Journal of Food Composition and Analysis 86 (2020) 103380

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Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Original Research Article

Comparison of colorimetric methods for determination of phytic acid T

content in raw and oil extracted flour samples of maize
Fatih Kahrımana,*, Umut Songura, Mehmet Şermenta, Şule Akbulutb, Cem Ömer Egeselb
a
Çanakkale Onsekiz Mart University, Faculty of Agriculture, Department of Field Crops, 17020, Çanakkale, Turkey
b
ÇanakkaleOnsekiz Mart University, Faculty of Agriculture, Department of Agricultural Biotechnology, 17020, Çanakkale, Turkey

ARTICLE INFO ABSTRACT

Keywords: There are different colorimetric methods, with various analysis principles and phases, for the estimation of
Phosphorus determination phytic acid content in agricultural products. Maize genotypes may possess a wide range of oil content, which is
Antinurtients considered as a factor affecting the results of phytic acid analyses. Elaborative studies are needed to examine
Zea mays these methods to clarify the effect of oil content on the results, especially in the sample sets with varying oil
Specialty maize
concentrations. We utilized 4 different colorimetric methods; namely, AOAC, Wade, Chen and Haug-Lantzsch
(H-L), to estimate phytic acid content in 19 maize genotypes, classified as having high (> 7 %, n = 7) normal
(3–5 %, n = 6) and low (< 3 %, n = 6) oil content. Phytic acid determination was carried out on 2 groups of
flour samples (raw: E1, and oil extracted: E2) using 3 replications. The results indicated that analysis methods
yielded rather different phytic acid values. They also differed significantly in time and cost, with the Chen
method being the cheapest and Haug-Lantzsch (H-L) the quickest. Oil extraction had significant effects on phytic
acid results, and these effects varied across the analysis methods and the oil content of the genotypes. Our data
suggest that either novel or improved colorimetric methods are necessary when analyzing phytic acid in special
maize genotypes, considering the dissimilarity of the results from the current methods.

1. Introduction nanoparticles, turbidimeter, and enzymatic reactions (Agostinho et al.,

Phytic acid (inositol hexaphosphate) is a compound found in many
different seeds at levels of 0.05–5 % (Agostinho et al., 2016). It plays an
active role in the storage of inorganic phosphorus in plant seeds. About
50–80 % of the total phosphorus is stored as phytic acid in cereal grains
(Coulibaly et al., 2011).
Phytic acid has been associated with a variety of undesirable as well
as desirable effects. Its ability to bind several nutritional factors in
food/feed renders an anti-nutritional influence (Coulibaly et al., 2011;
Adams et al., 2002). On the other hand, different researchers related
phytic acid with positive health effects such as prevention of cancers
(Liu et al., 2015) and lowering oxidative stress in seeds (Doria et al.,
2009). Obviously, this is a compound needed to be effectively detected
in many different agricultural products.
Today, several analysis methods are available that utilize different
instruments to estimate phytic acid content. These include the methods
developed for spectrophotometry/colorimetry, liquid chromatography,
gas chromatography, Ion Coupled Plasma-Optic Emissin Spectroscopy
(ICP-OES), Fourier Transform-Near Infrared (FT-NIR) instruments as
well as titration, molecular fluorescence, graphene quantum dots, gold
2016). Among all, colorimetric methods are the most preferred ones
since they are easy to use and cost effective. Especially, microplate-
based methods make it possible to analyze a high number of samples at
once, thereby making the these methods of choice.
Colorimetric methods for phytic acid analysis are based on the in-
direct measurement of color change in the metal complex, or mea-
surement of inorganic phosphorus composed of enzymatic or hydro-
lyzed compounds (Park et al., 2006; March et al., 1998). The Wade
method, widely used and accepted as a reference method for food and
seed analyses, measures the pink color, exposed by a complex that is
formed by phytic acid entering a reaction with iron chloride and sul-
phosalicylic acid, at 500 nm (Wade and Morgan, 1955). Another re-
cognized colorimetric method, namely Haug-Lantzsch method, is based
on the decrease of absorbance value at 519 nm, which originates as a
result of the complex of phytic acid with iron and bi-pyridine (Haug and
Lantzsch, 1983). Sureshkumar et al. (2015) developed a method using
Chen solution (6 N H2S04; 2.5 % ammonium molybdate; 10 % ascorbic
acid; water) by measuring the color change of phosphomolybdate
complex at 660 nm to determine phytic acid content on ground single
seed samples of maize. This method, which makes it possible to detect

