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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 Folin–Ciocalteu assay revisited: improvement of its

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

Zevallosc and Daniel A. Jacobo-Velázquez*a

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
Received 8th July 2013
Accepted 8th August 2013
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
DOI: 10.1039/c3ay41125g
assay procedure is proposed to quantify both the TPC value and the AA reducing activity in plant food
www.rsc.org/methods 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

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

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total protein concentration by measuring the content of tyro- determinations. Among those reducing compounds, AA, dehy-
sine and tryptophan. The principle of both methods is based on droascorbic acid (DHA), and reducing sugars (i.e. glucose and
the reaction between the oxidant reagent and tyrosine/trypto- fructose) have the highest impact on hampering the accuracy of
phan, resulting in blue color formation proportional to the the assay.12,21
concentration of protein. The main difference between the F–C The presence of AA is generally a problem when determining
and F–D assays is the proportion of molybdate (Mo) used to the total PC of extracts obtained from fruits such as orange,
prepare the reagent. Folin and Ciocalteu increased the Mo kiwifruit and strawberry, which have signicant concentrations
content to prevent the formation of a white precipitate observed of vitamin C.23 Under acidic conditions of the F–C reagent
in the F–D assay.17 The F–C assay is more sensitive and repro- (pH " 3), AA and DHA (both enediols) rapidly react with poly-
ducible than the F–D assay.18 phosphotungstate, giving a blue color right aer mixing the
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The application of both assays has been extended to deter- plant extract with the reagent (eqn (2)). Indeed, the observation
mine total PC in plant food extracts. Swain and Hillis19 adapted of blue color before the addition of the alkali indicates the
the F–D assay to determine the total PC content in presence of AA, DHA or other reducing compounds not
Prunus domestica, whereas Singleton and Rossi18 adapted the requiring the phenolate form to reduce the F–C reagent. Like-
F–C assay to determine total PC in wine. Both assays are widely wise, it has been suggested that AA could have an augmenting
used by the scientic community. Indeed, the procedures effect on the amount of F–C reagent reacting with PC, because it
developed by Swain and Hillis19 and Singleton and Rossi18 have may reduce the quinones formed during the assay. However,
been cited in scientic papers more than 1900 and 5500 times, this augmenting effect is not generally accepted, because during
respectively. Furthermore, the method has been recently adap- the assay, AA is rapidly oxidized before the addition of the alkali
ted to measure lipophilic antioxidants.20 and subsequent oxidation of phenolics takes place.21 DHA is the
The F–C reagent is prepared by rst dissolving 100 g of rst oxidation product of AA and it is naturally present in fruits
sodium tungstate (Na2WO4$2H2O) and 25 g of sodium Mo and vegetables, especially in those where the activity of poly-
(Na2MoO4$2H2O) in 700 mL of distilled water. Then, the phenol oxidase (PPO) is high and AA is used as a substrate to
solution is acidied with 50 mL of concentrated HCl and reduce quinones.24 DHA is an enediol and thus it also produces
50 mL of 85% phosphoric acid. The acidied solution is boiled blue color under acidic conditions during the F–C reaction.21
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 The presence of reducing sugars is a problem when deter-
nature of the F–C reaction that leads to a blue species [possibly mining the TPC of extracts obtained from fruits where the TPC
(PMoW11O40)4!] is unknown and likely to remain so due to its is low. In these cases most of the response (blue color forma-
complexity.21 However, it is assumed that the F–C reaction tion) may be generated by the sugars present in the sample. The
involves sequences of reversible one- or two-electron reduction interference produced by reducing sugars comes from the
reactions.11,21,22 From the components of the F–C reagent, enediol reductones formed under the alkali conditions of the
molybdates are more easily reduced than tungstates, and thus assay (eqn (3)).21
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
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
2.1 Interference during the analysis of total phenolic the AA reducing activity from the TPC quantied; and (iii) the
content (TPC) by the Folin–Ciocalteu (F–C) assay treatment of phenolic extracts with oxidative agents prior to F–C
As mentioned earlier, crude plant extracts may contain inter- assay performance, in order to oxidize interferants. In the
fering substances (other reducing compounds) that can react following sections each of these methodological approaches are
with the F–C reagent, skewing the results for TPC 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 Fig. 1 Estimation of total phenolic content (TPC) by subtraction of reducing
analytes (solubilized in a liquid phase) in a solid matrix. This interferants using solid phase extraction. The crude extract is introduced into the
SPE column where phenolics are retained due to interactions with the solid
interaction occurs based on the physicochemical and
matrix. The reducing interferants are eluted in the most polar fraction, which is
biochemical properties (hydrophobicity, electrochemical analyzed using the Folin–Ciocalteu (F–C) assay. The response from this fraction is
charge, size, etc.) of the solutes and the matrix.25 Therefore, the subtracted from the response of the crude extract, resulting in an estimation of
selection of the solid phase to be used is related to the char- the TPC.
