Food Chemistry 287 (2019) 338–345

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Statistical comparative study between the conventional DPPH% T

spectrophotometric and dropping DPPH% analytical method without
spectrophotometer: Evaluation for the advancement of antioxidant activity
analysis
⁎
Sridhar Kandia, Albert Linton Charlesa,b,
a
Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, 1 Shuefu Road, Neipu, Pingtung 912 01,
Taiwan
b
Faculty of Fisheries and Marine, Universitas Airlangga Campus C Universitas Airlangga, Mulyorejo, Surabaya 601 15, East Java, Indonesia

A R T I C LE I N FO A B S T R A C T

Keywords: Recently, new approaches for measuring antioxidant activity have been developed to eliminate or reduce the use
Conventional DPPH% method of a spectrophotometer. All analytical methods must provide consistent and reliable results, which should be
Dropping DPPH% method compared with statistical models. Therefore, we compared the conventional and dropping DPPH% methods with
Antioxidant activity three widely used standards. We employed and compared with three statistical methods. Good R-squared
Statistical studies
(R2 = ≥0.96) confirmed the similarity in comparison of using dropping DPPH% method compared with the
Bland-Altman method
conventional DPPH% method and reported lower deviations (ARD = −0.32 to 0.13; RMSD = 0.10 to 1.15).
Bland-Altman method (95% CI) demonstrated a good agreement between the two methods using standards and
grape extracts (skin and seed). These findings suggested that the dropping DPPH% method proved cheaper
(without spectrophotometer) and correlated well with the conventional DPPH% method. Therefore, this method
could be affordably conducted in research laboratories in developing and less developed countries.

1. Introduction
Research on free radicals has a long tradition and of great im-
portance in the field of science, especially in clinical medicine and
nutritional science (El-Kosasy, Hussien, & Abdel-Rahman, 2013). Free
radicals are generated by the oxidation reaction and incomplete re-
duction of an oxygen molecule, which leads to structural and functional
damage of biomolecules or cell organelles (Akar, Kucuk, & Dogan,
2017). Plant-based foods, especially fruits and vegetables have been
extensively used to scavenge these free radical generation by anti-
oxidant molecules present in fruits and vegetables. Grape skin and seeds
are by-products of grape processing industry and potential sources of
phenolic compounds. These by-products of grape (skin and seed) could
scavenge the free radicals by antioxidant mechanism. Thus, researchers
investigated in the field of free radicals and antioxidants stated that
“antioxidant is a term widely used but rarely defined” (Tirzitis &
Bartosz, 2010). The term antioxidants are defined as substances (even
at low concentration) that significantly delay or reduce oxidation of
that substrate including free radicals (Szabo, Idiţoiu, Chambre, &
Lupea, 2007; Tirzitis & Bartosz, 2010). However, the literature of the
last decade concerning the definition of antioxidant depends on the
environment (in vitro or in vivo) of its action (Tirzitis & Bartosz, 2010).
Hence, the evaluation of the antioxidant activity has been widely
adopted with different analytical methods, such as DPPH% (2,2 di-
phenyl-1-picrylhydrazyl), ABTS•+ (2,2'-azino-bis-3ethylbenzothiazo-
line-6-sulfonic acid), FRAP (ferric reducing antioxidant power), ORAC
(oxygen radical absorption capacity), cupric reducing antioxidant ca-
pacity (CUPRAC) assays, total reducing capacity (Chen, Bertin, &
Froldi, 2013), and inhibition of low-density lipoprotein (LDL) oxidation
(Szabo et al., 2007). Among all these methods, the DPPH% method is
one of the popular assays used for its ease, low cost, and efficiency.
The DPPH (Fig. 1) assay, developed by Blois in 1958 (Kedare &
Singh, 2011), investigates the stable free radical (α, α-diphenyl-β-pi-
crylhydrazyl (DPPH – C18H12N5O6, molecular mass of 394.33 g.mol−1))
However, due to a colorimetric mechanism, DPPH% assay requires the
use of a UV/Vis spectrophotometer. Research on DPPH% method has
gradually broadened and has led to the development of new approaches
for measuring the antioxidant activity. For instance, Milardović,

⁎
Corresponding author at: Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, 1 Shuefu
Road, Neipu, Pingtung 912 01, Taiwan.
E-mail address: alcharles@mail.npust.edu.tw (A.L. Charles).

https://doi.org/10.1016/j.foodchem.2019.02.110
Received 22 October 2018; Received in revised form 17 February 2019; Accepted 24 February 2019
Available online 02 March 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
S. Kandi and A.L. Charles Food Chemistry 287 (2019) 338–345

Fig. 1. The basic structures for diphenylpicrylhydrazyl (free radical) and diphenylpicrylhydrazine (non-free radical).

