Analytica Chimica Acta 427 (2001) 119–127

Approach to the content of total extractable phenolic compounds

from different food samples by comparison of chromatographic
and spectrophotometric methods
A. Escarpa, M.C. González∗
Departamento de Quı́mica Analı́tica, Facultad de Ciencias, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain
Received 19 April 2000; received in revised form 6 September 2000; accepted 7 September 2000

Abstract
A new approach to the error sources in the spectrophotometric determination of total phenols in foods has been performed.
The choice of the suitable phenolic standard and the influence of sugars and proteins as interfering compounds were carefully
studied. The results obtained by the spectrophotometric method were compared with those found from the chromatographic
method which was taken as reference method because it was free of interferences. The spectrophotometric method overesti-
mates the phenolic content except in some fruit samples with a high polyphenolic content. Sugars did not show interference
whereas protein showed a high influence on the total phenols at the concentration ranges found in the extracts. In green bean
samples both methods gave the same total phenols when the interference was masked. This fact could constitute an useful
way to find the real content of phenolics in foods. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: HPLC; Spectrophotometric; Phenolics; Foods

1. Introduction many cases. In other instances, isolation and identi-

Polyphenolic compounds are a complex group of
substances that have gained enormous attention in the
last years, especially within the analytical chemistry
field, because they exhibit important quality properties
[1–5] and antioxidant activity [6–15].
Many analytical procedures have been developed to
study polyphenolic compounds, which reflect their im-
portance for undertaking the analysis. In some cases,
profiling the phenolic content is necessary to exam-
ine process-related variability of phenolic composi-
tion, whereas the quantification is the ultimate goal in
∗ Corresponding author. Tel.: +34-91-885-50-98;
fax: +34-91-885-49-71.
E-mail addresses: alberto.escarpa@alcala.es (A. Escarpa),
cristina.gonzalez@uah.es (M.C. González).
fication of unknown phenolic compounds is done on
request. Therefore, the design of the analytical pro-
cedure will depend very much on the intent of the
ensuing analysis. In general, profiling and quantifica-
tion studies are undertaken as a preliminary step. Con-
sequently, the most successful approaches have been
based on both, chromatographic and spectrophotomet-
ric methods. Thus, HPLC has been the method of
choice because of its versatility, precision, and rela-
tively low cost. Most frequently, the method is used on
the reversed-phase C18 or C8 columns in conjunction
with aqueous mobile phases and methanol, acetonitrile
buffers as modifiers. In so much that spectrophotomet-
ric methods are employed for quantification purposes
only, where total phenols are indiscriminately obtained
from the extracts in both batch [16–19] and FIA vari-
ants [20,21]. Nonetheless, there are many insuperable