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 interactions between these compounds and the solid
phase SPE has been used for the fractionation and recovery of matrix.37,38 This unintended elution of phenolic acids along
PC in wine,28,29 plant tissues,30 fruits and vegetables,31 and with the interferants may cause an underestimation of TPC.13
juices,32 among others. The use of SPE-based strategies allows Therefore, the effectiveness of this SPE-aided TPC determina-
in most cases the elimination of reducing interferants (i.e. tion strategy would depend on factors related to the prole of
reducing sugars, organic acids) in food samples resulting in a PC and interfering compounds, creating the necessity for a
more accurate estimation of the content of PC (as well as other case-to-case optimization process. Furthermore, in order to
analytes of interest).13,32–35 prevent saturation of the solid matrix it is necessary to have a
With the aim to obtain a more accurate estimation of TPC priori information regarding the range of concentration of both
using the F–C assay, Georgé et al.13 developed a method to the phenolics and interferants. It is always possible to use
eliminate water-soluble interferants in plant food crude extracts unnecessarily high solid matrix volumes in order to prevent
using an Oasis HLB (hydrophilic-lipophilic balance) cartridge. saturation. However, this usually generates lower recovery
The procedure consisted in determining TPC in the crude yields of the product of interest while promoting nonspecic
extract by the F–C assay. Thereaer, the crude extract (con- interactions between undesirable solutes and the surface of the
taining phenolics and interferants) was passed through the HLB matrix. Additionally, depending on the solid phase selected
matrix. The interferants were eluted from the cartridge with and the solutes being fractioned, the use of SPE may require
water, and this eluted fraction was analyzed also by the F–C using a strong organic solvent during the elution stage.
assay. Based on the hypothesis of the authors, the difference Organic solvents (such as diethyl ether and ethyl acetate) need
between the response of the crude extract and the eluted polar to be removed using reduced pressure and/or high tempera-
fraction represented the TPC in the original sample (Fig. 1). ture before F–C assay is conducted since most of them are not
This subtraction approach aims at achieving a more accurate compatible with the F–C assay.33 Although a vacuum drying
estimation of TPC from biological samples. step may be used for solvent removal, this process induces
Certainly SPE is a well-characterized technique with losses in the products of interest due to hydrolysis, isomeri-
advantages such as selectivity,26,32 reproducibility,36 and a wide zation, and polymerization at temperatures above 40 # C.33
diversity of solid matrixes (i.e. C18, HLB, MCX, etc.) to separate Once the organic solvent is removed, the sample may be
compounds based on their physicochemical characteristics.27 reconstituted with methanol before analysis. Likewise, the
However, this method also has some disadvantages that need reuse of the cartridge can result in decreased reproducibility
to be considered. PC are very diverse in nature and vary within because some polymeric polyphenols can be retained in the
a wide range of hydrophobicity (from very polar to fairly matrix, resulting in weaker interactions between PC and the
hydrophobic), while in most cases the reducing interferants cartridge.27,39 Based on this, the cartridge may be used for a
usually found in biological samples are primordially hydro- limited number of times before being replaced,13 generating
philic. Therefore, it is difficult (or even impossible in some further drawbacks from the economical and methodological
cases) to select a solid matrix capable of fractionating polar point-of-view. These disadvantages imply that although the use
phenolic compounds from the polar interferants. For instance, of SPE-aided strategies may be effective in some cases, the
low retention yields are usually achieved for phenolic acids amount of work involved in their optimization and standard-
(hydroxycinnamic and hydroxybenzoic acids as well as their ization based on the characteristics of the sample being
derivatives) when using SPE.27 This is due to the weak analyzed is not trivial.