⁎
Corresponding author.
E-mail address: fkahriman@comu.edu.tr (F. Kahrıman).

https://doi.org/10.1016/j.jfca.2019.103380
Received 30 May 2019; Received in revised form 31 October 2019; Accepted 28 November 2019
Available online 29 November 2019
0889-1575/ © 2019 Elsevier Inc. All rights reserved.
F. Kahrıman, et al. Journal of Food Composition and Analysis 86 (2020) 103380

phytic acid indirectly, has been suggested to replace any direct detec-
tion methods. Most of the colorimetric protocols found in the literature
are the modified versions of above-mentioned methods. Efforts are
underway to introduce new reagents to bind phytic acid, to modify the
current methods, or develop new ones.
Different scientific studies have been carried out to compare col-
orimetric methods for determination phytic acid content (Raboy et al.,
2017). Some others compared the colorimetric methods with High-
Pressure Liquid Chromatography (HPLC), Anion Exchange Column
(AEC) and Nuclear Magnetic Resonance (NMR) based protocols (Gao
et al., 2007). It stands out that none of these studies considered oil
extraction as a pretreatment, although it was suggested, especially for
high oil samples, to improve the efficacy of phytic acid extraction
(Thavarajah et al., 2014). From this standpoint, it is a necessity to
clarify the effect of oil extraction pretreatment on phytic acid analysis,
especially for high oil maize genotypes. Another important aspect is the
possibility for the oil in the sample to react with the chemicals used for
the analysis, which has not been addressed so far.
The objectives of this study are to (i) compare the colorimetric
methods with regards to time and cost to determine the most ad-
vantageous method, (ii) clarify the effect of oil extraction pretreatment
on phytic acid analysis, (iii) evaluate how the oil level of the sample
interacts with phytic acid results from the raw and oil extracted samples
in different colorimetric methods.
2. Materials and methods
2.1. Materials
Nineteen maize genotypes with a range of oil content were used as
plant material (Table 1). These genotypes were classified into three
groups based on their oil content. In scientific literature, high oil maize
contains 6–7 % oil in kernel (Singh et al., 2014), normal oil had %3–5
(Lambert, 2001), and low oil genotypes had belowe the 3 % of oil
content. Based on this classification, nineteen maize genotypes which
are used in our maize breeding program were selected to conduct this
study. About 50 g seed was milled (Fritsch pulverisette 14, Germany)
from each genotype for laboratory analyses.
Ground samples were divided into two and the first group (E1) was
directly analyzed for phytic acid, while the samples in the second group
(E2) were subjected to oil extraction before the analysis. A modified
version of the Abbasi et al. (2008) method was utilized for oil extrac-
tion: About 10 g ground sample was kept in 100 mL dimethylether
overnight to extract oil from flour. The solvent was evaporated using a
rotary evaporator (Hannah, Kore), and the weight of the remaining
crude oil was used to calculate the oil ratio of the sample. The samples
free of oil (E2 group) were kept at room temperature to let the solvent
evaporate before being used for the phytic acid analysis. Both groups
(E1 and E2) were subjected to oven-drying for 24 h at 70 ℃ before
weighing the flours for the colorimetric analyses.
2.2. Colorimetric methods
Four different colorimetric methods were compared, which were
explained in detail below. To make a cost comparison, the expenses of
the chemicals and consumables were calculated (based on the Sigma
website, https://www.sigmaaldrich.com). To make comparison with
regards to the time spent, each step was timed separately for every
method, and analysis time per sample was calculated.
2.2.1. Method 1 (Wade Method)
Five grams ground sample was shaken in 100 mL 3.5 % HCl for an
hour at room temperature. The extract mixture was centrifuged at
3000 g for 10 min. and the upper phase was transferred into a clean
tube. From this sample, 5 mL upper phase was diluted in 25 mL distilled
water. Then, 10 mL diluted extract was injected through a glass column
stoppered with Teflon tape and a small piece of glass wool, packed with
0.5 g anion exchange resin (AG1 × 8, 200–400 mesh, Sigma). Inorganic
phosphorus and other interfering components were eliminated using
15 mL 0.1 M NaCl. The phytic acid in the column was eluted with 15 mL
0.7 M NaCl. 3 mL of this eluate was transferred into a clean glass tube,
and 1 mL Wade solution (30 mg of FeCl3.6H2O and 300 mg of sulfosa-
licylic acid in 100 mL distilled water) was added. It was vortexed and
centrifuged at 3000 g for 10 min, and the absorbance value at 500 nm
was recorded against Wade solution as blank. Phytic acid content was
determined by cross-reference to a calibration curve constructed by
using absorbance values of the phytic acid standard (Makkar et al.,
2007).
2.2.2. Method 2 (Chen Method)
A half gram of ground sample was extracted in 5 mL 0.4 M HCl for
12 h. Next, 100 μL extract and the same volume of Chen solution
(Chen's reagent- 6 N H2S04; 2.5 % ammonium molybdate; 10 % ascorbic
acid; H2O- 1:1:1:2 v/v/v/v) were mixed. The mixture was left to react
for 15 min. When it turned blue, the absorbance value was recorded at
660 nm to detect the amount of free phosphorus. Phytic acid contents of
the samples were estimated based on the curve that was created using
dipotassium hydrogen phosphate as standard (Sureshkumar et al.,
2015).