Iveković, and Grabarić (2006) developed a novel amperometric method
for antioxidant activity using DPPH%. More recently, new approaches
for measuring antioxidant activity have been based on different
sources, such as graphite sensor (El-Kosasy et al., 2013), TLC plates
(Akar et al., 2017), lamination method (Sirivibulkovit, Nouanthavong,
& Sameenoi, 2018), and HPLC method (Qiu et al., 2012). These
methods were developed to simplify the DPPH% assay. Nevertheless, the
DPPH% spectrophotometric method has had many applications in var-
ious academic and research fields, but requires high cost, which is the
major disadvantage of using the spectrophotometer. Many laboratories
in developing and less developed countries have failed to or unable to
use the spectrophotometer due to lack of funding sources or supporting
agencies, which is a common problem in many laboratories of devel-
oping and less developed countries. To overcome this problem, recent
methods have been focused by proposing reduced cost antioxidant bio
assays, which could replace use of spectrophotometers (Akar et al.,
2017). To date, few studies report on investigating antioxidant activity
using thin-layer chromatography (TLC) plates in bioautography and
spot assays, which are alternatives to spectrophotometric and spectro-
scopic methods (Hosu, Cimpoiu, Sandru, & Seserman, 2010).
Image transformation technologies, using freely available software
sources, has been used in recent years to determine antioxidant activity.
For example, Akar et al. (2017) developed a new dropping DPPH%
scavenging activity method without the need of the spectrophotometer.
It is clear that antioxidant activity could be easily determined by
without spectrophotometer; moreover, this technique eliminates the
use of a costly spectrophotometer and a high amount of chemical use
(solvents and reagents). This method also compared with natural (plant
extracts) and synthetic (BHT, gallic acid, and caffeic acid) antioxidants,
but failed to compare with widely used synthetic antioxidant standards
(ascorbic acid, BHT, and propyl gallate) as these are commonly used
antioxidants for comparing the antioxidant activity (Sharma & Bhat,
2009). Moreover, another study by Olech, Komsta, Nowak, Cieśla, and
Waksmundzka-Hajnos (2012) reported a new analytical method for the
investigation of antiradical activity of plant extracts by TLC with image
processing software. There is no previous research that reports on the
comparison between conventional DPPH% method and colorimetric
DPPH% method without spectrophotometer, using standard synthetic
antioxidants (ascorbic acid, BHT, and propyl gallate). It is our interest
to know whether this dropping DPPH% method produces accurate re-
sults compared with that of conventional DPPH% method using statis-
tical studies.
Therefore, the study objectives were to compare the conventional
DPPH% method and dropping DPPH% method (proposed by Akar et al.,
2017), thus eliminating the need of a spectrophotometer. Moreover, in
order to assess the comparison of the methods, we employed the sta-
tistical models (correlation analysis, linear regression model, and
Bland–Altman method). The contributions of this comparative study
might have wide applicability in developing and less developed coun-
tries, where a spectrophotometer is unaffordable.
2. Materials and methods
2.1. Reagents and instruments
2,2 diphenyl-1-picrylhydrazyl (DPPH%), butylated hydroxytoluene
(BHT), methanol, ethanol, TLC (Thin Layer Chromatography) plates –
5 × 10 cm (RP-18 modified silica gel coated with fluorescent indicator
F254s), and propyl gallate were obtained from Merck Chemicals
(Darmstadt, Germany). Ascorbic acid was purchased from Thermo
Fisher Scientific (Massachusetts, United States). Fresh stock solutions
were prepared before each analysis: stock solutions of DPPH were
prepared in methanol; other solvents and reagents were of the highest
analytical/food grade; and used without any further purification; and
chromatographic grade water was obtained by Milli-Q water (Merck
Millipore Water System, Germany) purification unit.
Spectrophotometric measurements were taken at 517 nm using a DU
730 UV/Vis spectrophotometer (Beckman-Coulter, United States).
Images were taken using Canon Digital Camera (600 D) – 5 × 10 cm
(EOS, DS126311, Canon Inc. Japan) at a shuttle speed of 1/1000 (lens:
18–55 mm, brightness: ± 0 without flash). Data was processed on
computer using Windows 10 Education 64-bit, (ASUSTek computer
INC.) with a 2.80 GHz processor configuration (4096 MB RAM).
Detailed information on the general properties of the chemical reagents

Table 1
Detailed information on the general properties of the chemicals used in this study.
Chemical Molecular formula Molecular weight (g.mol−1) Purity (%w)b ChemSpider ID CASRNc

DPPHa C18H12N5O6 394.33 ≥90 2016757 1898-66-4

Butylated hydroxytoluene C15H24O 220.35 ≥95 13835296 128-37-0
Propyl gallate C10H12O5 212.19 ≥98 4778 121-79-9
Ascorbic acid C6H8O6 176.12 99 10189562 50-81-7
Methanol CH4O 32.04 ≥99 864 67-56-1
Ethanol C2H5OH 46.07 ≥90 682 64-17-5
Water H2O 18.01 none 937 7732-18-5

a
2,2 diphenyl-1-picrylhydrazyl.
b
The purity of chemicals were stated by the suppliers.
c
Chemical Abstracts Service Registry Number (CASRN).