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3.2 Calculation of a corrected total phenolic content (TPC) conditions. This methodology has not been explored yet by
value by subtracting the reducing activity of interferants other research groups to the best of our knowledge. In this
present in the plant crude extract regard, preliminary data evaluating the performance of the
proposed two-stage approach for the simultaneous quantica-
The subtraction of reducing activity of interferants from the
tion of AA and TPC are presented in the following sections.
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 3.3 Treatment of phenolic extracts with hydrogen peroxide
vitamin C13 during TPC determinations, which are the most (H2O2) to oxidize interferants
important interfering compounds present in fruits and vegeta- An additional approach that may be used to improve the spec-
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bles that react in the F–C assay.13,21,33 icity of the F–C assay for TPC determinations is the treatment
As suggested by Slinkard and Singleton,40 the corrected TPC of plant food extracts or fruit juices with oxidative agents before
values in sweet wines can be obtained by adding sugar to the performing the F–C assay. By treating plant extracts with
standard solutions (i.e. gallic acid solution) equivalent to the oxidative agents, the interfering compounds would be oxidized,
level in the samples. Corrections to be subtracted from the TPC decreasing their response to the F–C reagent. This approach has
found in sweet wine were determined by preparing standard been recently proposed by Ford et al.15 and is still under
curves of gallic acid (GA) added with glucose–fructose (1 : 1) at investigation. The authors suggested that AA interferants could
different levels. In addition, those sugars were added to dry be eliminated by treating fruit juices with ascorbate oxidase
wines prior to analysis. The authors observed that fructose was (AO) followed by the addition of H2O2. In this method, AA is
the sugar that produced more blue color formation during the transformed to DHA by AO, and the remaining DHA is oxidized
F–C assay. Unfortunately, using correction factors to eliminate into non-reducing compounds by H2O2 ("600 ppm). H2O2
the response given by sugars when performing the F–C assay treatments affected by "10% the total PC content of juice and
would be quite impractical because it would be necessary to non-juice PC model systems, losses that are not signicant
determine the exact concentration of the different sugars in compared with the large errors commonly exerted by AA
each biological sample. In order to determine the individual ("100% of error) or DHA (20–40% error) present in orange juice
sugars content it is usually necessary to utilize sophisticated samples. In addition, H2O2 can oxidize reducing sugars.42
laboratory equipment such as an HPLC coupled to a refractive Further experiments are required to evaluate if the application
index detector. Once they are determined, standard solutions of oxidative agents prior to performing the F–C assay is a suit-
containing the exact concentrations of sugars present in the able approach to eliminate interferants from diverse plant food
extract must be prepared. extracts.