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 3 - 2 6 7 0 ( 0 0 ) 0 1 1 8 8 - 0
120 A. Escarpa, M.C. González / Analytica Chimica Acta 427 (2001) 119–127
difficulties associated with these determinative tech-
niques and their usefulness is often questioned. Firstly,
because the exhaustive extraction with alcoholic and
aqueous alcoholic solvents is likely to exclude much
of the tannin and other phenolics bounded to the cell
wall, in which case measurement of total phenols is
actually confined to the soluble fraction. Secondly, the
diversity of phenolics implies that the selection of a
reagent and/or absorbing wavelength is rather arbi-
trary. An important analytical disadvantage of direct
spectrophotometric measurements could be attributed
to the lack of selectivity proper of these methods,
mainly because they overestimate the phenolic content
[15]; nonetheless, the spectrophotometric methods are
still widely employed in the analytical chemistry labs
[18,19].
On the other hand, there is a scarcity of scientific
references that deal in depth with analytical evaluation
of polyphenolic determinations when undertaken by
both chromatographic and spectrophotometric meth-
ods. In fact, the interferences due to reducing sugars
and SO2 have been evaluated only in wines [12]
whereas in other instances, i.e. apple samples, inter-
ference studies were never conducted [21]. Likewise,
only on a few occasions, the total phenols obtained
by both methods were ever scrupulously compared
[22–24] and the resulting divergences analyzed or
discussed. Furthermore, the quantitative comparison
among the results as reported in the literature is not an
easy task because much too often, the employed ex-
traction and chromatographic conditions seem to vary
from one report to other. Taking into account all the
before-mentioned inconveniences and drawbacks, it is
only reasonable to assume that the comparison of ob-
served differences during simultaneous employment
of both methods could supply valuable information
concerning viability and advantages when it comes to
quantifying polyphenols.
The aim of this work was to investigate the
best approach for studying interferences in the
Folin–Ciocalteu method with respect to chromato-
graphic method, in order to find out the differences
between both methods with quantitative purposes.
This approach could serve as an excellent tool for
total polyphenolic content determination. To fulfill
this objective we have relied on the results obtained
through HPLC method, which were taken as reference
values since this method is free of interferences.
2. Experimental
2.1. Materials and reagents
Methanol (MeOH) (HPLC grade; Scharlau,
Barcelona, Spain), phosphoric acid (H3 PO4 ) (Merck)
and HPLC grade water (Milli-Q system, Milli-
pore, Bedford, MA) were used in the prepara-
tion of mobile phases. The standards, arbutin,
gallic, chlorogenic, caffeic and coumaric acids,
(+)-catechin and (−)-epicatechin, phlorizdin, rutin,
quercetin, myricetin, daidzein, genistein, apigenin
and kaempferol were acquired from Sigma (Chemical
Co. St. Louis, MO, USA). (+)-Merck’s glucose and
albumin were used as spectrophotometric standards.
2.2. Samples
Fresh red wine pomace (Vitis Vinifera) was acquired
from Valdepeñas (Spain). The apples used in analy-
sis belonged to the following varieties: (Malus domes-
tica) Golden and Reineta, while pears (Pyrus comu-
nis) came from Pasagrana and Decana varieties; veg-
etables were represented by fresh green beans (Phase-
olus vulgaris) from two varieties (1 and 2) and dried
lentils (Lens culinaris). All samples were studied as
representative polyphenolic sources in the optimiza-
tion process.
2.3. Procedures
Samples were acquired in different local super-
markets from Alcalá de Henares (Alcalá de Henares,
Madrid, Spain). Both apples and pears were peeled
and the peel was separated from the pulp. Whole
green beans and peel fractions were carefully ho-
mogenized and the pulp was cut up into little pieces.
Lentils were ground to a particle size of 1 mm.
Five gram samples of fresh red wine pomace, 5
and 10 g of peel and pulp (apples and pears), re-
spectively, 5 g of fresh green beans and 2 g of dried
lentils were extracted at room temperature and in the
absence of light with methanol (pure methanol for
fruits and 80% v/v aqueous methanol for red wine
pomace, fresh green beans and lentils) containing 1%
2,6-di-tert-butyl-4-methylphenol (BHT) as an antiox-
idant agent in an ultrasonic bath. The conventional
extraction procedure was carried out according to the
 A. Escarpa, M.C. González / Analytica Chimica Acta 427 (2001) 119–127
following method in order to obtain a quantitative
extraction (higher than 95% in all polyphenols of
interest): samples were extracted twice with 10 ml of
solvent for 1 h, 10 ml for 30 min, and then with 5 ml
for 30 min. The extracts were combined to a final
volume of 25 ml. Solutions for HPLC analysis were
filtered through membrane filters (0.5 ␮m pore size)
prior to the injection. Immediately after the sampling,
tissues were extracted under non-oxidative conditions
and the extracts were stored at −20◦ C before chro-
matographic and spectrophotometric analysis (n = 5).
All the preparations (solutions and extracts) were
filtered through 0.45 ␮m membranes (Millipore) and
degassed in an ultrasonic bath right before analysis.
All food samples were checked with respect to their
chromatographic profile after the final optimization of
the chromatographic method and before quantification
studies designated as analytical quality control.
2.3.1. High-performance liquid chromatography
A HPLC Varian model system consisting of ternary
solvent delivery system (9012), an autosampler
(9100), and a photodiode array detector (9065) cou-
pled to an analytical workstation were employed. A
Waters system consisting of two pumps (510), gra-
dient controlled and UV–VIS detector (481) were
also employed. Polyphenols were detected at 280 nm.
The injection volume was 20 ␮l. The separation
was carried out with a Nucleosil 120 C18 column
(25 cm × 0.46 cm i.d.) with 5 ␮m packing. The elution
solvents were classified as A (aqueous 0.01 M phos-
phoric acid) and B (100% methanol), respectively.
The samples were eluted according to the following
binary gradient: 5% in B as initial conditions; 50%
in B during 10 min; 70% in B during 5 min, 80% in
B during 5 min and finally 100% in B during 5 min.
The flow rate was 1 ml/min.
Identification of compounds was made by compar-
ing their tR values and UV spectra against those of
standards stored in a data bank whenever these were
commercially available. In cases where the polyphe-
nolic compounds were commercially unavailable, the
provisional identification was made from the spectra
characteristics, hydrolysis and isolation studies, as
well as by comparing the most relevant bibliographical
data. Procyanidins were isolated from apples by using
Sephadex LH-20. Two grams were swelled in water
beforehand and introduced into the column (0.7 cm ×
121
30 cm). The bed was washed with methanol (20%)
and 3 ml of apple extracts were carefully applied into
the column. Phenolic acids were eluted with methanol
20% and separated from the procyanidin fraction.
These compounds were eluted from the column with
methanol. The isolation procedure was carefully con-
trolled by DAD and no impurities were observed. The
hydrolysis studies were performed on 5 g of samples
of interset were extracted with ethyl acetate, and af-
terwards 3 ml of these extracts were hydrolyzed in
2 N HCl at 100◦ C for 30 min under constant shaking
conditions and the final mixture was passed through
a C18 Sep-Pak. Aglycons retained on the Sep-Pak
were eluted with methanol, and then analyzed at
350 nm.
2.3.2. Spectrophotometric procedures
A Hewlett Packard (8452A) spectrophotometer was
used. In the case of the Folin–Cicalteu method [25],
a mixture of 0.5 ml of each extract or spectrophoto-
metric standard, 0.5 ml of Folin–Ciocalteu reagent and
10 ml of Na2 CO3 1 M were introduced in a 25 ml vol-
umetric flask. After letting them react for 1 h, the solu-
tions were measured at 750 nm. The Anthrone method
[26] was employed for the determination of soluble
sugars. For this purpose 0.5 ml of each extract was
mixed with Anthrone reagent (prepared in the same
day) and the mixtures were heated at 100◦ C in concen-
trated H2 SO4 media to be then measured at 650 nm.
Coincidentally, the protein content was determined
by relying on the Kjeldahl method [27].
2.4. Recovery studies
The recovery efficiency was determined by adding
measured amounts of seven representative standards
(arbutin, gallic acid, (+)-catechin, (−)-epicatechin,
rutin and quercetin) to the samples prior to extraction
of sample-tissues. The samples were prepared as de-
scribed above and 20 ␮l of the filtrate was injected
into the HPLC column. Controls from all studied
samples were prepared and subjected to the same ex-
traction procedure. The recoveries were determined
by subtracting the values obtained for the control ma-
trix preparation from those of the samples prepared
with the added standards. The recovery experiment
was performed at three concentration levels, taking
into account variety and matrix in a large number of
122 A. Escarpa, M.C. González / Analytica Chimica Acta 427 (2001) 119–127

