Journal of Chromatographic Science, 2017, 1–7

doi: 10.1093/chromsci/bmx062
Article

Article

Simultaneous Determination of Soyasaponins

and Isoflavones in Soy (Glycine max L.) Products
by HPTLC-densitometry-Multiple Detection
Eman Shawky* and Shaimaa M. Sallam
Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt
*
Author to whom correspondence should be addressed. Department of Pharmacognosy, Faculty of Pharmacy,
Alexandria University, Alexandria 21521, Egypt. Email: shawkyeman@yahoo.com
Received 26 September 2016; Revised 5 June 2017; Editorial Decision 25 June 2017

Abstract
A new high-throughput method was developed for the simultaneous analysis of isoflavones and soya-
saponnins in Soy (Glycine max L.) products by high-performance thin-layer chromatography with den-
sitometry and multiple detection. Silica gel was used as the stationary phase and ethyl acetate:
methanol:water:acetic acid (100:20:16:1, v/v/v/v) as the mobile phase. After chromatographic develop-
ment, multi-wavelength scanning was carried out by: (i) UV-absorbance measurement at 265 nm for
genistin, daidzin and glycitin, (ii) Vis-absorbance measurement at 650 nm for Soyasaponins I and III,
after post-chromatographic derivatization with anisaldehyde/sulfuric acid reagent. Validation of the
developed method was found to meet the acceptance criteria delineated by ICH guidelines with
respect to linearity, accuracy, precision, specificity and robustness. Calibrations were linear with corre-
lation coefficients of >0.994. Intra-day precisions relative standard deviation (RSD)% of all substances
in matrix were determined to be between 0.7 and 0.9%, while inter-day precisions (RSD%) ranged
between 1.2 and 1.8%. The validated method was successfully applied for determination of the studied
analytes in soy-based infant formula and soybean products. The new method compares favorably to
other reported methods in being as accurate and precise and in the same time more feasible and cost-
effective.

Introduction (acetyldaidzin, acetylgenistin and acetylglycitin), according to their

Soybean (Glycine max (L.) Merill) is a highly utilized crop through-
out the world. Several reports are available in the literature regard-
ing the protective effects of soy against heart disease, osteoporosis
and various cancers including breast and colon cancers [1, 2]. Soy
contains different phytochemicals including isoflavones, anthocya-
nins and soyasaponins. Many studies have shown that the beneficial
effects of soybeans can be attributed to these phytochemicals [1–4].
Soybean products are numerous and include, flour, curd, milk, tofu,
miso (fermented soybean), sprouts, soy sauce, soybean oil, textured
soy proteins and soy protein drinks [2].
The major isoflavones (phytoestrogens) of soy can be classified as
glycosides (daidzin, genistin and glycitin), malonyl glycosides (malo-
nyldaidzin, malonylgenistin and malonylglycitin) or acetyl glycosides
conjugated functional groups, the most abundant being the glyco-
sides daidzin, genistin and glycitin [2, 3].
There is a growing interest in these compounds due to their bio-
logical effects, including estrogenic and fungi-toxic activities.
Isoflavones have been associated with a decreased risk of breast,
prostate and colon cancers and are under investigation for the pre-
vention of menopausal symptoms and osteoporosis. They are
thought to contribute to the cholesterol-lowering effect observed
when these products are consumed by humans [1–4].
Meanwhile, group B saponins are another ethanol-soluble frac-
tion associated with isoflavones which have also been associated
with cholesterol-lowering abilities. Relatively high amounts of soyasa-
ponins were reported in soybeans and different soy products [5–9].