Isabelle et al.14 proposed the subtraction of AA contribution
to TPC results in order to obtain a more accurate quantication 4 Comparison between methodological
of TPC in common vegetables. In this approach, AA was rst approaches to improve the specificity of the
determined by a HPLC method. In addition, an AA standard was
F–C assay for total phenolic content (TPC)
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
determinations
AA. To obtain the corrected TPC of the vegetables analyzed, the Different approaches to eliminate interferants during the
authors multiplied the AA content of the sample by 0.872 and quantication of TPC in plant food extracts by the F–C assay
the result was subtracted from the TPC obtained. This strategy have been discussed earlier herein. In this section of the paper,
could be simpler if a specic spectrophotometric method for AA the feasibility of applying two simple methodologies to elimi-
determinations would be utilized for AA quantication instead nate the contribution of interferants to TPC is evaluated and
of an HPLC method. Okamura41 described a spectrophoto- compared with those previously proposed based on the SPE
metric method that would be suitable for this purpose. strategy13 and based on calculation of a corrected TPC value.14
As mentioned earlier herein, when performing the F–C assay The methodologies evaluated in the present paper consist of: (i)
before the addition of the alkali, AA rapidly reacts with poly- the application of H2O2 to plant food extracts prior to F–C assay
phosphotungstate from the F–C reagent. Therefore, the blue in order to oxidize interfering compounds and (ii) the simul-
color formation observed under the initial acidic conditions of taneous quantication of vitamin C and TPC using the F–C
the F–C assay may be attributed to the AA content in the plant assay to obtain a corrected TPC value. Both methodological
food extract (eqn (2)). It would be interesting to explore the approaches proposed to eliminate the contribution of inter-
feasibility of subtracting the absorbance value at 765 nm fering compounds to the F–C assay as well as the methodologies
obtained under the acidic conditions of the F–C assay (blue described by Georgé et al.13 and Isabelle et al.14 are explained in
color developed by AA) from that obtained under alkaline detail in the following sections of the paper.
conditions (blue color developed by PC) to obtain a corrected
TPC value. By using such an approach, the AA content of plant 4.1 Experimental approach
food extracts would be also quantied by comparing the
4.1.1 Chemicals. AA, DHA, a-D-glucose (GLU), D-(!)-fruc-
absorbance obtained under acidic conditions of the F–C assay
with the absorbance of AA standard solutions under the same 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 Fig. 2 Procedure to oxidize interfering compounds in crude extracts by treating
approaches described herein to improve the specicity of the extracts with hydrogen peroxide (H2O2) prior to total phenolic content determi-
F–C assay for TPC quantication. 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
4.1.3 Preparation of model systems containing phenolic
mixture is vortexed and subjected to the F–C assay. Absorbance values are
and interfering compounds. Model systems containing AA, compared against a standard curve of chlorogenic acid treated with H2O2.
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- food extracts or model systems where TPCs were to be deter-
anol. The concentrations of the interferants used in the model mined (15 mL) were diluted with distilled water (240 mL) in a
systems were established based on their concentration expected 96-well microplate well. Thereaer, the F–C reagent (0.25 N, 15
in methanol extracts of fruits rich in AA, DHA (i.e. strawberry, mL) was added. The mixture was incubated for 3 min, and
kiwifruit),23 and reducing sugars (banana, apple, pineapple) as Na2CO3 (1 N, 30 mL) was added. The nal mixture was incu-
reported in the USDA nutritional database. The nal concen- bated for 2 h at room temperature in the dark. Spectrophoto-
tration of individual PC, AA, and DHA (commercial standards) metric readings at 765 nm were collected using a plate reader
in the model system treated with H2O2 was 312.5 mM, whereas (Epoch, BioTek Instruments, Inc. Winooski, VT). Absorbance
the nal concentration of GLU and FRU was 625 mM and values were compared against a standard curve of CHA and
468 mM, respectively. results were reported in mg of CHA equivalents per 100 g fresh