Table 1
2.2.3. Method 3 (Haug-Lantzsch Method)
The codes and classification of the genotypes used in the experiments.
A half gram of ground sample within 10 mL 0.2 N HCI was placed in
Genotype Code Speciality for Oil Content Oil Content (%) boiling water bath for 1 h. Then, 0.25 mL of extract was added into a
test tube and 2.25 mL of 0.2 N HCI and 5 mL of iron solution were
H1 High Oil (> 7 %) 8.42
H2 High Oil (> 7 %) 8.63 added. The tubes were incubated in a boiling water bath for 30 min.
H3 High Oil (> 7 %) 9.67 After that, they were transferred to an ice bath till they reached room
H4 High Oil (> 7 %) 10.02 temperature. Then, they were centrifuged for 20 min. at 3000 g and
H5 High Oil (> 7 %) 10.47 100 μL of upper phase was taken and mixed with 150 μL H-L reagent
H6 High Oil (> 7 %) 10.95
(400 mg bipyridine +400 μL TGA +40 mL distilled water). Absorbance
M1 Normal (3–7 %) 3.04
M2 Normal (3–7 %) 3.26 values of the samples were recorded at 519 nm. With the help of the
M3 Normal (3–7 %), Opaque 3.42 curve that has been constructed with the phytic acid standard, which
M4 Normal (3–7 %), Opaque 3.54 was subjected to the same procedure, phytic acid values of the samples
M5 Normal (3–7 %) 3.61
were determined (Raboy et al., 2017).
M6 Normal (3–7 %), Opaque 3.65
M7 Normal (3–7 %), Opaque 3.79
L1 Low Oil (< 3 %) 1.16 2.2.4. Method 4 (AOAC reference method 986.11)
L2 Low Oil (< 3 %) 1.83 A half gram of ground sample was shaken in 2.4 % HCI room
L3 Low Oil (< 3 %) 1.94 temperature for 3 h. Equal volumes (1 mL) of the acquired extract and
L4 Low Oil (< 3 %) 2.64
Na2EDTA-NaOH (10.23 g Na2EDTA and 7.5 g NaOH for 250 mL) solu-
L5 Low Oil (< 3 %) 2.70
L6 Low Oil (< 3 %) 2.94 tion were mixed and diluted to 25 mL. Then sample passed through an
anion exchange column (0.7 cm × 30 cm) packaged with 0.5 g anion