samples. Mean values with standard deviations are Wallis test) and no parametric method for two samples
reported. (Kolmogorof test) (P ≤ 0.05).

2.5. Quantitative determination of interferences

To quantify sugar and protein interferences, stan-
dard solutions corresponding to interference com-
pounds were prepared and then subjected to the
spectrophotometric analysis. Linearity studies of cali-
bration slopes for both interferences were conducted.
The response was determined for each interference
concentration (previously quantified) and then re-
calculated from the gallic acid’s calibration curve.
The corrected phenolic concentration was expressed
as the difference between the total phenolic con-
tent (including interference) and the total phenolic
content obtained only from the interference com-
pound. The regression equation for albumin was
A = 0.018 + 12.4 × 10−3 C(r = 0.9990), linear
range 5–200 ␮g ml−1 . The regression equation for
(+)-glucose was A = 0.001 + 9.4 × 10−6 C(r =
0.9990). In both cases A is absorbance and C concen-
tration in ␮g ml−1 .
2.6. Statistical analysis
The statistical analysis was performed using no
parametric method for multiple samples (Kruskal–
3. Results and discussion
To achieve the previously discussed goal, the fol-
lowing steps in the working strategy were carried out:
different food samples were chosen as representative
samples of the main polyphenolic classes: hydroxy-
benzoic acids, hydroxycinnamic acids and flavonoids
(flavan-3-ols, chalcones and flavonols). Conventional
ultrasonic extraction was performed in all investi-
gated fruit and vegetable samples. Solvent, extraction
time and number of extractions were optimized using
HPLC and spectrophotometric methods. After quanti-
tative extraction (extraction higher than 95%) the ex-
tracts were analyzed by using spectrophotometric and
chromatographic methods. Finally, a study of inter-
ferences in relation to the spectrophotometric method
was carried out and both methods were compared
when the found interferences proved to be masked.
3.1. Analysis by chromatographic method
Table 1 summarized the retention times including
standard deviation (S.D.) for five replicates of each
phenolic standard used to quantify phenolics in each