339
S. Kandi and A.L. Charles
used in this study are presented in Table 1.
2.2. Conventional DPPH% spectrophotometric method
The electron donation abilities of standard compounds
(100–1000 µM) were determined based on the measurement of free
radical scavenging capacity of antioxidant compounds using DPPH%
according to Sridhar and Charles (2018) with some modifications.
Standard solution (0.10 mL) containing different concentrations
(100–1000 µM) was added to 3 mL methanolic solution of DPPH
(100 µM). Tubes were allowed to stand at 27 °C for 20 min after vig-
orous shaking. A control without sample was prepared as described
above; absorbances were measured at 517 nm against methanol as
blank.
2.3. Dropping DPPH% method
This assay was carried out according to Akar et al. (2017). Standard
solution (0.10 mL) containing different concentrations (100–1000 µM),
methanolic solution of DPPH, and incubation was performed as de-
scribed in DPPH% spectrophotometric method. After incubation, 15 µL
of the incubation mixture was dropped onto the adsorbent surface of
the TLC plate according to Akar et al. (2017) with some modification
and left at room temperature for 5 min (TLC plates were activated by
heating at 105 °C for 1 h). At the end of incubation, TLC plates were
dried in a hood for 30 min. Dried TLC plates were documented using a
compact digital camera (approximately 10–20 s). Moreover, we fol-
lowed Olech et al. (2012) procedure for image processing requirements
for TLC plates. The images were captured and saved as JPEG (Joint
Photographic Experts Group) files for the best quality (smallest com-
pression) and accessed them for further image processing using non-
commercial ImageJ software. Color images were turned into 8-bit type
images by applying following steps: Image – Type – 8-bit. Denoised
images were obtained by built-in algorithm used in ImageJ software
without plugins (Process – Filter – Median – Radius: 5 pixels). Subse-
quently baseline drift (caused by inhomogeneous illumination) was
removed by the high-pass 2D filters implemented in ImageJ software
(Process/FFT/Bandpass Filter/Filter large structures down to: 40 pixels;
filter small structures up to: 0 pixels; tolerance of direction: 5%). After
denoising and removing the baseline drift, elliptical brush selection tool
was employed to select the desired track on the color images. The color
values (arbitrary units – AU) on TLC plates (images) were determined as
area (Analyze – Set measurements: Area) by non-commercial free pro-
gramming ImageJ software (https://imagej.nih.gov/ij/download.html)
developed at the National Institute of Health, the United States of
America (Cieśla, Kowalska, Oleszek, & Stochmal, 2013). The color va-
lues (area) obtained from ImageJ were plotted against the concentra-
tions.
2.4. Statistical fit of conventional and dropping DPPH% method
Statistical fit of the methods was calculated based on the average
relative deviations (ARD) (Baluja, Alnayab, & Hirapara, 2017; Chen
et al., 2017; Kandi & Charles, 2018; Sunsandee, Hronec, Štolcová,
Leepipatpiboon, & Pancharoen, 2013), and root-mean-square devia-
tions (RMSD) (Kandi & Charles, 2018; Shakeel, Haq, & Siddiqui, 2015)
by the following Eqs. (1) and (2). These methods are frequently used
methods to measure the differences between the methods or values.
i=1
⎡1 X cs − Xnd ⎤
ARD = ⎢
N
∑ X cs ⎥
⎣ N ⎦ (1)
i=1
⎡1 Xnd − X cs ⎞ ⎤
RMSD = ⎢N ∑⎛ ⎜
X cs
⎟
⎥ method of solvent extraction using an ultrasonic bath (Delta Ultrasonic