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2 Shawky and Sallam
Soyasaponin groups A and B are found in soybeans. The group B
soyasaponins are the primary soyasaponins present in soybeans [10].
A number of studies have shown the soybean saponins ability to
inhibit different stages of cancer development or progression, in vivo
and in vitro [11–14].
Moreover, Soy-based infant formulas have been marketed
worldwide for over 60 years as safe and important feeding options
for infants. Since several studies have shown that soy-based infant
formulas are very rich in both classes of bioactive compounds, con-
cerns related to their potential biological effects on infants have
been aroused [15, 16]. Even though soyasaponins have low bioavail-
ability [17, 18], there is always a need for comprehensive analysis of
both isoflavones and soyasaponins composition of infant formulas.
The plethora of recent studies available on the different biologi-
cal actions of isoflavones and soysaponins phytochemicals support
the need for monitoring of these constituents from human intake to
excretion is considered crucial [1–10].
Several methods for soy isoflavones analysis have been reported,
such as high-performance liquid chromatography (HPLC)-coupled
with ultraviolet (UV), diode-array detector or mass spectrometric
(MS) detectors, high-performance thin-layer chromatography (HPTLC)
[24, 25] and gas chromatography (GC)-coupled with flame ionization
detector or MS detector [19–24]. LC–MS [22–23] has also been widely
used, especially for clinical studies of isoflavone metabolites in animals
and humans.
On the other hand, the quantitative determination of saponins in
plant material is considered challenging as they do not have chromo-
phores in their structure which hampers their detection in UV light,
allowing only for non-specific detection at 200–220 nm. Some
improved techniques for quantification of soyasaponins in plant
material have been reported [26], which include the application of
evaporative light-scattering detection for soyasaponins quantifica-
tion [27]. Some progress in applications utilizing LC–electrospray
MS (LC–ESI/MS) for soyasaponins determination has also been
done [7, 17]. The combination of thin-layer chromatography and
densitometry to determine soyasaponin content in different legumes
has been reported in Curl et al. [28] where the described method
was not validated and its sensitivity was unknown, and in Gurfinkel
et al. [29], which described the determination of total soyasaponins
without development of the TLC plates in a mobile phase.
Only few methods for the simultaneous determination of isofla-
vones and saponins in soy products are reported [30, 31] using LC/
electrospray ionization-MS and reversed-phase LC with evaporative
light-scattering and UV detection.
Meanwhile, a HPTLC method is a preferred analytical technique
for phytochemicals analysis because of its several advantages which
include the small amount of mobile phase required, the simple sample
preparation, and the possibility of the simultaneous quantification of
a large number of samples in shorter time compared with already
published analytical methods including HPLC, GC, etc. [32, 33]. The
automated sample applicator in HPTLC facilitates sample application
and repeated detection (scanning) of chromatogram which can be per-
formed with the same or different parameters.
In view of the above mentioned points, the objective of this work
was to develop a high-throughput analytical method can simulta-
neously detect isoflavones and soyasaponins in different Soy (Glycine
max L.) products. Due to its flexibility regarding detection, a planar
chromatographic method, i.e., HPTLC/UV/Vis was designed. It is, for
the first time, a simple, accurate and rapid HPTLC method has been
developed for the simultaneous quantification of isoflavones and
soyasaponins in a single run with single mobile phase. This method
offers a good alternative for other reported methods for routine analy-
sis due to its simplicity, low cost and at the same time reliability.
Experimental
Reagents and samples
Ethyl acetate, methanol, acetic acid and sulfuric acid all purchased
from Merck (Merck, Darmstadt, Germany), were of HPLC grade.
Soyasaponins I and III for quantitative analysis were isolated in the
Department of Pharmacognosy, Faculty of Pharmacy, Alexandria
University, Egypt. They were isolated using preparative TLC and
identified using different spectroscopic techniques (1 H-NMR, 13 C-
NMR and MS analysis). They were found to be 99% pure.
Genistin, daidzin and glycitin (>99.5%) were obtained from Sigma
(Steinheim, Germany).
Instrumentation
A CAMAG (CAMAG, Switzerland) HPTLC system consisting of a
sample applicator Linomat5, and a TLC Scanner was used for the
analyses. All instruments were controlled via the software platform
winCats 1·4·2 Planar Chromatography Manager (CAMAG).
HPTLC silica gel 60 W F254, 200 × 10 cm, (Merck, Darmstadt,
Germany) were used and developed in a CAMAG 20 × 10 cm hori-
zontal double twin trough chamber.
Chromatographic procedure
Standards and sample solutions were applied band-wise using the
following settings: band length 6 mm, distance between bands
4 mm, distance from left plate edge 10 mm, distance from right plate
edge 10 mm and from the lower plate edge 10 mm, resulting in
