Research Article

Received: 14 January 2014 Revised: 16 April 2014 Accepted article published: 22 April 2014 Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6706
Intra-laboratory validation of microplate
methods for total phenolic content
and antioxidant activity on polyphenolic
extracts, and comparison with conventional
spectrophotometric methods
Gloria Bobo-Garca, Gabriel Davidov-Pardo,
*
Cristina Arroqui, Paloma
Vrseda, Mara R Marn-Arroyo and Montserrat Navarro
Abstract
BACKGROUND: Total phenolic content (TPC) and antioxidant activity (AA) assays in microplates save resources and time,
therefore they can be useful to overcome the fact that the conventional methods are time-consuming, labour intensive and
use large amounts of reagents. An intra-laboratory validation of the FolinCiocalteu microplate method to measure TPC and
the 2,2-diphenyl-1-picrylhydrazyl (DPPH) microplate method to measure AA was performed and compared with conventional
spectrophotometric methods.
RESULTS: TocomparetheTPCmethods, thecondenceintervals of alinear regressionwereused. Intherangeof 1070 mgL
1
of
gallic acid equivalents (GAE), both methods were equivalent. To compare the AAmethodologies, the F-test and t-test were used
in a range from220 to 320 mol L
1
of Trolox equivalents. Both methods had homogeneous variances, and the means were not
signicantively dierent. The limits of detection and quantication for the TPC microplate method were 0.74 and 2.24 mgL
1
GAE and for the DPPH 12.07 and 36.58mol L
1
of Trolox equivalents. The relative standard deviation of the repeatability and
reproducibility for both microplate methods were 6.1%. The accuracy ranged from88%to 100%.
CONCLUSION: The microplate and the conventional methods are equals in a 95%condence level.
2014 Society of Chemical Industry
Keywords: FolinCiocalteu; DPPH; grape seed extract; apple extract; green tea extract
INTRODUCTION
Interest in the research of polyphenols from dierent natural
sources has grown because polyphenols can be utilised as antiox-
idants in the food industry, and they benet human health in
various ways. The benecial eects of polyphenols on human
health could be due to their free radical scavenger properties,
blocking the deleterious action of these molecules on cells.
1
In
addition to their antioxidant capacity polyphenols can bene-
t human health in other metabolic ways such as prevention
of cardiovascular diseases, anti-inamatory eects and cancer
prevention.
24
Due to all the above mentioned, antioxidants may
be the most promising functional ingredient to add to a product.
5
Considering that the amount of polyphenols and their antiox-
idant activity is closely related to their action as food additives
or functional ingredients, a fast, cheap and accurate method to
measure the total phenolic content and antioxidant activity of
plant extracts is needed. Nowadays, the most common method
to measure the total phenolic content of all types of sample is
the FolinCiocalteu method, which is based in the reduction of
the phospho-molybdate heteropoly acids Mo(VI) centre in the
heteropoly complex to Mo(V), resulting in a blue colouration
which is measured at around 750 nm.
6
To measure the antiox-
idants scavenging activity DPPH

is considered a valid; and

easy method, because the radical compound (2,2-diphenyl-
1-picrylhydrazyl, DPPH) is stable and does not have to be gen-
erated hours before the analysis, as in other radical scavenging
assays.
7
The quantication of the antioxidant activity is based on
decolourisation of the reagent at around 515 nm.
8
Generally, the
conventional methods to assess the total phenolic content and
antioxidant activity of a sample are time-consuming, labour inten-
sive and use large amounts of reagents.
9,10
To overcome these
drawbacks, the assays in microplates have been done with posi-
tive results on a wide variety of samples: seaweeds,
10
sorghum,
9

Correspondence to: Gabriel Davidov-Pardo, Public University of Navarra, Food

Technology Department, Campus Arrosadia s/n, Pamplona 31006, Spain.
E-mail: aenoltec@unavarra.es
Public University of Navarra, Food Technology Department, Campus Arrosadia
s/n, Pamplona 31006, Spain
J Sci Food Agric (2014) www.soci.org 2014 Society of Chemical Industry
www.soci.org G Bobo et al.
berries
1113
and Cyphostemma digitatum.
14
In some cases after
analysing a determined number of samples, they showed the
values obtained with the new and the reference method. They
showed the recovery percentage, precision and reproducibil-
ity of the new method.
9,15
But statistical comparison between
the results obtained with the microplate and the conventional
methods have not yet been reported.