4.1.4 Treatment of model systems and plant food extracts weight (FW).
with hydrogen peroxide (H2O2). The H2O2 treatments were 4.1.7 Simultaneous quantication of total ascorbic acid
performed by mixing either the model system or plant food (AA) and total phenolic content (TPC) by the Folin–Ciocalteu
extracts (500 mL) with H2O2 solution (300 mL). H2O2 solutions (F–C) assay. As described earlier herein, AA reacts at the
(0.25–2.00 M) were prepared in methanol. The TPC of samples beginning of the F–C assay when the plant food extract is mixed
treated with H2O2 was determined by the F–C assay. Likewise, to with the F–C reagent (eqn (2)). At that point, under the acidic
evaluate the effect of H2O2 treatments on the stability of conditions of the F–C assay PC do not react with the F–C reagent
phenolics, the compounds were detected and quantied by because they need to be in the form of phenolate ions to be able
HPLC-photodiode array (PDA) aer treating the samples with to donate electrons (eqn (1)), which occurs only under alkaline
H2O2. This methodology is summarized in Fig. 2. conditions. Therefore, to quantify AA in the plant food extract,
4.1.5 Partial purication of phenolic compounds (PC) by blue color formation was spectrophotometrically determined
using solid phase extraction (SPE). PC in plant food extracts aer the addition of the F–C reagent at the beginning of the F–C
were partially puried using the SPE procedure reported by assay. For this, the plant food extract (15 mL) was diluted with
Georgé et al.13 The SPE cartridge used in this procedure was an distilled water (240 mL) in a 96-well microplate well and the F–C
Oasis HLB (Waters, Milford, MA, USA). Before passing the plant reagent (0.25 N, 15 mL) was added. The mixture was incubated
food extract through the cartridge, it was conditioned with 4 mL for 3 min and absorbance values were determined at 765 nm
of pure methanol and rinsed with 2 $ 4 mL of water. Then, the using a plate reader. The absorbance values obtained were
extract (3 mL) was passed through the cartridge and the water- attributed to the presence of AA in the extract and thus were
soluble compounds were recovered with 2 $ 2 mL of distilled compared against a standard curve prepared with AA solutions
water. The nal volume of the water-soluble compounds was (0.1–3.0 mM) in order to quantify total AA in the extract.
adjusted to 10 mL. The TPC was quantied in the crude extract To obtain the TPC value in the sample, the assay was
and in the water-soluble extract eluted from the column. To continued by adding Na2CO3 (1 N, 30 mL) to the mixture used to
obtain the corrected TPC calculations were performed as shown determine AA (with extract, water and F–C reagent) and it was
in Fig. 1. incubated for 2 h at room temperature in the dark. Spectro-
4.1.6 Quantication of total phenolic content (TPC) by the photometric readings at 765 nm were collected using a plate
Folin–Ciocalteu (F–C) assay. The TPC was determined with the reader and absorbance was compared against a CHA standard
F–C assay18 adapted to a 96-well microplate.43 Briey, plant curve.

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4.1.8 Quantication of total ascorbic acid (AA) by the a, a0 - from the TPC obtained in the plant food extract to calculate the
bipyridyl method. Total AA in plant food extracts was quantied corrected TPC. This methodology is summarized in Fig. 3.
by the a, a0 -bipyridyl method41 adapted to the 96-well micro- 4.1.10 Identication and quantication of phenolic
plate format.44 Briey, the extract (100 mL) was placed in a 2 mL compounds (PC) by HPLC-PDA. In order to determine the effect
tube and mixed with DTT solution (20 mM, 100 mL). The of H2O2 treatments on individual PC, the qualitative and
mixture was incubated for 10 min at room temperature and in quantitative analysis of PC was performed by high performance
the dark. Thereaer, NEM solution (0.5%, 100 mL) was added to liquid chromatography with photodiode array detection (HPLC-
the mixture and incubated for 30 s. Finally, TCA (10%, 500 mL), PDA) as previously described.45 The HPLC system used was
H3PO4 (43%, 400 mL), a, a0 -bipyridyl (4%, 400 mL) and FeCl3 (3%, composed of two 515 binary pumps, a 717-plus autosampler,
200 mL) solutions were added to the assay tubes. The assay tubes and a 996-photodiode array detector (Waters Corp, Mildford,
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were incubated at 37 # C for 1 h. Then, 200 mL of the reaction MA). The PC were separated on a 4.6 mm $ 250 mm, 5 mm, C18
solutions from the assay tubes were placed in a well of a clear reverse phase column (Luna, Phenomenex, Torrance, CA, USA).