⎣ N ⎝ ⎠⎦ (2)
in Eqs. (1) and (2), xcs represents the antioxidant activity values of
340
Food Chemistry 287 (2019) 338–345
conventional DPPH% spectrophotometric method, xnd represents the
antioxidant activity values of dropping DPPH% radical scavenging ac-
tivity method, and N is the number of experimental points in the ex-
periment.
2.5. Methods of measurements: Statistical studies
Statistical studies between conventional DPPH% spectrophotometric
and dropping DPPH% analytical methods were evaluated using three
methods of measurements: (i) correlation analysis (Armstrong, Eperjesi,
& Gilmartin, 2005; Bland & Altman, 2003), (ii) linear regression model
(Lai & Shiao, 2005), and (iii) Bland–Altman method (Lai & Shiao, 2005;
McAlinden, Khadka, & Pesudovs, 2011). We used data from 1000 µM
concentration of ascorbic acid, BHT, and propyl gallate (n = 10) to
calculate correlation analysis and Bland–Altman method.
2.5.1. Correlation analysis
Correlation analysis is a statistical method used to analyze the re-
lationship between two variables using ‘product moment correlation
coefficient’ – PMCC (referred as “r”) first introduced by Karl Pearson in
1902 (Armstrong et al., 2005). Pearson’s correlation coefficient be-
tween any two variables (x and y) is represented as rxy and calculated as
shown in equation (3) according to IBM® SPSS® Statistics version 22.0
(2013) (2013) (2013) following the procedure of Analyze – Correlate –
Bivariate (Pearson’s correlation coefficient).
⎡ C xy ⎤
rxy = ⎢
C xx C yy ⎥ (3)
⎣ ⎦
where, Cxy = covariance of x and y, Cxx = variance of ×, and
Cyy = variance of y.
2.5.2. Linear regression model
A linear regression model is statistical method used to explain linear
association between two or more continuous variables using a straight
line. The linear association between conventional and dropping DPPH%
method calculated (Passing & Bablok, 1983; Sridhar & Charles, 2019) as
shown in equation (4) following the procedure of Microsoft Excel® –
Insert – Charts (Scatter – to compare at least two sets of values).
Yi = β0 + β1 Xi (4)
in which, Xi and Yi = variables, β1 = slope of the line, and
β0 = intercept.
2.5.3. Bland–Altman method
Bland – Altman method or the limits of agreement method origin-
ally developed by Altman and Bland in 1983 and widely used in the
clinical comparison of two analytical methods (McAlinden et al., 2011).
According to Bland–Altman method, the differences between two ana-
lytical methods (y-axis) plotted against the average of the two analy-
tical method (x-axis) values (Ludbrook, 2002). We constructed graphs
using Microsoft Excel® version 2016 following the procedure of Excel –
Insert – Charts (Scatter – to compare at least two sets of values).
2.6. Evaluation of two analytical methods in grape extracts
The best statistical method was used for the evaluation of agreement
between two analytical methods using Kyoho grape skin and seed ex-
tracts (n = 10). The grape samples of Vitis labruscana (Kyoho) were
collected from the local market in Pingtung county of Taiwan (February
2019) and washed with deionized water (dH2O). Sample preparation
for Kyoho skin and seed samples were performed according to the
method previously described by Sridhar and Charles (2019). The
Cleaner, Taiwan) was employed for the preparation of grape skin and
seed extracts (Sridhar & Charles, 2019) with a combination of green
S. Kandi and A.L. Charles Food Chemistry 287 (2019) 338–345