2
F. Kahrıman, et al.
exchange resin (AG1 × 8, 200–400 mesh, Sigma). The fraction con-
taining phytic acid was eluted using 0.7 M NaCl and sample was di-
gested using 0.5 mL H2SO4 and 3 mL HNO3 in a Kjeldahl system
(Gerhardt, Germany). Digested sample was transferred to a 50 mL flask
and cooled at room temperature. Then the sample was mixed with 2 mL
molybdate reagent and 1 mL sulfonic acid reagent. After dilution the
volume to 50 mL, colorimetric reading was done at 640 nm using a
spectrophotometer. The KH2PO4 (80 μg/mL) was used to create the
standard curve (AOAC, 2012).
2.3. Statistical analyses
The data were analyzed using R software (R Development Core
Team, 2012). Variance analysis was done on the combined data based
on the following statistical model in order to evaluate the effect of
phytic acid analysis methods and oil extraction.
Yijkl = μ + αi + βj + γk + (αβ)ij + (αγ)ik + (βγ)jk + (αβγ)ijk + ril +
εijkl
where; Yijkl: observed value, μ: grand mean, αi: effect i. genotype (i = 1,
2…..19), βj: effect of j. method (j = 1, 2, 3, 4), γk: effect of k. extraction
(k = 1, 2), (αβ)ij: effect of genotype × method interaction, (αγ)ik: effect
of genotype × extraction interaction, (βγ)jk: effect of method × ex-
traction interaction, (αβγ)ijk: effect of genotype × method × extraction
interaction, rl: l. replication effect (l = 1, 2, 3), εijkl: random error term.
Variance analyses were done for each method separately and by ex-
cluding method effect and related interactions to more clearly under-
stand the effect of interaction of G × E according to the analysis
methods. Differences between E1 and E2 were compared using t-test to
evaluate the effect of oil extraction on the results of phytic analysis
methods.
The relations between the results obtained from raw and oil ex-
tracted samples with the same colorimetric method were analyzed
using Pearson Correlation Analysis. The relations of the oil level of the
sample and the difference between the results of raw and oil extracted
samples were evaluated by using Regression and Decision Tree Analysis
(Breiman et al., 1984).
3. Results and discussion
3.1. Comparison of the methods for analysis cost and duration
The results of the calculations for analysis cost and duration per
sample for each method are given in Table 2. In the extraction phase,
the Chen method required the longest analysis time per sample set
(720 min.), while the Wade method was the most expensive (0.15 €).
Considering the total analysis cost, the most expensive method per
sample was the AOAC method (28.60 €) while the cheapest method was
Chen method (0.65 €). AOAC method offered some advantages for
analysis time, but it was about 44 times as expensive as the Chen
method (Table 2). Overall data suggested that one would opt for the
Chen method in terms of expenses, while the Wade method would be
the method of choice in terms of duration. Nevertheless, this evaluation
takes only cost and time into consideration; precision and robustness
Table 2
Comparative time and cost assessment for phytic acid analysis methods.
Step 1-Extraction Step 2- Analysis
Time requirement (min) Chemical cost (€) Time requirement (min)
H-L Method 45 0.08 65
Chen Method 720 0.02 15
Wade Method 60 0.15 34
AOAC Method 180 0.05 86
Note: Cost calculations are based on the prices listed on https://www.sigmaaldrich.com as of 18.12.2018.
3
Journal of Food Composition and Analysis 86 (2020) 103380
are discussed later.
Large differences among the methods for their duration stems from
the procedure steps applied. The Chen method includes sample
weighing, extraction, and colorimetric reading steps; while H-L method
uses sample weighing, extraction, centrifuging, heating for sedimenta-
tion, centrifuging, and colorimetric reading steps in sequence. AOAC
(sample weighing, extraction, NaCI cleaning, AEC column step, wet
digestion and colorimetric reading), and Wade methods (sample
weighing, extraction, NaCI cleaning, centrifuging, and colorimetric
reading) use 6 and 5 steps, respectively. Although the Chen method
includes the least number of steps, it is not the method of choice in
terms of analysis duration since the extraction step lasts too long. The
range of chemicals and consumables used in the procedure steps as well
as their prices are determinant factors. The methods that require the use
of columns are costly due to heavy use of extra chemicals and appa-
ratus. Requirement of several types of chemicals in Wade and AOAC
methods increase their cost of analysis.
3.2. Variation of phytic acid values based on analysis methods
Variance analysis results for individual methods as well as the
combined data are presented in Table 3. According to the combined
analysis; genotype, oil extraction, and the analysis method had sig-
nificant effects on the variation of phytic acid obtained with colori-
metric methods. Interactions of these main effects also affected the
variation of phytic acid content.
There were statistically significant differences among the AOAC
method, Chen, and H-L methods (Fig. 1). Considering the distributions
on boxplot graphics, it is seen that the variation of phytic acid values in
Wade and H-L methods is higher than the variation in the other
methods. It can be said that oil extraction generally caused a significant
(p = 0.0092) increase in the determined phytic acid value (Fig. 1).
However, as can be seen from Fig. 1, this is not observed in all geno-
types. In other words, the effect of the extraction process on the phytic
acid content varies depending on the analysis method used and the
genotype of the sample being analyzed.
The variation between the results of the phytic acid obtained from
the methods used in the study can be associated with the basic differ-
ences in the protocols. Firstly, in addition to using HCL at different
concentrations for extraction, the extraction time is different for each
method. The amount of sample weighed in the Wade method (5 g) is
more than in the other 3 methods (0.5 g). In addition, the amount of
acid used per unit weight is also different in the four methods. Wade
and AOAC methods require 20 mL, while Chen and H-L methods require
10 and 16.7 mL acid per gram sample, respectively. These two aspects
may have a significant effect on the extraction efficiency of the
methods. As a matter of fact, it was shown earlier that acid con-
centration, extraction time and extraction temperature affected the
phytic acid extraction in rice (Canan et al., 2011). The other issue af-
fecting the results stems from the theoretical approaches. Wade and
AOAC are the methods of analysis based on the Anion Exchange
Column (AEC) principle. On the other hand, in the Wade method phytic
acid is determined based on the reaction of Fe-III and sulfosalicylic acid,
so the determination of phytic acid is accomplished indirectly
Total
Chemical cost (€) Time requirement (min) Chemical cost (€)
0.78 110 0.86
0.63 735 0.65
26.31 94 26.46
28.55 266 28.60
F. Kahrıman, et al. Journal of Food Composition and Analysis 86 (2020) 103380