96-well microplate and absorbance readings were collected at The mobile phases consisted of water (phase A) and meth-
525 nm. Absorbance values were compared against an AA anol:water (60 : 40, v/v, phase B) adjusted to pH 2.4 with formic
standard curve (0.15–10 mM) prepared in methanol. AA calcu- acid. The gradient solvent system was 0/100, 3/70, 8/50, 35/30,
lated by this method was used to obtain corrected values of TPC 40/20, 45/0, 50/0, and 60/100 (min/% phase A) at a constant ow
as described in the following section. rate of 1 mL min!1. Chromatographic data were processed with
4.1.9 Calculation of a corrected total phenolic content the Millennium soware V3.1 (Waters Corp, Mildford, MA). The
(TPC) value based on total ascorbic acid (AA) quantication. As identication of individual phenolics was based on their PDA
suggested by Isabelle et al.14 a corrected TPC value can be spectra characteristics as compared with authentic standards of
obtained if AA is quantied in the plant food and based on its the compounds. For the quantication of individual PC, stan-
AA content its contribution to the F–C assay can be subtracted dard curves of CHA, RES, Q 3-O-G, and FA were prepared at a
from the TPC of the extract to obtain a corrected value. range of 0.5–100 mM.
Following this approach, to obtain the corrected TPC value, the
total AA content quantied (in mg mL!1) by either the a, a0 - 5 Results and discussion
bipyridyl method or the F–C assay (as described earlier) was
5.1 Treatment of model systems and plant food extracts with
used to estimate the AA reducing activity in the plant food
hydrogen peroxide (H2O2)
extract. For this, a standard solution of AA was evaluated
following the F–C assay for TPC determination, and it was The effect of H2O2 pre-treatments on the development of blue
found that 1 mg of AA had a reducing activity equivalent to color response (absorbance @765 nm) during the determina-
1.43 mg of CHA. Therefore, the total AA content in the plant tion of total PC in model systems containing pure individual PC
food extract was multiplied by 1.43. The value obtained repre- and interferants is shown in Table 1. The methodological
sented the AA reducing activity in the extract and was subtracted 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
Fig. 3 Proposed Folin–Ciocalteu (F–C) assay procedure for the simultaneous determined by HPLC-PDA (Table 2). H2O2 pre-treatment did not
quantification of ascorbic acid (AA) and total phenolic content (TPC) in plant affect the concentration of individual PC. Therefore, the higher
crude extracts, and for the calculation of a corrected TPC value. To quantify AA, or lower blue color formation shown by individual PC when pre-
blue color formation is spectrophotometrically determined after the addition of
treated with H2O2 and subjected to the F–C assay cannot be
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)
attributed to degradation. Regarding the interferants, H2O2
with 1.43 and the value obtained is subtracted from the TPC. Results calculated resulted in an effective approach to decrease their response
are expressed in mg of chlorogenic acid (CHA) equivalents per mL. during the F–C assay (Table 1). Reducing sugars (FRU and GLU)

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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 for total PC determinations. However, it is important to take
and DHA. H2O2 pre-treatments on model systems containing a into consideration that H2O2 can increase the sensitivity of
mixture of interferants reduced up to 75% the development of certain PC to donate electrons during the F–C assay. In order to
response in the F–C assay. Furthermore, the model system compensate this effect the standard curve used to calculate total
containing individual PC + interferants showed a lower PC must be performed with the standard compound pre-treated
response to the F–C assay when pre-treated with H2O2. Mixing with H2O2 under the same conditions of the sample. Likewise, it
500 mL of this model system (individual PC + interferants) with is important to consider that if the extract contains trace tran-
1.50 M H2O2 (300 mL) reduced by approximately 20% the sition metal ions, they can catalyse the oxidation of PC in the
absorbance values obtained in the non-treated model system presence of H2O2, decreasing their response in the F–C assay.46
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 5.2 Calculation of a corrected total phenolic content (TPC)
development observed in the H2O2 treated model system con- value based on the simultaneous quantication of total
taining individual PC + interferants can be attributed to the ascorbic acid (AA) and phenolic compounds (PC) in plant food
degradation of the interferants. extracts
These results demonstrate that indeed H2O2 pre-treatments As mentioned earlier, plant food methanol extracts (strawberry,
may result in an improvement of the specicity of the F–C assay 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 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 5.3 Comparison between methodological approaches to
reagent to the plant food extract at the beginning of the F–C assay. improve the specicity of the Folin–Ciocalteu (F–C) assay for
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
total phenolic compound (TPC) determinations
N, 15 mL) was added. The mixture was incubated for 3 min and The TPC values for strawberry, kiwifruit, and carrot obtained in
absorbance values were taken at 765 nm using a plate reader. The
absorbance values obtained were attributed to the presence of the methanol extracts and aer the application of the different
ascorbic acid in the extract and thus were compared against a approaches to improve the specicity of the F–C assay for TPC
standard curve ( y ¼ 0.4758x + 0.0048; R2 ¼ 0.9992) prepared with AA determinations are shown in Table 4. As expected, the different
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 procedures (SPE, H2O2 pre-treatment, subtraction of AA
in the samples. In this method, the plant extract was compared reducing activity from TPC) to eliminate the interferants
against a standard curve of AA ( y ¼ 5.9603x + 0.0111; R2 ¼ 0.9999). 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
the F–C assay using the traditional procedure and the methods interferants with H2O2 (Fig. 2) resulted in "54% lower quanti-
reviewed and proposed herein to improve the specicity of the cation of TPC, followed by the corrected TPC value (based on
method. One of the strategies evaluated to improve the speci- the total AA content, Fig. 3) and by the TPC quantied in the
city of the assay, consisted of the quantication of total AA to methanol extract using the SPE approach (Fig. 1).