solvents (75% ethanol: water, 4:1 v/v). The combination of green sol-
vents was used to establish an eco-friendly extraction method for the
efficacy of antioxidant activity using two analytical methods. The an-
tioxidant activities of Kyoho skin and seed extracts were determined
according to the aforementioned protocol described for conventional
and dropping DPPH% methods.

2.7. Statistical analysis

The results are presented as the mean values ± SD of three mea-

surements. Correlation analysis was performed using IBM® SPSS®
Statistics version 22.0 and values of p < 0.05 was assumed as statis-
tical differences between the experimental points. Graphs were plotted
by Microsoft Excel® version 2016 (www.office.com).

3. Results and discussion

3.1. Conventional DPPH% and dropping DPPH% method

In this study, the antioxidant activity of each standard (ascorbic

acid, BHT, and propyl gallate) was determined by applying the con-
ventional DPPH% method and a dropping DPPH% method. The visual
appearance of the reaction mixture obtained from each method showed
clear color changes (Supplementary Fig. S1). The intensity of the yel-
lowish color of the reaction mixture in both methods increased with
increase in the concentrations of standard compounds (100–1000 µM),
which reflected the dose-dependent antioxidant activity of standard
compounds. Moreover, a similar color change from purple to yellowish
based on concentration in both methods further confirmed the simi-
larity of the reaction mixture (color change) in the dropping DPPH%
Fig. 2. Calculation of antioxidant activity of standard compounds. (A)
method to the color change (reaction mixture) in the conventional Concentration – absorbance measured by the conventional DPPH% method and
DPPH% method (Supplementary Fig. S1). The color change in conven- (B) Concentration – area measured by dropping DPPH% method. Error bars
tional DPPH% method was estimated by measuring absorbance with a represent the standard deviation of the mean.
spectrophotometer at 517 nm and recorded as 0.87 ± 0.03 for the
control sample, whereas TLC plates were processed with ImageJ for the
confirmed the relationship between concentration and absorbance or
determination of areas of the spot (reaction mixture on TLC plate). The
area. Moreover, the best fit of the regression line was explained by good
antioxidant activity of spots increases with a decrease in spot area
R-squared values in both methods. For example, all the standard com-
(Hosu et al., 2010) based on the concentration used (100–1000 µM).
pounds reported good R-squared values (R2 = ≥0.96) for both
The area measured for the control spot was in the range of
methods. Good R-squared values have also been explored in a prior
74384 ± 152.42. A decrease in areas was observed with increase in
study by Hosu et al. (2010), who investigated the antioxidant activity of
concentration (100 – 1000 µM), which ranged from 62,268 to 22,120
juices using conventional and the dropping DPPH% methods. Our re-
(ascorbic acid), 37,699 to 14,592 (BHT), and 62,343 to 25,505 (propyl
ported R-squared values agreed with a recent analytical study by Olech
gallate). The decreased area with an increased concentration in this
et al. (2012) on antiradical activity of plant extracts using TLC –image
study corroborated the earlier findings by Hosu et al. (2010), who
processing technology and reported R-squared values of 0.95 (gallic
conducted studies on the antioxidant activity of juices by thin-layer
acid), 0.96 (rose extracts), and 0.98 (protocatechuic acid). Therefore, R-
chromatography and image processing technology.
squared values above 0.95 are acceptable to confirm the similarity of
A calibration curve was constructed for the conventional DPPH%
any two analytical methods in a comparative study. However, minor
method by plotting the absorbance of the difference between control
variations in R-squared values might be the consequences of errors in
and each standard (ascorbic acid, BHT, and propyl gallate) against each
an instrument (spectrophotometer) for the conventional DPPH% method
standard concentration (100–1000 µM) as shown in Fig. 2A. On the
and dropping volume of the sample (amount of sample used for an
other hand, a calibration curve was also constructed for the dropping
experiment) in the case of the dropping DPPH% method (Akar et al.,
DPPH% method by plotting area against each standard concentration
2017). Moreover, there are other reasons for dropping DPPH% method
(Fig. 2B). The plotted areas were obtained by the difference between
including the requirements of the compact digital camera and photo
mean area of control sample and the mean area of each standard. The
resolution (Olech et al., 2012). Based on the available literature, we
linear calibration curve for ascorbic acid (y = 0.00005x + 0.80,
proposed that R-squared values above 0.95 is acceptable to produce
R2 = 0.99), BHT (y = 0.0004x + 0.39, R2 = 0.99), and propyl gallate
similar result for any analytical method. Hence, from the above study as
(y = 0.00005x + 0.79, R2 = 0.98) were constructed using the conven-
well as our study confirms the similarity in data using the dropping
tional DPPH% method (Fig. 2A). In addition, linear calibration curves for
DPPH% method with that of the conventional DPPH% method.
ascorbic acid (y = 0.48.91x + 7400.60, R2 = 0.97), BHT
(y = 24.20x + 36.13, R2 = 0.96), and propyl gallate
(y = 39.81x + 10154, R2 = 0.97) are presented using the dropping 3.2. Statistical fit of conventional and dropping DPPH% method
DPPH% method (Fig. 2B), constructed similarly to calibration curves for
conventional DPPH% (concentration versus absorbance) and dropping We calculated the ARD and RMSD values in order to estimate the
DPPH% (concentration versus area) methods, respectively reported in deviations between the conventional and dropping DPPH% methods
Hosu et al. (2010). (Supplementary Table S1). The ARD values ranged from −0.32 to 0.13
Conclusively, both methods demonstrated linear responses that for standard compounds between the conventional and dropping DPPH%