In the light of this background, the aim of this study was to vali-
date FolinCiocalteu and DPPHmicroplate methods and compare
themwith the conventional ones, using statistical methodologies.
MATERIAL ANDMETHODS
Samples
Grape seed extract (GSE) was provided by Puleva Biosearch
(Granada, Spain). Apple extract (AE) was purchased from Princip-
ium (Viganello, Switzerland). GSE and AE are commercial extracts,
obtained through a hydro-alcoholic extraction. Green tea (Camel-
lia sinensis) extract (GTE) was made by infusing 10 g of green
tea (SoriaNatural, Gargay, Spain) in 60 mL of deionised water at
55

C for 15 min. Then, the plant material was separated using a

cheesecloth and the infusion was vacuum ltered.
16
The samples
were stored at 4

C until used.
Chemicals
Methanol, ethanol water (96:4, v/v) and sodium carbonate
pharmaceutical grade and gallic acid 1-hydrate analytical grade
were purchased from Panreac (Barcelona, Spain). 2,2-Diphenyl-
1-picrylhydrazyl (DPPH) and FolinCiocalteu reagent analytical
grade were purchased from Sigma Chemical Co (St. Louis, MO,
USA). 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
(Trolox; 970 gkg
1
) analytical grade was purchased from Aldrich
Chemical (Steinheim, Germany).
Total phenolic content
Conventional method
The conventional total phenolic content (TPC) method is based on
the FolinCiocalteu European Commission Regulation method.
6
In a100- mL volumetric ask, 1 mL of the diluted extract, 50 mL of
deionised water type I and 5 mL of the FolinCiocalteu reagent
were added and left to react for 300 s. To complete the reaction,
20 mL of a sodiumcarbonate solution (200 gL
1
) were added, and
the volumetric ask was lled to its volume with deionised water.
After 30 min at room temperature, the absorbance of the samples
at 750 nm was measured in polystyrene cuvettes in a Thermo Sci-
entic Multiskan GOspectrophotometer (ThermoFisher Scientic,
Vartaa, Finland). The measured absorbance of the same reaction
with water instead of the extract or standard was subtracted from
the absorbance of the reaction with the sample. The phenolic
content was expressed in gallic acid equivalents per litre after
the preparation of a standard curve of gallic acid from 10 to
200 mgL
1
.
Microplate method
The microplate TPC method was based on the 96-well microplate
FolinCiocalteu method given by Al-Duais et al.
14
and Mller
et al.
17
with some modications. A total of 20 L of the diluted
extract were mixed with 100 L of 1:4 diluted FolinCiocalteu
reagent and shaken for 60 s in a at-bottom 96-well microplate
(NUNC, Roskilde, Denmark). The mixture was left for 240 s and
then 75 L of sodium carbonate solution (100 gL
1
) were added
and the mixture was shaken at medium-continuous speed for
1 min. After 2 h at room temperature, the absorbance was mea-
sured at 750 nmusing the microplate reader of a Thermo Scientic
Multiskan GO spectrophotometer (ThermoFisher Scientic). The
absorbance of the same reaction with water instead of the extract
or standard was subtracted from the absorbance of the reaction
with the sample. Gallic acid dilutions (10200 mgL
1
) were used
as standards for calibration.
Antioxidant activity
DPPHconventional method
The conventional antioxidant activity (AA) was evaluated based
on the technique by Rivero-Prez et al.
8
In a polystyrene cuvette,
2940 L of DPPH dissolved in methanol (60 mol L
1
) were mixed
with 60 L of the diluted extract. The absorbance at 515 nm was
measured after 60 min in the dark using a Thermo Scientic Mul-
tiskan GO spectrophotometer (ThermoFisher Scientic). The %
DPPH quenched was calculated using Eqn 1 and reported as mol
L
1
of Trolox equivalents after the construction of a standard curve
of Trolox (50500 mol L
1
):
% DPPH quenched =
[
1
(
A
sample
A
blank
A
control
A
blank
)]
100 (1)
where A
sample
is the absorbance at 515 nm of 60 L of extract
or standard with 2940 L of DPPH solution after 60 min, A
blank
is
the absorbance at 515 nm of 3000 L of methanol, A
control
is the
absorbance at 515 nm of 60 L of water with 2940 L of DPPH
solution after 60 min.
DPPHmicroplate method
The microplate AA methodology was based on the 96-well plate
assay described by Herald et al.