obtain a corrected TPC value by subtracting from the TPC the AA A similar behaviour was observed for kiwifruit. However,
reducing activity in the extract. Therefore, the total AA content the application of the different methodologies to eliminate
in the plant food extract was quantied by the a,a0 -bipyridyl interferants reduced in a higher percentage the TPC values
method. Likewise, the procedure proposed herein involving the obtained in the methanol extracts. For instance, H2O2 treated
simultaneous quantication of AA and TPC based on the F–C methanol extracts of kiwifruit showed "84% lower TPC values.
assay was evaluated (Fig. 3). The results showed no signicant This can be attributed to the lower TPC and higher reducing
difference ( p > 0.05) between the total AA values obtained by sugar content reported for kiwifruit as compared with straw-
either of the two methods utilized (F–C assay or the a,a0 -bipyr- berry.23,48 Interestingly, for carrot the treatment that showed
idyl method) (Table 3). the lower TPC value when compared with the methanol extract
These results conrm that the F–C assay-based strategy was the elimination of interferants by SPE. This approach
proposed herein indeed can be applied for the simultaneous reduced by "89% the TPC quantied in the crude extract.
quantication of total AA and TPC in plant food extracts. This 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).

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the main PC in the plant tissue, the main PC in carrots are which can be obtained by subtracting the AA reducing response.
phenolic acids.43,45 Phenolic acids have been reported to have In addition, the method proposed for the simultaneous quan-
low retention yields when using SPE cartridges.27 This is due to tication of AA and TPC using the F–C assay may be used for
the weak interactions between these polar compounds and the this purpose. This approach represents a remarkable advantage
solid matrix.37,38 This unwanted elimination of phenolic acids because with the simple F–C assay it is possible to quantify both
along with the interferants resulted in an underestimation of the TPC and total AA without additional requirements of
the TPC when using the SPE strategy (Fig. 1).13 Therefore, the chemicals and equipment.
use of this strategy to eliminate interferants from plant food
extracts is only recommended when most of the PC are Acknowledgements
amphipathic in nature, such as avonoids. On the other hand,
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the corrected TPC value obtained (by subtracting AA reducing This study was supported by research funds from the Tec-
activity, Fig. 3) showed "20% lower TPC value and the appli- nológico de Monterrey – Research Chair Initiative (CAT 161) and
cation of H2O2 pre-treatments in the extract reduced by "40% Cátedra de Nutrigenómica-FEMSA. Author J.C.S.-R. also
its TPC value. acknowledges the scholarship (169222) from the Consejo
Results suggest that the calculation of a corrected TPC value, Nacional de Ciencia y Tecnologı́a (CONACYT, México).
based on the calculation of the reducing activity of AA present in
the extract, is the best approach to obtain a better estimation of Notes and references
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This journal is ª The Royal Society of Chemistry 2013 Anal. Methods, 2013, 5, 5990–5999 | 5999

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