18 tracks per plate, dosage velocity 120 nL/s, as sample application
volumes, they were 4 μL for soybean and 6 μL for soy-based infant
formula and as standard application volumes 1–6 μL.
Plates were developed with ethyl acetate:methanol:water:acetic
acid (100:20:16:1, v/v/v/v) as the mobile phase (migration distance
85 mm). UV and Vis in situ spectra were recorded using the TLC
Scanner 3 (CAMAG). Multi-wavelength detection was performed
by TLC Scanner 3 with a slit dimension of 4 mm × 0.2 mm and a
scanning speed of 100 mm/s. In UV-absorbance mode, isoflavones
were measured at 265 nm and in Vis-absorbance mode, saponins
were detected at 650 nm, after post-chromatographic derivatization
by immersion into anisaldehyde/H2SO4 reagent solution and heating
on a plate heater for 2 min at 100°C.
Sample preparation
For the development and validation of the analytical method for the
simultaneous analysis of isoflavones and soyasaponins in soy-based
foods, a sample of soybean was acquired in a local supermarket in
Alexandria, Egypt. The soy-based infant formula sample Isomil 1
(Abbott Laboratories, the Netherlands) was purchased from a local
pharmacy located in Alexandria, Egypt.
Samples were extracted in triplicate according to a modification of
the methods of Genovese and Lajolo [34], Fang et al. [35]. About 1 g
of each sample and 25 mL of aqueous methanol 80% was extracted
in an Ultra-sonic extractor 40 min at 20°C. The obtained extract was
centrifuged for 10 min at 3,000 rpm, the supernatant collected and
the residue re-extracted twice following the same procedure. Next,
Simultaneous Determination of Soyasaponins and Isoflavones
supernatants were combined and placed in an ultrasound bath for
15 min. The organic solvent was removed with the aid of a rota-
evaporator at 150 rpm (Büchi, Switzerland). The concentrated extract
was transferred to a 25 mL volumetric flask and filled up to 25 mL
with methanol. All samples were stored at 4°C.
Standard and derivatization solutions
Five milligrams of each of genistin, daidzin and glycitin were
weighted in 50 mL volumetric flask and filled up to 50 mL with
methanol. Two milligrams of each of Soyasaponins I and III
(Figure 1) were weighted in 10 mL volumetric flask and filled up to
10 mL with methanol. The solution stored refrigerated and pro-
tected from light was at least stable for 10 days. As derivatization
solution (for immersion), 0.5 mL p-anisaldehyde was mixed with
50 mL glacial acetic acid and 1 mL conc. sulfuric acid (v/v).
Method validation
The procedure was validated according to the International
Conference on Harmonization guidelines (ICH, 2005) [36].
Selectivity
The selectivity, defined as the ability to distinguish the analyte from
other substances, was confirmed by comparing the Rf values of
Genistin, daidzin, glycitin, Soyasaponins I and III in samples with
that of the reference standards.
Linearity and range
The calibration curves were generated using six data points. Volumes
of 1, 2, 3, 4, 5 and 6 μL for isoflavones and soyasaponins standard
solution were analyzed by the proposed method in triplicate. Linear
calibration plots were obtained over the calibration range belonging
to 75–125% range from target value for each compound.
LOD and LOQ
Limit of detection (LOD) and limit of quantification (LOQ) were calcu-
lated by the use of the equations LOD = 3 SD/σ and LOQ = 10 SD/σ,
Figure 1. Chemical structure of (a) glycosidic isoflavones, (b) Soyasaponin I and (c) Soyasaponin III.
3
respectively, where SD is the standard deviation of the response
(y-intercept) and σ is the slope of the calibration curve.
Precision
The precision was tested as repeatability (intra-day precision) and
intermediate precision (inter-day precision). Intra-assay precision
and inter-assay precision were expressed as relative standard devia-
tion (RSD) of six individual spots of 100% of the test concentration
for each compound on the same day and six repeated assay of refer-
ence compounds at 100% of the test concentration over a period of
6 days, respectively.
Accuracy
The accuracy of the method was tested by performing recovery stud-
ies in terms of standard addition technique. Known quantities of
each reference standard compounds plotting were added to pre-
quantified sample solution in the range of low (75%), medium
(100%) and high (125%) nominal analytical concentrations. Each
experiment was performed in triplicate and the accuracy was calcu-
lated as recovery and RSD% of the compound in the sample.
Robustness
As is specified in ICH guidelines [36], the robustness of an analytical
procedure is a measure of its capacity to remain unaffected by small,
but deliberate variations in method parameters and provides an indi-
cation of its reliability during normal usage. Minor changes in chro-
matographic conditions were made in mobile phase composition
position (±2%), chamber saturation period, development distance
and the effect of these changes on retention factor was measured.
Statistical analyses
Data are presented as mean ± standard deviation. Quantitative eval-
uation was by peak area measurement. The contents of isoflavones
and soyasaponins in the analyzed samples were determined using
analysis of variance (one-way ANOVA). All statistical analyses were
performed using Excel 2010® (Microsoft Office 2010). Differences
were considered significant when P < 0.05.
4 Shawky and Sallam

