids are

Table 4 Comparison between dierent methodological approaches to improve the specicity of the FolinCiocalteu 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 FC assay. c Values shown in this column
represent the TPC determined in the plant extract aer 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 FC
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 FC 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 FC assay, Table 3) with 1.43 (Fig. 3). f Dierent letters in the same row indicate statistical dierence by the LSD test ( p < 0.05).

This journal is The Royal Society of Chemistry 2013 Anal. Methods, 2013, 5, 59905999 | 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 FC 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 FC 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 ecient 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 specicity
of the FC assay for TPC determinations is summarized in
Fig. 3, where by using the FC assay the simultaneous quanti-
cation of total AA and TPC can be achieved.
6 Conclusions
In this paper, dierent methods to improve the specicity of the
FC assay for TPC determinations were reviewed. These
dierent 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 FC assay,
and the quantication 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 modication to the FC
assay procedure that allows the simultaneous quantication 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 FC 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, 59905999
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Paper
which can be obtained by subtracting the AA reducing response.
In addition, the method proposed for the simultaneous quan-
tication of AA and TPC using the FC assay may be used for
this purpose. This approach represents a remarkable advantage
because with the simple FC 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-
nologico de Monterrey Research Chair Initiative (CAT 161) and
Catedra de Nutrigenomica-FEMSA. Author J.C.S.-R. also
acknowledges the scholarship (169222) from the Consejo
Nacional de Ciencia y Tecnologa (CONACYT, Mexico).
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