Table 1
Phenolic compounds standards employed in the validation of chromatographic method
Peak no. Phenolic compound Phenolic structure Purpose tR ± S.D. (min) UV bands (nm) Recovery (%)
1 Arbutin Hydroquinone – 7.47 ± 0.02 281 98.2 ± 3.1
2 Gallic acid Hydroxybenzoic acid Hydroxybenzoic (der.)a 9.75 ± 0.02 270 101.3 ± 5.4
3 (+)-Catechin Flavan-3-ol Procyanidinsa B1, B2, B3 12.96 ± 0.02 277 100.4 ± 2.1
4 Chlorogenic acid Hydroxycinnamic acid Hydroxycinnamica (der.) 13.96 ± 0.02 240; 326 (max) 99.3 ± 3.0
5 (−)-Epicatechin Flavan-3-ol Procyanidinsa B1, B2, B3 14.45 ± 0.03 277 N.D.
6 Caffeic acid Hydroxycinnamic acid – 15.22 ± 0.03 238; 324 (max) 98.6 ± 2.3
7 Coumaric acid Hydroxycinnamic acid – 17.00 ± 0.03 224; 309 (max) 102.2 ± 1.9
8 Phloridzin Dihydrochalcone Phloretin xyloglucosidea 17.88 ± 0.03 283 N.D.
9 Rutin Flavonol glycoside Quercetin glycosidesa 18.18 ± 0.04 255 (max); 356 100.9 ± 3.0
10 Myricetin Flavonol aglycone Isorhamnetin glycosides 19.21 ± 0.04 251; 367 (max) N.D.
11 Daidzein Isoflavonoid aglycone – 20.06 ± 0.05 246 (max); 299 N.D.
12 Quercetin Flavonol aglycone Hydrolysisb 20.78 ± 0.06 253; 367 (max) 97.9 ± 4.3
13 Genistein Isoflavonoid aglycone Isoflavonoid glycosidesa 21.49 ± 0.06 258 (max); 341 N.D.
14 Kaempferol Flavonol aglycone Kaempferol glycosides 22.46 ± 0.06 262; 367 (max) N.D.
15 Apigenin Flavona aglycone – 23.01 ± 0.06 265; 335 (max) N.D.
a Phenolic structures quantified.
b Hydrolysis identification.
 A. Escarpa, M.C. González / Analytica Chimica Acta 427 (2001) 119–127 123

Table 2
Phenolic structures provisionally identified, spectroscopic parameters and variables involved in the extraction procedure
Sample Phenolic UV bands UV bands VIS bands Solvent Extraction Extraction
structures (nm)a (nm)b (nm)c time (min.) numbers
Red wine Hydroxybenzoic acids 272 278 750 MeOH (80% v/v) 45 3
pomace
Flavan-3-ols 278
Flavonols 255; 354
Apple peels Hydroxycinnamic acids 240; 326 280, 350 750 MeOH 60 3
and pulps
Flavan-3-ols 277
Chalcones 283
Quercetin glycosides 255; 356
Pear peels Arbutin 281 280, 350 750 MeOH 60 3
and pulps
Chlorogenic acid 240; 326
Flavan-3-ols 277
Quercetin glycosides 253; 353
Isorhamnetin glycosides 255; 357
Green beans Single phenols 254 278, 348 750 MeOH (80% v/v) 60 3
Hydroxycinamic 234; 313
Flavan-3-ols 277
Quercetin glycosides 255; 356
Kaempferol glycosides 262; 348
Lentils Hydroxybenzoicderivative 270 278, 350 750 MeOH (80% v/v) in 90 3
HCl media (1% v/v)
Caffeic acid 231; 330
Flavan-3-ols 277
Kaempferol glycosides 263; 344
a Spectroscopic bands obtained for each phenolic structure using DAD.
b HPLC method (spectra obtained in whole extract).
c Folin–Ciocalteu method (spectra obtained in whole extract).