341
S. Kandi and A.L. Charles
methods. The highest value of ARD was observed in butylated hydro-
xytoluene (0.13), whereas the lowest value recorded as −0.32 for as-
corbic acid. The RMSD values between the conventional and dropping
DPPH% methods were recorded in the range of 0.10–1.15. The highest
RMSD value was observed in ascorbic acid (1.15), followed by propyl
gallate (0.11) and butylated hydroxytoluene (0.10). Lower values of
ARD and RMSD indicated lower deviations between the two methods.
Moreover, lower values of ARD and RMSD found clear support from
previous findings (Kandi & Charles, 2018), for the comparison between
experimental and calculated solubilities. Based on the results obtained
from deviations, we concluded that the results of a dropping DPPH%
method agreed with the conventional DPPH% method, and therefore
was a good fit with the dropping DPPH% method.
3.3. Methods of measurements: Statistical studies
3.3.1. Correlation analysis
Correlation analysis of the two analytical methods (conventional
and dropping DPPH%) using correlation coefficient indicates the
agreement between two methods (Magari, 2002). We assumed that
there was no relation between difference and magnitude of the two
analytical methods (conventional and dropping DPPH% method). The
linear dependence between the two methods was evaluated and sum-
marized in Table 2. Conventional DPPH% method (X) exhibited sig-
nificantly higher linear correlations with difference (X – Y), r = 0.83,
p < 0.01 and mean (X + Y/2), r = 0.71, p < 0.05, whereas the
dropping DPPH% method (Y) showed negatively strong correlation
(−0.74) with difference at the significant level of 0.05 (2-tailed). The
correlation between individual data points of the conventional DPPH%
method (X) and dropping DPPH% method (Y) negatively correlated
(r = −0.24), which was not significant (p < 0.05). The results de-
monstrated a good correlation between the two methods. A series of
recent studies have indicated good correlation coefficients between
these two analytical methods. For example, an analytical study con-
ducted by Huang Foen Chung et al. (2004) reported a correlation
coefficient of 0.84, which revealed the agreement between the two
methods. Nevertheless, many researchers argued that the definition and
function of a correlation coefficient are to measure the strength of as-
sociation between two variables, and therefore inappropriate to inter-
pret it as a measurement of the agreement between two methods (Bland
& Altman, 2003; Hollis, 1996; Magari, 2002). Moreover, the application
of correlation has also been well acknowledged and Hollis (1996) has
commented on the irrelevant use of correlation coefficient for the limit
of agreement studies; moreover, Hollis (1996) stated that the range of
experimental data and a number of experimental points inflates the
correlation coefficient. Similar conclusions were also stated by Magari
(2002) on correlation analysis in his study on comparing statistics for
laboratory methods. Several authors have documented the bias in the
correlation method and reported consistent values of the correlation
coefficient (Bland & Altman, 2003; Magari, 2002). Nonetheless, we
observed the use of a correlation method in many scientific journals for
agreement studies (between the two analytical methods), which has led
to the wrong interpretation of statistical method applications in re-
porting scientific research. Based on arguments presented in the lit-
erature and our study, the use of the correlation coefficient to compare
Table 2
Pearson’s correlation coefficient (r) for the level of agreement between the conventional and dropping DPPH% method with their difference and mean.
Conventional DPPH% method (X)
Conventional DPPH% method (X) 1 −0.24
Dropping DPPH% method (Y)
Difference (X-Y)
Mean (X + Y/2)
** Correlation is significant at the 0.01 level (2-tailed).
* Correlation is significant at the 0.05 level (2-tailed).
342
Food Chemistry 287 (2019) 338–345
the agreement between the two methods was determined irrelevant.
The results from our study provided evidence for the inappropriate use
of a correlation method for the study of agreement between different
methods of measurement.
3.3.2. Linear regression model
The linear regression model is the basic and commonly applied
predictive analysis to determine the relationship between the conven-
tional DPPH% and dropping DPPH% methods. We applied a regression
model to the relationship and to determine the strength of both
methods as shown in Fig. 3. Based on the regression approach, the two
methods yielded straight lines for ascorbic acid (y = 1.01x + 3.91,
R2 = 0.96), BHT (y = 0.89x + 5.33, R2 = 0.95), and propyl gallate
(y = 1.022x + 85.77, R2 = 0.92) as illustrated in Fig. 3A. The straight
line and R-squared (R2 = ≥0.92) values demonstrated a good agree-
ment between the two analytical methods. Although these two methods
were related, they failed to exhibit perfect agreement between the
conventional and dropping DPPH% methods using straight line and R-
squared (R2 = ≥0.92) values. A possible explanation for this dis-
agreement was attributed to the method that we used. However, similar
results have previously been described by Bland and Altman (2003) in
their study on the application of right statistics for measurement stu-
dies. They argued that the straight line in linear regression failed to
explain the perfect agreement between any two analytical methods