Table 3
Mean squares for phytic acid estimations for different analysis methods.
Source of variation dfϯ Combined dfϯ H-L Method Chen Method Wade Method AOAC Method

Replication 2 0.0013 2 0.0075 0.0221 0.0012** 0.0593**

Genotype (G) 18 1.3606** 18 0.7136** 0.1969** 2.9194** 4.6459**
Method (Y) 3 5.2348** – – – – –
Extraction (E) 1 0.9132** 1 1.2898** 2.9981** 0.4537** 0.0797**
GxY 54 0.9097** – – – – –
YxE 3 1.3028** – – –
GxE 18 1.3203** 18 0.3507** 0.3422** 3.0109** 3.5697**
GxYxE 54 0.8607** – – –
Error 302 0,0073 74 0.8321 0.9156 0.1287 0.2188

*Significant at the 0.05 probability level.

** Significant at the 0.01 probability level.
ϯ
Degrees of freedom.

(Sivakumaran and Kothalawala, 2018). In the Chen method, the de-
termination of phytic acid is carried out indirectly by measuring the
total phosphorus content. In the H-L method, the estimation is per-
formed based on the phosphorus content of the phytic acid standard
without anion exchange column. These differences may have an impact
on the analysis results. In fact, it has been reported that the AOAC
method overestimates phytic acid values, which has been related with
the reasons arising from the theory of the method (Lehrfeld and Morris,
1992). This supports our findings that phytic acid results obtained by
the AOAC method were found to be higher than the other analysis
methods. In a study analyzing an infant formula, Park et al. (2006)
reported higher values from the AOAC method compared to Wade
method. Another important point in the methods is the differences in
protocol steps and the principle of analysis. Wade and H-L are methods
based on the ability of the phytic acid in the sample to bind iron. The
AOAC method, on the other hand, is based on the determination of
inorganic phosphorus by means of wet ashing, which eliminates organic
phosphorus in a sample. These differences may partly explain the var-
iation of the results obtained in our study.
One of the important findings from this study is the variable re-
sponses of maize genotypes with different biochemical properties (e.g.,
oil, protein quality) across the method of analysis. Since no study was
encountered in the literature with a similar setup as the current study,
making a literature-based discussion was not possible, except for
comparing the results obtained from the genotypes. Queiroz et al.
(2011) found a range of 0.48–1.04 mg/g for phytic acid content within
22 tropical maize lines. Lorenz et al. (2007) reported phytic acid values
between 2.40–4.09 mg/g for 50 different maize lines, using modified
Wade method. Although the results of this study are similar to the
earlier studies in general, extreme values were detected for some gen-
otypes. Genotypic effects and seed characteristics may be the reason for
these differences. Because most of the phytic acid is located in the
embryo (O’Dell et al., 1972), genotypes with large embryos may have
higher phytic acid values. Raboy et al. (1989) showed that high oil

Fig. 1. Boxplots demonstrating the variation of phytic acid variation as affected by oil extraction (a) analysis methods (b) and genotypes (c).