sample as well as UV absorption maxima of each
peak obtained by DAD and recovery efficiency. Like-
wise, Table 1 shows the phenolic structure studied and
the identification and quantitation purposes. Phenolic
structures provisionally identified in the samples as
well as their corresponding absorption maxima band
for each compound are summarized in Table 2.
On the other hand, the chromatographic method was
carefully evaluated with regard to the precision and
accuracy. The precision was evaluated as the repro-
ducibility of the calibration slopes at different time in-
tervals and operators that yielded R.S.D. values within
5%. The accuracy was evaluated by relying on results
from the recovery studies concerning main phenolic
classes and carried out at three different concentra-
tions levels. After taking into account all matrices and
the added phenolic structures, the resulting recoveries
ranged between 92 and 106%. Since the number of re-
covery studies was very high and the recovery percent-
age was satisfactory, we were able to conclude that the
chromatographic method was highly accurate, and at
the same time independent of the sample, matrix, con-
centration and type of investigated phenolic. After the
evaluation of chromatographic method, we proceeded
to determine the concentration of each phenolic, and
then, to calculate the HPLC index as the sum of all
phenolics previously quantified from each sample.
3.2. Extraction procedures
A conventional ultrasonic extraction was developed
and the variables involved in the extraction procedure
were tested to obtain a quantitative extraction of in-
vestigated phenolics. Non-oxidative conditions (pres-
124 A. Escarpa, M.C. González / Analytica Chimica Acta 427 (2001) 119–127

Table 3
Analytical characteristics of the spectrophotometric method
Phenolic Intercept Slope (±Sb) Correlation Detection Determination Linear range
compound (±Sa) (×103 ) (×103 )a coefficient (r) limit (␮g/ml) limit (␮g/ml) (␮g/ml)
Gallic acid 16 (±9) 12.0 (±0.3) 0.9990 0.02 0.08 0.04–8.0
Chlorogenic acid 5 (±4) 6.5 (±0.2) 0.9990 0.04 0.14 0.12–9.5
Caffeic acid −2 (±3) 11.5 (±0.2) 0.9995 0.02 0.08 0.08–8.5
(+)-Catechin 18 (±10) 10.0 (±0.3) 0.9990 0.03 0.10 0.06–9.0
(−)-Epicatechin 14 (±7) 10.5 (±2.5) 0.9990 0.03 0.09 0.07–9.0
Rutin 15 (±9) 5.5 (±2.4) 0.9990 0.05 0.18 0.11–11.0
a Expressed as cm−1 ␮g−1 ml.

ence of BHT and the absence of light during extrac-
tion process) were scrupulously observed in all cases.
The extraction conditions were optimized using HPLC
method. Table 2 lists also the quantitative extraction
conditions of all investigated samples. As it can be de-
duced from Table 2, methanol and aqueous methanol
permitted a quantitative extraction with equilibrium
times that ranged between 45 and 90 min and multiple
extractions were needed in all the examined cases.
3.3. Analytical characteristics of the
spectrophotometric method
the viability of the discussed method to determine
the mentioned compounds in real extracts. As far as
sensitivity (represented by calibration slope) was con-
cerned, different values were obtained for each studied
polyphenolic structure. In fact, sensitivity decreased
in the following order: gallic acid > (+)-catechin >
chlorogenic acid > rutin. Caffeic acid exhibited an
identical slope as that found for gallic acid, while
(−)-epicatechin showed the sensitivity identical to that
found for (+)-catechin. Based on these results the to-
tal polyphenolic content was determined as a function
of the standard employed in the calibration curves.