(conventional and dropping DPPH% method). Thus, it is difficult to
explain the agreement between these two analytical methods using
linear regression model. Therefore, to demonstrate the agreement be-
tween the two analytical methods, we constructed the equality line
(determined by slope and intercept) as shown in Fig. 3B. The purpose of
equality line was to indicate a lack of agreement between the conven-
tional and dropping DPPH% method. In Fig. 3B, all the data points were
scattered close to the line of equality and indicated a minor bias be-
tween the two methods. For instance, data points for ascorbic acid and
BHT lie close to the line of equality, whereas a minor deviation from the
line of equality was observed for propyl gallate (Fig. 3B). We propose
that this might be due to the structure of the standard compound propyl
gallate that we used in the study. However, several clinical and statis-
tical scientists/researchers suggested that the application of the re-
gression line and line of equality was an inappropriate method to prove
the agreement between the two methods (Bland & Altman, 2003).
Hollis (1996) reported errors with the regression and line of equality
approach and recommended the elimination of errors in variables to
produce symmetric results in a linear regression model. In regression
analysis, experimental data points should cluster around the regression
line or line of equality for good agreement between the two analytical
methods (Bland & Altman, 2003). A study by Magari (2002) in-
vestigated the bias and variations in regression parameters. Several
assumptions for regression analysis was pointed out in a study by Hollis
(1996) on the analysis of method comparison concluded that regression
analysis assumes to measure the dependent variable with an error. On
this basis, we concluded that the linear regression model was an in-
appropriate model for the measurement of agreement between the two
methods (conventional and dropping DPPH% method). Therefore, re-
searchers investigated the different methods to compare the agreement
between the two methods. In 1983, Altman and Bland provided a valid
Dropping DPPH% method (Y) Difference (X-Y) Mean (X + Y/2)
0.83** 0.71*
1 −0.74* 0.51
1 0.20
1
S. Kandi and A.L. Charles
Fig. 3. Linear regression analysis of the conventional DPPH% and dropping DPPH% method. (A) conventional DPPH% and dropping DPPH% method with regression line
and (B) conventional DPPH% and dropping DPPH% method with equality line.
and alternate method of analysis (Hollis, 1996). This cornerstone ap-
proach had a considerable impact on the comparative studies of two
analytical methods. Therefore, we used this method in order to explain
the perfect agreement between the conventional DPPH% and dropping
DPPH% method.
3.3.3. Bland–Altman method
Altman and Bland proposed this method to compare the agreement
between two methods. We employed this method with a minimum
sample size (n = 10) to compare agreement between the conventional
DPPH% and dropping DPPH% method (Mathews, 2010). According to the
fundamentals in statistics, there is no relationship between difference
and average of data from the conventional DPPH% and dropping DPPH%
methods. Therefore, we summarized the limit of agreement between
these methods using difference and average (Bland–Altman method) as
illustrated in Fig. 4. Based on the Bland–Altman method, we calculated
the mean difference (conventional DPPH%– dropping DPPH% method)
and means of the two methods (((conventional DPPH% + dropping
DPPH% method)/2)). As a result, we expected the differences between
the two methods to lie between the limit of agreements (mean ± 1.96
standard deviation). For the data shown in Fig. 4A, the mean of dif-
ferences for ascorbic acid was −44.24 with a standard deviation of
8.15, and the limit of agreement (95% confidence interval) was re-
ported as −44.24 ± 15.97. The mean of differences for BHT and
propyl gallate was observed as 26.19 and −27.96 with a standard de-
viation of 5.85 and 8.35, respectively. The limit of agreements was
observed as 26.19 ± 11.46 and −27.96 ± 16.36 for BHT and propyl
gallate, respectively. These results suggested that the data points for
ascorbic acid and BHT were within the limits as presented in Fig. 4(A
and B) except for propyl gallate (Fig. 4C). However, we were not able to
decide the acceptable limit of agreement. Interestingly, Hollis (1996)
reported no statistical conclusion for an acceptable limit of agreement
and suggested that it can be decided by clinical judgment. Generally,
the acceptable limit of agreement between two methods should be se-
lected based on the study plan and before the collection or calculation
of experimental results (a priori). Moreover, researchers need to define
the limit of agreement in the methods to be compared with it (Babaei
Jandaghi et al., 2015). Therefore, the Bland-Altman method explained
the better limit of agreement between the two methods. However, se-
lection of a right method for the agreement between two methods is of
great importance in an analytical comparative study. Prior study have
noted the Bland-Altman analysis as the best statistical method for re-
porting agreement between two methods (Babaei Jandaghi et al.,
2015). Therefore, Altman and Bland clearly demonstrated the agree-
ment between two analytical methods (conventional DPPH% and drop-
ping DPPH%) compared with correlation and linear regression model.
343
Food Chemistry 287 (2019) 338–345