Results The selectivity of the method was confirmed by comparing the

Mobile phase optimization and wavelength selection
Several solvent mixtures for single or multiple separation of isoflavones as
described in literature [25, 37] were investigated to evaluate the separation
between the isoflavones and soyasaponins. A satisfactory separation was
not obtained with any of the mobile phases described. After several trials,
the mixture of ethyl acetate:methanol:water:acetic acid (100:20:16:1,
v/v/v/v) was selected. The maximum absorption wavelengths described
for genistin, daidzin and glycitin are 264, 270 and 260 nm, respectively
[20, 21]. In this work, the optimal wavelength selected for isoflavones
was 265 nm. In the case of soyasaponins, the optimum absorption
wavelength after derivatization was 650 nm since there was no inter-
ference from the isoflavones which did not show any readings at this
wavelength, though they were detectable under visible white light.
Method validation
The proposed HPTLC method was validated according to ICH
guideline [36] regarding selectivity, linearity and range, LOD and
LOQ, precision, accuracy and robustness.
Rf of analytes and genistin, daidzin, glycitin, Soyasaponins I and III
standards (Rf = 0.39, 0.47, 0.59, 0.27 and 0.34, respectively)
(Figure 1) and it has been observed the absence of interfering or
overlapping bands in the analyzed samples (Figures 2 and 3), while,
the specificity was assessed with the peak purity test of the main
three isoflavones, genistin, daidzin and glycitin and the two
Soyasaponins I and III, performed in each sample comparing the UV
overlaid spectra. The scan-densitogram obtained from a mixture of
all standards (Figure 4) showed selective baseline separation
between genistin, daidzin and glycitin (scan wavelength 265 nm)
and Soyasaponins I and III (after derivatization with anisaldehyde/
sulfuric acid followed by scanning at 650 nm).
For each compound a calibration was established using six
analyte concentrations in triplicate, applying different volumes
of the standard solution. The calibration plots showed linear regres-
sions with correlation coefficients R2 > 0.994 (Table I). For routine
analysis, a three-point calibration was used, applying in duplicate the
lowest-, middle- and highest-point of each calibration on the HPTLC
plate.

Figure 2. HPTLC scan densitogram of different soy samples and isoflavones and soyasaponins standards mixture at 265 nm, where only isoflavones are de-
tected, and chromatogram photographed at UV 254 nm.

Figure 3. HPTLC scan densitogram of the same samples and standards tracks in figure 2, after derivatization with anisaldehyde/H2SO4, at 650 nm, where only
soyasaponins are detected, and chromatogram photographed under visible daylight.
Simultaneous Determination of Soyasaponins and Isoflavones 5

Figure 4. Scan densitogram of the same tracks, containing a mixture of all the standards, showing selective base line separation between, (a) genistin (1), daid-
zin (2) and glycitin (3) (at 265 nm) and (b) soyasaponin I (4) and soysaponin III (5) (at 650 nm after derivatization with anisaldehyde/sulphuric acid).