The main difficulty in the quantification of the total
phenols stems from choosing a suitable spectrophoto-
metric standard because, except on rare occasions, the
total phenolic composition is very complex, and more
than often due to the fact that different structures
are present in the real extracts. In the current study,
the initial effort was geared at choosing a suitable
standard for the determination of the total phenolic
content. Consequently, the calibration curves for six
polyphenolic compound standards, representative of
all polyphenolic structures in studied samples were
investigated. Table 3 shows the calibration characteris-
tics for gallic and chlorogenic acids (as the representa-
tive sample of hydroxybenzoic and hydroxycinnamic
acids), caffeic acid in form of non-esterified hydrox-
ycinnamic acid, (+)-catechin and (−)-epicatechin as
flavan 3-ols isomers and rutin as flavonol glycoside.
Judging from the results, a satisfactory correlation
was obtained within the linear range of all investi-
gated compounds. The detection and determination
limits ranged between 0.02 and 0.05 ␮g ml−1 and 0.08
and 0.18 ␮g ml−1 , respectively. These results proved
3.4. Determination of total extractable phenolics
Table 4 lists the total phenolic content as obtained
by both, HPLC and Folin–Ciocalteu methods. The de-
terminations have been carried out while employing
four phenolic compounds with different calibration
slopes and as representative of studied samples with
a high, intermediate and low phenolic content. As
it can be observed, the spectrophotometric method
overestimated the phenolic content and it was, (as ini-
tially forecasted) highly dependent on the employed
standard. The overestimation factor (FC/HPLC) is
also included in Table 4 and it was defined as the
ratio between the total polyphenol content obtained
by the spectrophotometric method (FC) and the total
polyphenol content obtained by the chromatographic
method (HPLC). The mentioned factor decreased
at the rate of the calibration slope’s increase. The
ensuing results, listed in Table 4, were helpful in
establishing two important conclusions:
First, the relative variation in the total phenol con-
tent obtained by the spectrophotometric method was
 A. Escarpa, M.C. González / Analytica Chimica Acta 427 (2001) 119–127 125

Table 4
Total polyphenolic index by using of chromatographic and spectrophotometric methods employing different standards and interference
contents in representative samples
Sample HPLCa Gallicb FC/HPLC (+)-Catechind Chlorogenic Rutinf Sugarg Proteinh
(mg/kg) (mg/kg) ratioc (mg/kg) acide (mg/kg) (mg/kg) (g/kg) (g/kg)
(R.S.D.) (R.S.D.) (R.S.D.) (R.S.D.) (R.S.D.)
Red wine pomace 1790 (1.3) 7967 (5.6) 4.5 9267 (6.1) 14622 (5.6) 16160 (5.6) 13 1.8
Dried lentils 1740 (0.8) 6346 (1.8) 3.6 7487 (1.7) 11955 (1.6) 12895 (1.7) 47 49.0
Green beans (variety 1) 171 (4.0) 449 (5.6) 4.0 523 (5.2) 920 (6.7) 920 (5.8) 15 8.7
Green beans (variety 2) 242 (5.2) 458 (5.4) 2.7 575 (5.3) 932 (4.4) 935 (4.8) 15 9.8
Golden apple (peel) 663 (0.7) 2379 (2.4) 3.5 2769 (2.7) 4445 (2.7) 4821 (2.8) 82 1.0
Golden apple (pulp) 176 (1.1) 847 (2.7) 5.0 983 (2.8) 1067 (2.8) 1717 (2.5) 59 0.6
Reineta apple (peel) 3990 (1.5) 3346 (1.6) 1.0 4151 (1.8) 6597 (1.8) 7224 (1.8) 65 1.1
Reineta apple (pulp) 891 (0.6) 1051 (2.3) 1.2 1686 (2.2) 2679 (2.4) 2923 (2.4) 65 0.7
Pasagrana pear (peel) 1491 (2.3) 2510 (1.4) 1.7 2950 (1.3) 4862 (1.9) 5111 (1.5) 68 1.1
Pasagrana pear (pulp) 133 (1.5) 381 (1.3) 2.9 446 (1.3) 764 (1.5) 778 (1.4) 56 0.5
Decana pear (peel) 2172 (3.4) 2378 (3.4) 1.1 2809 (3.5) 4450 (3.2) 4830 (3.4) 47 1.9
Decana pear (pulp) 82 (2.4) 376 (1.6) 4.6 414 (1.4) 760 (1.0) 769 (1.5) 34 0.5
a Total polyphenol index obtained by HPLC method.
b Total polyphenolic index by using the Folin–Ciocalteu method with gallic acid as spectrophotometric standard.
c Total polyphenol index ratio between Folin–Ciocalteu (FC) and chromatographic (HPLC) methods.
d Total polyphenolic content by using the Folin–Ciocalteu method with (+)-catechin as spectrophotometric standard.
e Total polyphenolic content by using the Folin–Ciocalteu method with chlorogenic acid as spectrophotometric standard.
f Total polyphenolic content by using the Folin–Ciocalteu method with rutin as spectrophotometric standard.
g Sugar yields obtained from Anthrone method.
h Protein yields obtained from Kjeldahl method.