4
F. Kahrıman, et al. Journal of Food Composition and Analysis 86 (2020) 103380

Table 4
Phytic acid content (mg/g) of raw flour (E1) and oil extracted (E2) samples of genotypes in different analysis methods.
Genotype AOAC Method Chen Method H-L Method Wade Method

E1 E2 Diff E1-E2 E1 E2 Diff E1-E2 E1 E2 Diff E1-E2 E1 E2 Diff E1-E2

H1 2.60 2.04 0.56** 0.98 1.93 −0.95** 1.92 1.93 0.01 2.44 2.51 −0.07
H2 1.67 1.56 0.11 1.53 1.92 −0.38* 1.24 1.58 −0.35* 1.39 0.00 1.39**
H3 1.86 1.82 0.05 1.13 1.87 −0.74** 2.02 1.87 0.15 2.53 0.00 2.53**
H4 2.28 1.98 0.30** 1.38 2.38 −1.00** 1.70 1.96 −0.26 0.27 2.42 −2.15**
H5 1.87 1.96 −0.08 1.40 1.96 −0.56** 2.22 2.54 −0.32 0.03 0.00 0.03
H6 1.83 1.92 −0.09 0.91 1.58 −0.67** 0.44 2.02 −1.58** 0.93 2.46 −1.53**
L1 1.77 2.06 −0.29** 1.87 1.21 0.66** 2.00 2.19 −0.19 2.03 2.19 −0.16**
L2 1.90 1.82 0.08 0.66 2.03 −1.37** 0.78 1.28 −0.50** 0.71 1.12 −0.41**
L3 1.70 2.23 −0.52** 1.82 1.88 −0.06 1.45 1.59 −0.14 0.62 0.42 0.19**
L4 1.90 1.92 −0.02 1.84 1.97 −0.13 2.18 1.91 0.27** 2.30 2.47 −0.17**
L5 1.86 1.88 −0.02 1.74 1.76 −0.02 1.84 2.95 −1.12** 1.94 1.90 0.04
L6 1.78 1.97 −0.19 1.63 1.85 −0.23 1.20 1.80 −0.60** 1.91 1.42 0.49**
M1 3.20 1.97 1.23** 1.58 1.60 −0.02 1.71 1.60 0.11** 2.50 1.57 0.93**
M2 1.81 1.91 −0.10 1.69 1.89 −0.20 1.85 1.81 0.05 1.10 2.43 −1.33**
M3 2.00 1.94 0.06 1.48 1.56 −0.08 1.76 1.83 −0.07 2.45 2.11 0.34**
M4 1.91 1.97 −0.07 1.74 1.63 0.11 1.78 1.54 0.24 2.50 0.00 2.50**
M5 1.76 1.86 −0.10 1.72 1.67 0.06 1.71 1.76 −0.06 0.35 0.58 −0.22**
M6 1.77 1.89 −0.12 1.74 1.78 −0.04 1.73 1.83 −0.10 0.08 2.48 −2.40**
M7 2.00 1.79 0.21* 1.22 1.76 −0.54* 1.96 1.54 0.42** 2.40 0.00 2.40**
Mean 1.97 1.92 0.05** 1.48 1.80 −0.32** 1.66 1.87 −0.21** 1.50 1.37 0.13**

* Statistically significant at 0.05 level.

** Statistically significant at 0.01 level. Diff E1-E2 shows the differences between raw and oil axtracted sample of the same genotype.