9
with some modications. A total
of 20 L of the diluted sample was added to 180 L of DPPH solu-
tion (150 mol L
1
) in methanol water (80:20, v/v) and shaken
(a)
(b)
Figure 1. Standard curves for microplate assays. (a) Total phenolic content
(FolinCiocalteu assay); (b) antioxidant activity (DPPH assay).
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for 60 s in a 96-well microplate (NUNC). After 40 min in the dark
at room temperature, the absorbance was measured at 515 nm
in the microplate reader of a Thermo Scientic Multiskan GO
spectrophotometer (ThermoFisher Scientic). Trolox was used as
a standard at 50500 mol L
1
to generate a calibration curve. The
%DPPHquenched was calculated using Eqn 1, where A
sample
is the
absorbance at 515 nm of 20 L of extract or standard with 180 L
DPPH solution after 40 min; A
blank
is the absorbance at 515 nm
of 20 L of water with 180 L methanol water (80:20, v/v) after
40 min, and A
control
is the absorbance at 515 nm of 20 L of water
with 180 L DPPH solution after 40 min.
Limit of detection and quantication
The limit of detection (LOD) of an individual analytical procedure
is the lowest amount of analyte in a sample which can be detected
but not necessarily quantied as an exact value.
18
The LOD was
calculated using Eqn (2):
LOD =
3.3
S
(2)
where is the standard deviation of the blank and S is the slope of
the calibration curve.
(a)
(b)
(c)
Figure 2. Linear regression for total phenolic content methods compari-
son. (a) Grape seed extract; (b) apple extract; (c) green tea extract.
The limit of quantication (LOQ) of an individual analytical pro-
cedure is the lowest amount of analyte in a sample which can be
quantitatively determined with suitable precision and accuracy.
18
The LOQ was calculated using Eqn (3):
LOQ =
10
S
(3)
where is the standard deviation of the blank and S is the slope of
the calibration curve.
Statistical comparison
The condence intervals of the slope and the constant of a linear
regression equation were used to compare the TPC conventional
and microplate methods.
19
A linear regression of ve dierent
concentrations of the GSE, AE and GTE was performed, where the
results of the conventional TPC method represent the indepen-
dent variable while the results of the microplate TPC represent the
dependent variable. To compare the conventional and microplate
AA a statistical comparison based on the F-test and t-test of three
concentrations of the GSE, AE and GTE was performed. To consider
both methods statistically equal with a condence of 95%, the
P-value for the F-test and t-test has to be higher than 0.05.
19
For the TPC method comparison, ve concentrations of GSE (12,
24, 36, 48 and 60 mgL
1
) and ve concentrations of AE (24, 48,
54, 60 and 72 mgL
1
) were prepared in ethanol water solution
(20:80, v/v). Five dilutions (1:10, 1:5, 1:4, 1:3.3 and 1:2.5) from a
1:20 diluted GTE were also used to compare the TPC methods. To
compare the AA methods, three concentrations of GSE (35, 40 and
45 mgL
1
) and AE (50, 60 and 70 mgL
1
) were prepared as in TPC
assays and three dilutions (1:2.5, 1:2 and 1:1.7) from a 1:50 diluted
GTE were used.
Precision and accuracy
Precision is based on the repeatability and reproducibility, which
can be expressed as the relative standard deviation (RSD). Accu-
racy expresses the closeness of a result to a true value.
19,20
In this work, the repeatability was the RSD calculated from
ve repetitions of the analysis of the same sample on the same
day and conditions. The reproducibility was the RSD calculated
from ve repetitions of the analysis in dierent days and daily
prepared reagents. The accuracy was calculated as the percentage
of recovery.
9,15
Statistical analyses
The statistical analyses were performed using Statgraphics Cen-
turion XVI (Statpoint Technologies, Virginia, USA) and Minitab 16
(Minitab Inc., State College, PA, USA) software.
Table 1. Condence intervals at 95% for the estimated coecients
Condence levels
Extract Parameter Estimated coecient Lower Upper
Grape seed Slope 0.9982 0.9551 1.0412
Constant 1.1455 2.7246 0.4336
Apple Slope 0.9656 0.9248 1.0064
Constant 1.3315 2.8692 0.2061
Green tea Slope 0.9838 0.9674 1.0002
Constant 0.2523 1.0406 0.5361
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www.soci.org G Bobo et al.