These classical approaches, such as correlation and linear regression
were not recommended for agreement studies as these describe the
association of two variables instead of agreement, which are misleading
in the use of statistical models (Altman & Bland, 1983). Hence, we
conclusively recommend the Bland–Altman method for any agreement
studies. This is an important finding in the understanding of the com-
parison of statistical methods to measure the agreement between con-
ventional DPPH% and dropping DPPH% method. Therefore, this method
was employed for the evaluation of agreement between conventional
and dropping DPPH% methods using Kyoho grape skin and seed extracts.
3.4. Evaluation of two analytical methods in grape extracts
Kyoho skin and seed extracts were treated with DPPH% working
solution using conventional and dropping DPPH% methods and the vi-
sual color appearance of reaction mixtures are shown in Supplementary
Fig. S2. The color change for Kyoho skin and seed extracts was recorded
as 0.078 ± 0.02 and 0.086 ± 0.007 using the conventional DPPH%
method. The areas of the spot for Kyoho skin and seeds were observed
as 60691.60 and 59887.77, which were in the range of measured areas
of control sample (111052 to –138930) using dropping DPPH% method.
The color change (pink to yellow) in both methods demonstrated that
the Kyoho skin and seed extracts had the capacity to inhibit the gen-
eration of free radicals (Supplementary Fig. S2). The agreement be-
tween the analytical methods was further confirmed by using the ap-
propriate statistical Bland–Altman method. The Bland–Altman scatter
plot using Kyoho grape skin and seed extracts were constructed for the
evaluation of agreement between conventional and dropping DPPH%
methods (Fig. 4D and E). Fig. 4(D and E) showed the possible agree-
ment between the conventional DPPH% and dropping DPPH% methods. It
was obvious to expect that the differences between the two methods
ranged within the limits of agreement (mean ± 1.96 standard devia-
tion). The mean differences for Kyoho skin extracts were recorded as
288.72, whereas Kyoho seed extracts documented a difference of
195.03. These results were within the limits of agreement and sug-
gested the similarity in the analysis of both analytical methods. These
results further indicated that the data points for Kyoho skin and seed
extracts were within the limits (95% confidence interval) as illustrated
in Fig. 4(D and E). Similar observations were noticed for ascorbic acid
and BHT Bland–Altman scatter plots (Fig. 4A and B) except for propyl
gallate (Fig. 4C). These results agreed with those obtained by Akar et al.
(2017), who reported similar trends using standards and plant extracts
in the conventional and dropping DPPH% analytical method. Similarities
within the limits of agreement between the two analytical methods
using standards and Kyoho grape extracts (skin and seed) projected a
wider applicability of the dropping method over the conventional
S. Kandi and A.L. Charles
Fig. 4. Bland–Altman method measured by the conventional DPPH% and dropping DPPH% method with the mean difference (solid line) and limits of agreement
(dashed line). (A) Ascorbic acid, (B) Butylated hydroxytoluene, (C) Propyl gallate, (D) Kyoho seed extracts, and (E) Kyoho skin extracts. SD represent the standard
deviation of the mean.
DPPH% method. Conclusively, these findings suggested that the drop-
ping DPPH% method reported similar results as the conventional DPPH%
method and therefore could be an alternate method for the determi-
nation of DPPH% scavenging activity. These results confirmed that the
dropping DPPH% method could be of practical use in an analytical la-
boratory where a spectrophotometer is unaffordable.
4. Conclusions
The aim of the present study was to compare the conventional
DPPH% method and dropping DPPH% method, thus eliminating the de-
pendence on spectrophotometry. Lower ARD (−0.32 to 0.13) and
RMSD (0.10–1.15) values for statistical fit further confirmed the
agreement between the two methods with the widely used antioxidant
standards (ascorbic acid, BHT, and propyl gallate) in a dose-dependent
manner. However, correlation and linear regression analysis failed to
explain the agreement between the two methods; moreover, our results
provided the evidence for the inappropriate use of correlation and
linear regression methods for agreement studies. The Bland-Altman
method successfully confirmed the agreement between the two analy-
tical methods (conventional DPPH% and dropping DPPH%) with the limit
of agreement (95% confidence interval) using standards and grape
extracts (skin and seed).
The evidence from this study suggested that the results of a drop-
ping DPPH% method were a good fit with the conventional DPPH%
344
Food Chemistry 287 (2019) 338–345
method. A suitable TLC plate, camera, and image processing software
(for example, non-commercial ImageJ) sufficiently estimated the ca-
pacity of standard antioxidants. This method is cheaper (without
spectrophotometer), uses user-friendly non-commercial image proces-
sing software (free access), and is easy to operate under any environ-
mental conditions.
Acknowledgements
The authors gratefully acknowledge the TaiwanICDF for scholarship
to Mr. Kandi Sridhar and Mr. Ribesh Dhungana for his technical sup-
port.
Conflict of interest
The authors declare no conflict of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2019.02.110.
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