genotypes from the Illinois Long Term Experiment possessed higher
levels of phytic acid. The results obtained from high oil genotypes in
our study are similar to these results (Table 4). However, the significant
differences between the analysis methods were striking. AEC requiring
methods such as AOAC and Wade did not meet this expectation. The
phytic acid content of high oil genotypes was found to be relatively
high compared to the other genotypes with Chen and H-L methods,
which require no use of AEC. It was remarkable that H-L and Wade
methods, which are based on iron precipitation, could not determine
the phytic acid content in some genotypes when raw samples were
used. Odd results were observed for genotypes with high oil content as
well as opaque type samples when analyzed with these methods. Even
though this study focused on the determination of phytic acid content
in the samples with different oil concentrations, the results indicated
that there is a need to carry out research to investigate the effect of
other biochemical attributes of maize kernels on the phytic acid ana-
lyses. Park et al. (2006) reported that when the sample contained
protein, it could react with the iron found in the Wade solution and
cause precipitation. This may be a reason for obtaining low values from
the opaque genotypes with Wade method in this study (Table 4).
3.3. Effect of oil extraction and oil ratio on phytic acid analysis results
The correlation analysis results among the phytic acid values from
raw and oil extracted samples using different colorimetric methods are
presented in Table 5. Correlation analyses were performed on the data
sets of genotypes separated into three classes (high > 7 %,
Table 5
Pearson correlation coefficients among phytic acid values from raw (E1) and oil
extracted samples (E2) using different colorimetric methods.
Samples with Low Samples with Samples with
Oil Content (< %3) Normal Oil Content High Oil Content
(%3–5) (> %7)
AOAC Method −0.78** 0.37 0.65**
Chen Method −0.45 −0.09 0.43
H–L Method 0.62** −0.23 0.35
Wade Method 0.90** −0.38 −0.04
** Statistically significant at 0.01 level.
normal = 3–5 %, and low < 3 %) according to their oil content. The
aim was to compare the values calculated over the combined data set to
the values calculated from the three oil classes. The correlation coeffi-
cients for phytic acid values estimated from the raw and oil extracted
samples varied across the colorimetric methods and oil classes
(Table 5). For example, a significant negative correlation (r=−0.78;
p < 0.01) was found between the phytic acid results that were ana-
lyzed from oil extracted (E2) and raw (E1) samples of low oil (< 3 %)
genotypes with AOAC method; while there was a positive relationship
between high and normal oil containing genotype groups (r = 0.65;
p < 0.01 for high, R = 0.44; p > 0.05 for normal). The correlations
between the phytic acid results obtained from raw and oil extracted
samples were not significant in all oil classes for Chen and H-L methods.
It is remarkable that all correlation coefficients were positive (Table 5).
Wade method yielded a positive and significant correlation between the
raw and oil extracted samples of low oil genotypes (r = 0.90;
p < 0.01), but for high and normal oil genotypes the correlation values
were negative and nonsignificant (Table 5). These results suggest that
there were significant differences in phytic acid values obtained from
E1 and E2 groups when using the same method. Besides, these differ-
ences varied significantly depending on the method used and the oil
classes of the genotypes.
Studies in the literature have focused on the similarity of phytic acid
results obtained by different methods, rather than the raw vs. oil ex-
tracted samples (Gao et al., 2007; Raboy et al., 2017). In this respect, a
comparative discussion on the effect of oil extraction on the phytic acid
analysis was not possible. Nevertheless, our results may provide some
insight into this matter. Chen and H-L methods, which do not use AEC
separation, showed a positive correlation between the phytic acid va-
lues from the raw and oil extracted samples; while, this was not the case
in AEC using AOAC and Wade methods. It is noteworthy that the cor-
relations calculated in low oil genotypes had the opposite signs and
were found to be statistically significant when AOAC and Wade
methods were used. In these methods, organic phosphorus is collected
with 0.7 NaCI after passing the column, with the AOAC method using
an additional combustion process, and the detection is carried out via
total phosphorus with KH2PO4 standard. Such differences may have
caused the calculated correlations with these two methods to be in
opposite directions in low oil genotypes; and this may be valid for the
other genotype groups just as well.