RESULTS ANDDISCUSSION
Calibration curves and limits of detection and quantitation
The standard calibration curves for the microplate methods are
presented in Fig. 1. The curves are linear when the concentration
of gallic acid is in the range of 10200 mgL
1
(R
2
=0.9998) and
the concentration of Trolox is in the range of 50500 mol L
1
(R
2
=0.9999). When comparing these curves with those obtained
by Herald et al.,
9
who used similar methodologies and the same
concentration ranges, it can be seen that the slight changes in the
methodology had more inuence upon the slope of the AA assay
than on the slope of the TPC assay. The slope of the calibration
curve for the AA in this study was 0.1647, while the slope in
their study was 0.1512; for the calibration curves of the TPC the
slopes were 0.0076 and 0.0073, respectively. However, comparing
the slopes of this work with those obtained by Horszwald and
Andlauer,
13
TPC=3.1761 and DPPH=0.0591, it can be seen that
greater dierences in the TPC and DPPH microplate methods had
a signicant impact on the sensitivity of the methods. That work
had also calculated the limits of detection and/or quantication
for their methods.
Inthis work, the LODandLOQfor the FolinCiocalteumicroplate
method were 0.74 and 2.24 mgL
1
GAE, respectively. In the case of
the DPPH microplate method the LOD and LOQ were 12.07 and
36.58 mol L
1
of Trolox equivalents, respectively. As expected,
because of the greater sensitivity of the Horszwald and Andlauer
method,
13
the LOQ (0.015 mgL
1
GAE) of their TPC microplate
method is lower than the LOQ of this work. On the contrary,
the greater sensitivity of DPPH microplate method of this work,
resulted in a lower LOD than that obtained by Horszwald and
Andlauer,
13
which was 50 mol L
1
of Trolox.
Statistical comparison
Preliminary studies
The dilutions to perform the TPC and the AA comparisons were
established to obtain 10 to 70 mgL
1
of gallic acid equivalents
and mol L
1
concentrations from 220 to 320 Trolox equivalents,
respectively. These were the broadest concentration ranges in
which the three extracts in the microplate method gave determi-
nation coecients higher than 0.999. The determination coe-
cients were obtained by a linear regression of the dilutions of each
extract and the resulted gallic acid or Trolox equivalents concen-
trations. The regression residues were tested for a normal distri-
bution and homoscedasticity. The RyanJoiner coecient of cor-
relation (P >0.05) indicated a normal distribution; the Levene test
(P >0.05) conrmed homoscedasticity; nally the DurbinWatson
test (P >0.05) showed independence of the residues.
Total phenolic content
To statistically compare the TPC conventional and microplate
methods, the condence intervals of a linear regressionwere used.
To generate the graphic and the linear regression equation, the
abscissa represents the values of the reference method (conven-
tional TPC method) and the ordinate shows the values of the
method to evaluate (microplate TPC method). Figure 2 shows the
linear regressionof the TPCcomparisonusingthe GSE, AE andGTE.
Table 1 shows the condence values for the slope and the
constant. If the slope condence intervals donot include a value of
1, it means that there is an error proportional to the concentration
of the analyte. If the constant condence intervals do not include
a value of zero, it means that there is a systematic excess or
defect error.
19
The methods in the studied range of concentrations
can be considered equivalent because, as shown in Table 1, the
condence intervals of the slope and the constant meet the above
mentioned requirements.
The changes made in the TPC method used by Al-Duais et al.
14
and Mller et al.
17
were made to increase the concentration of
the FolinCiocalteu reagent compared to the concentration of
the sample. This increment intended to bring the proportions of
the microplate method closer to those used in the conventional
method. Due to the increase in the FolinCiocalteu reagent the
desired statistical values were obtained to consider both method
equivalents. It is important to remark that the proportions of
Table 2. Validation of DPPH conventional and microplate methods by using F of the Fisher test and t of the Student test
Fisher test Student test
Extract Concentration or dilution DPPH method
a
Mean SD (mol L
1
Trolox) F P-value t P-value
Grape seed 35 mgL
1
C 222.7 6.9 7.7484 0.07 0.1159 0.91
M 222.3 2.5
40 mgL
1
C 250.5 2.7 2.2287 0.46 0.5255 0.61
M 251.3 1.8
45 mgL
1
C 274.6 8.4 1.7686 0.59 0.5899 0.57
M 277.4 6.3
Apple 50 mgL
1
C 229.1 3.0 1.0325 0.98 1.7240 0.12
M 225.8 3.0
60 mgL
1
C 264.9 3.3 1.9642 0.53 0.2590 0.80
M 265.4 2.3
70 mgL
1
C 302.3 3.6 0.6138 0.65 1.2998 0.23
M 305.6 4.6
Green tea 1:2.5 C 253.9 2.9 0.1378 0.08 0.0815 0.94
M 254.2 7.8
1:2.0 C 308.1 5.7 0.1420 0.08 0.6061 0.56
M 304 15
1:1.7 C 324.9 8.5 0.2225 0.17 0.8542 0.42
M 317 18
a
C, conventional; M, microplate.