very similar to that found by using its chromatographic
counterpart. In fact, both methods indicated that the
fruits were samples with high polyphenol content,
followed by legumes as samples with intermediate
polyphenol content and with green beans as samples
with a low polyphenolic content at the end of the list.
Likewise, both methods proved that fruits possessed
higher polyphenolic content in peels than pulps. On
the other hand, after careful analysis of the results
listed in Table 4, we have came to conclusion that both
methods were similarly effective for analyzing sam-
ples with a high level of polyphenolics (i.e. peels from
Reineta apple and Decana pear). This fact was also
observed in another matrix corresponding to Reineta
apple (peel and pulp). The mentioned apple variety
has been already studied on previous occasions, but to
lesser extent. Insomuch that, in yet another, recently
published study [28], this variety showed high levels
of polyphenols. In those samples the FC/HPLC ratio
ranged between 0.8 and 1.1 whereas in samples with
low polyphenol content FC/HPLC ratio was higher
than 5 (Table 4).
So far, we were able to find in bibliography only a
sketchy comparative study that deals with spectropho-
tometric and chromatographic methods as applied to
the analysis of total polyphenols [22–24]; however, the
mentioned bibliography does not include any study
about the divergences that may exist between both
methods.
3.5. Interference studies
The study of interferences was performed with
the spectrophotometric method in mind, because this
technique showed a low selectivity in relation to the
chromatographic method. Two interferences were
investigated, sugars and proteins. We have concen-
trated on these compounds since they could appear
in the final composition of methanol extracts. The
interferences studies were carried out in represen-
tative samples such as: legumes (high protein and
low sugar contents), vegetables (intermediate protein
and sugar contents) and fruits (low protein and high
sugar contents). Coincidentally, these samples showed
a different polyphenolic content. Subsequently, we
have carried quantitative determination of sugar and
protein by relying on Anthrone and Kjeldahl meth-
ods, respectively. The obtained results are also listed
126 A. Escarpa, M.C. González / Analytica Chimica Acta 427 (2001) 119–127

Table 5
Total polyphenolic index by using of chromatographic and spectrophotometric methods in representative samples
Sample HPLCa Gallicb Yields ratioc Total polyphenolic Protein FC/HPLCf
(mg/kg) (mg/kg) FC/HPLC index corrected correctione (%) corrected ratio
(R.S.D.) (R.S.D.) (mg/kg)d
Red wine pomace 1790 (1.3) 7967 (5.6) 4.4 7345 8 4.1
Dried lentils 1740 (0.8) 6346 (1.8) 3.6 5133 19 3.0
Green beans (variety 1) 171 (4.0) 449 (5.6) 2.6 207 54 1.2
Green beans (variety 2) 242 (5.2) 458 (5.4) 1.9 238 48 1.0
Golden apple (peel) 663 (0.7) 2379 (2.4) 3.6 2311 3 3.5
Golden apple (pulp) 176 (1.1) 847 (2.7) 4.8 811 4 4.6
Reineta apple (peel) 3990 (1.5) 3346 (1.6) 0.8 3276 2 0.8
3990 (1.5) 4151 (1.8)g 1.0 4073 2 1.0
Reineta apple (pulp) 891 (0.6) 1051 (2.3) 1.2 1013 4 1.1
Pasagrana pear (peel) 1491 (2.3) 2510 (1.4) 1.7 2440 3 1.6
Pasagrana pear (pulp) 133 (1.5) 381 (1.3) 2.9 346 9 2.6
Decana pear (peel) 2172 (3.4) 2378 (3.4) 1.1 2292 4 1.0
Decana pear (pulp) 82 (2.4) 376 (1.6) 4.6 341 9 4.0
a Total polyphenol index obtained by HPLC method.
b Total polyphenolic index by using the Folin–Ciocalteu method with gallic acid as spectrophotometric standard.
c Total polyphenolic index ratio between Folin–Ciocalteu (FC) and chromatographic (HPLC) methods.
d Total polyphenolic content corrected by using the Folin–Ciocalteu method with gallic acid as spectrophotometric standard and taking

account the protein interferent as albumin equivalents.