5
F. Kahrıman, et al.
Fig. 2. Regression and decision tree plot showing the effect of oil content and analysis method used on the phytic acid content differences between raw and oil-
extracted samples.
The regression and decision tree analysis results, carried out to
elucidate the effect of oil extraction on phytic acid analysis results, are
highly informative. A model was developed to evaluate the relationship
between the change in the amount of oil in a sample and the difference
in the amount of phytic acid between the raw and oil extracted samples.
In Fig. 2, we tried to explain the relationship of the phytic acid differ-
ence of raw and oil extracted samples with oil content of the samples
and the analysis method used. A total of 13 nodes were formed in the
regression and decision tree. The average of the difference between the
phytic acid values obtained from the samples (n = 76) is −0.089 mg/g.
In these samples, the difference in the phytic acid values of the samples
(n = 12) with an oil ratio of more than 9.8 % was negative (−0.66 mg/
g). As these samples are classified only in terms of oil content, it is
understood that the analysis method used has no effect on the differ-
ence in phytic acid analysis results. The difference between the phytic
acid values of the samples (n = 64) with an oil content of less than 9.8
% and classified under node 3 was found to be 0.018 mg/g. These
samples are divided into two different groups according to the analysis
method used, and phytic acid difference between the raw and oil ex-
tracted samples were calculated as 0.38 mg/g for those that were not
analyzed with AOAC, Chen or H-L methods (n = 16). In other words, in
the case of using the Wade method in these samples, it can be stated
that removing oil causes a difference in the phytic acid content esti-
mated. In the samples classified under the third node, it was observed
that the number of the samples analyzed by AOAC, Chen and H-L
methods included 63 % of all samples. A negative difference was ob-
served in phytic acid values from raw and the oil extracted samples
when these three methods were used. The samples classified at the sixth
knot were assigned to two sub-classes based on the analysis method
applied. The samples analyzed with Chen or H-L method (n = 32) got a
share of 42 % within the total number of samples and the phytic acid
difference of these samples were in a negative direction. Under the
same knot, the differences between the phytic acid results were positive
(0.055 mg/g) for the methods other than Chen or H-L methods. Con-
sidering the knots within this class, it could be said that the above-
mentioned group contains the samples analyzed with the AOAC
method. Hence, we can argue that the method applied had a significant
impact on the difference between the analysis results from raw and oil
extracted samples. In addition, based on regression and decision tree
analysis, it can be stated that an oil ratio above 9.8 % in the samples has
6
Journal of Food Composition and Analysis 86 (2020) 103380
a notable effect on the phytic acid analysis result. Our results clearly
indicate that the sample’s oil concentration affects the phytic acid
analysis results, and this effect varies across the methods applied.
Sivakumaran and Kothalawala (2018) stated earlier that phytic acid
analysis should be performed after removing oil in the samples with oil
content above 15 %. Our findings agree with this earlier report in that
there is critical value of oil content, while suggesting a lower cutoff
point specifically for maize samples.
4. Conclusion
The colorimetric methods compared here showed significant dif-
ferences in terms of cost and duration of analysis. The AOAC method
was the most expensive, while the Chen method offered the most cost-
effective option. The data indicated that oil extraction had significant
effects on phytic acid results. This effect varied based on the oil level of
the genotype as well as the analysis method. Regression and classifi-
cation tree analysis results suggest that the oil content over a limit value
(9.8 %) affect the results of phytic acid analysis. It was also found that
the oil content of the sample interacts with phytic acid results from the
raw and oil extracted samples in different colorimetric methods, as it
can be deduced from the correlation analysis. Another point is that
other biochemical components may also affect the phytic acid analysis
and magnified the difference between raw and oil extracted samples
based on the method used. It is considered that new methods should be
developed, or existing methods should be revised in order to eliminate
the deviation in the high oil samples exceeding a limit value. The dif-
ferent results obtained from the analysis methods may be due to the
interaction of the extracted and unextracted samples with the seed
content and the chemicals used in the method and the applications. The
presence of oil in the sample may prevent the separation of phytic acid
and phosphorus from the sample which allows indirect detection of this
component in the extraction step. Since the methods used directly or
indirectly yield results based on the amount of organic or inorganic
phosphorus in the sample, this has changed the result of phytic acid in
the degreased and unextracted samples.
In conclusion, our results may help to select the appropriate method
for phytic acid analysis when dealing with high, normal, or low oil
maize genotypes. However, since all of these are spectrophotometric
methods, it would be necessary to conduct comparative studies with
F. Kahrıman, et al.
alternative methods such as chromatography and NMR spectroscopy for
decisive results. Further studies should also focus on optimization of the
factors such as the acid volume and concentrations used in the ex-
traction procedure and explore other agricultural products possessing
significant variation in terms of the biochemical constitution.
Acknowledgment
This work was supported by Çanakkale Onsekiz Mart University
Scientific Research Coordination Unit, Project number: FHD-2018-
2476.
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