DPPH, 2,2-Diphenyl-1-picrylhydrazyl.
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reagents and sample of both methods could not be equal because
there was bubbling and precipitation during the whole assay and
the measurements were unstable. This phenomenon was also
reported by Herald et al.
9
when they used the FolinCiocalteu
reagent undiluted.
Antioxidant activity
To compare the AA conventional and microplate methodologies
three dierent concentrationlevels of grape seed, apple andgreen
tea extracts were used. Table 2 shows the resulting P-values of
the F parameter of the Fisher test and the t parameter of the
Student test. In all cases the P-values for the F parameter of the
Fisher test were above 0.05, which indicates that the variances
among the measurements for each concentration (n =5) were
not signicantly dierent at a 95% condence level. The lack of
signicant dierences in the variance indicates that both methods
are equally precise and allows the performance of the t parameter
of the Student test.
19
All the P-values for the t parameter of
the Student test were above 0.05. It indicates that there are not
signicant dierences between the mean values for both methods
at a 95% condence level.
19
Based on the P-values resulting from
both statistical tests, it can be said that the conventional and the
microplate methods were equivalent in the concentration ranges
studied.
The equivalency of both methods to measure the AA of the
samples indicates that the increase in the concentration of the
sample for the microplate analysis was compensated with the
increase in the molar concentration of the DPPH. The higher ratio
of the sample and the reagent in the microplate methodology
could contribute to reduce the time to reach a stable absorbance
compared to the time required with conventional method, which
is one of the requirements for a correct AA analysis.
11
Finally, the
addition of 20% of water to the DPPH reagent in the microplate
methodology did not aect the results when compared to the
conventional methodology and together with the reduction of
time, prevented the evaporation of the solvent leading to more
stable measurements.
11
Precision and accuracy
The RyanJoiner and Levene test (P >0.05) conrmed a normal
distributionandhomoscedasticity for eachday andconcentration.
Table 3 shows the repeatability, reproducibility and percentage of
recovery for ve gallic acid and Trolox concentrations. The con-
centrations were chosen within the ranges used to compare the
conventional andmicroplatemethods. It canbeseenthat theRSDs
for the repeatability were 3.6%andthe RSDs for the reproducibil-
ity were 6.1% for both methods. RSD values below 10% indicate
good precision.
9
The repeatability and reproducibility results and
the fact that in general, the RSDs of the TPC are higher than those
of the AA, are in agreement with the ndings by Cheng et al.
15
and Herald et al.
9
The percentages of recovery (87.8100.3%) sug-
gest an excellent accuracy for both methods within the studied
concentrations.
19
CONCLUSIONS
In the range of 1070 mgL
1
GAE and 220320 mol L
1
of Trolox
equivalents for the FolinCiocalteu and the DPPH techniques,
respectively, the microplate and the conventional methods are
equal at a 95% condence level. The repeatability, reproducibil-
ity and percentage of recovery for the TPC and AA microplate
Table 3. Repeatability, intra-laboratory reproducibility and percent-
age of recovery for total phenol content and antioxidant activity with
the conventional and microplate methods
Method Standard Repeatability Reproducibility
Recovery
(%)
FolinCiocalteu
microplate
12 3.6 5.2 87.8
24 2.2 2.9 97.9
36 2.4 6.1 98.3
48 2.7 5.3 99.3
60 2.1 3.9 100.3
DPPH microplate 224 3.4 3.2 98.3
246 2.4 2.7 99.2
268 1.0 1.9 97.8
293 2.2 2.4 96.4
315 2.1 3.2 95.6
For the FolinCiocalteu method, the standard was gallic acid
(mgL
1
). For the DPPH method, the standard was Trolox (mol L
1
).
Repeatability andreproducibility are givenas %relative standarddevi-
ation for both methods.
methods showed a precision below 6% and accuracy between
88%and100%. The use of the microplate methods saves resources
and time, which makes it more suitable than the conventional
ones.
ACKNOWLEDGEMENTS
We thank Exxentia (Spain) for kindly providing the samples. This
work was partially nanced by the Navarra government (Spain)
and by the Public University of Navarre (Spain).
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