e Effect on the polyphenolic total index of the albumin as interference substance.
f Total polyphenolic index ratio between Folin–Ciocalteu (FC) and chromatographic (HPLC) methods taking account the protein index

interference.
g Total polyphenolic index by using the Folin–Ciocalteu method with (+)-catechin as spectrophotometric standard.

in Table 4. Hence, when it comes to sugar content,
high levels of this compound were obtained for fruits
and legumes, whereas green bean samples proved to
possess low total sugar content. With regard to the
protein content, the investigated samples could be
classified into three groups with a high (legumes),
intermediate (vegetables) and low (fruits) protein
content, respectively.
On the other hand, to quantify the interferences,
each interference compound was analyzed by employ-
ing the spectrophotometric method. (+)-Glucose, used
primarily as an interference standard, exhibited a very
low calibration slope (9.2 × 10−6 cm−1 ␮g−1 ml) and
it appeared as a low sensitivity polyphenolic. The stud-
ies carried out with albumin, used primarily as an
interference standard, exhibited an important interfer-
ence in all studied cases, since the obtained calibration
slope was 12.4 × 10−3 cm−1 ␮g−1 ml, and it appeared
as a high sensitivity polyphenolic with characteristics
similar to those found in gallic and caffeic acids. By
relying on these results, the total polyphenolic index
was corrected for all samples and, the FC/HPLC ra-
tio was recalculated while employing gallic acid as
a spectrophotometric standard. The final results are
summarized in Table 5. As it can be observed, these
results confirmed the validity of the suggested ap-
proach for finding out the divergences between both
methods. The correction for protein in the phenolic
contents ranged between 2 and 60%. This correction
resulted in the overestimation of about 50% in green
bean samples, which allowed to calculate the simi-
lar phenolic content in these samples. However, the
correction was not efficient in fruits, since these sam-
ples presented low protein content. Finally, the to-
tal polyphenolic quantification in some apple samples
(when using of (+)-catechin as standard), allowed se-
curing the same values when employing chromato-
graphic and spectrophotometric methods, probably,
because these phenolic structures were dominant in
the studied samples.
3.6. Statistical analysis
The statistical analysis was carried out in two steps.
First, the no normal distribution fitting of the results,
the HPLC index and the total of all phenols obtained
 A. Escarpa, M.C. González / Analytica Chimica Acta 427 (2001) 119–127
when using different spectrophotometric standards
were examined by Kruskal–Wallis test. Significant
differences among averages were obtained at 95%
of significance level. As it was to be expected, only
HPLC index differed from the total phenols index.
However, no statistically significant differences were
perceivable among total phenols obtained from the
all employed spectrophotometric standards. In con-
tinuation, we have compared the HPLC index with
total phenols lacking interference and found none of
statistically significant differences. This fact could
be also interpreted as a proof of validity of the re-
cently discussed approach for differentiating between
the two analytical methods, since both methods have
shown similar behavior.
4. Conclusions
The method developed in this paper could constitute
an excellent approach for determining the real content
of the polyphenols in complex extracts, since neither
official methods nor reference data are currently avail-
able. The spectrophotometric method overestimated
the polyphenolic content with respect to the chromato-
graphic method, mainly because of non-phenolic ma-
terials present in the investigated extracts interfered
in the spectrophotometric analysis. Non-interfered to-
tal soluble sugars and partially dissolvable protein
showed an important interference in the majority of
the studied extracts. The corrected values allowed de-
termining that the polyphenolic content was similar in
green bean samples. In some cases where sampling
yielded high polyphenolic content, both methods al-
lowed to obtain similar results, probably because the
interference was masked. Nonetheless, more studies
are needed to gain a better understanding about the
divergences found in both methods during current in-
vestigation.
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