Table I. The Chromatographic and Calibration Parameters are LOD and LOQ

Compounds Rf Regression equation R2 LOD (μg/mL) LOQ (μg/mL)

Genistin 0.39 y = 6,861.9x + 2,004.3 R2 = 0.9974 0.0318 0.106

Diadzin 0.47 y = 12,401x + 3,196.7 R2 = 0.9936 0.0502 0.167
Glycetin 0.59 y = 4,532.7x + 1,240.9 R2 = 0.9949 0.0449 0.149
Soyasaponin B-I 0.27 y = 2269.3x + 3,845.4 R2 = 0.9946 0.1143 0.381
Soysaponin B-III 0.34 y = 3559.8x + 1,914.9 R2 = 0.9942 0.096 0.32

As recommended by ICH (ICH, 2005) the LOD and LOQ were Table II. Precision Results
calculated based on the calibration curve. The LOD and LOQ were
0.032 and 0.106 μg/μL for genistin, 0.050265 and 0.167 μg/μL for Intra-day precision Inter-day precision
(n = 6) (μg/mL) (n = 6) (μg/mL)
daidzin, 0.044 and 0.149837 μg/μL for glycitin, 0.114336 and
0.381122 μg/μL for Soyasaponin I and 0.096002 and 0.320006 μg/μL Compound Mean ± SD RSD% Mean ± SD RSD%
for Soyasaponin III, respectively.
Genistin 15.06 ± 0.124 0.827 14.98 ± 0.263 1.761
The results concerning the precision of the method are presented
Diadzin 6.98 ± 0.059 0.847 7.065 ± 0.132 1.872
in Table II. Inter-day precision of the validated method ranged from
Glycetin 2.505 ± 0.0187 0.746 2.49 ± 0.046 1.849
1.2 to 1.8%, while intra-day precision ranged from 0.5 to 0.9%. All Soyasaponin I 124.7 ± 0.742 0.595 124.65 ± 1.51 1.212
values were acceptable according to the ICH (2005) guidelines. Soysaponin III 29.78 ± 0.287 0.965 29.83 ± 0.536 1.798
The results of accuracy and recovery investigations are shown in
Table III. The average recovery for genistin, daidzin, glycitin,
Soyasaponins I and III depending on the concentration tested varied
detection. Table IV summarizes the values found in the respective
from 99.4 to 101.8%, 98.1 to 102.5%, 98.6 to 100.8%, 99.4 to
samples for each compound. Isoflavones and Soyasaponins in infant
101.2% and 99.3 to 100.3%, respectively. The recoveries and RSD
formula ranged between 0.01–0.28 mg/g and 0.26–1.3 mg/g while in
for each studied compound revealed that the method is highly accu-
soybean they ranged between 0.06–0.39 and 0.74–3.1, respectively.
rate and suitable for the future use.
In general, these results were in accordance with data previously
During method development several parameters were evaluated
published in the literature [16, 18, 34, 38].
regarding robustness. The results obtained on the robustness study
showed that slight variations in the chromatographic conditions had
no significant effect on the Rf values of the analytes.
Discussion
A HPTLC method for the simultaneous quantification of three iso-
Analysis of soy samples flavones and two soyasaponins in Soy (Glycine max L.) products
The applicability of the validated method was checked by analyzing was developed and validated in this study. To the best of our knowl-
two different Soy (Glycine max L.) samples. Figures 2 and 3 show edge, the separation in one run and the simultaneous detection of
the densitograms of two samples obtained by multi-wavelength genistin, daidzin glycitin, Soyasaponins I and III as well as the
6 Shawky and Sallam

Table III. Accuracy of the Proposed Method

Compounds Amount in Level Amount Amount found Recovery RSD %

sample (μg/mL) % added (μg/mL) (μg/mL) %

Genistin 15.6 75 10 25.4 99.44 1.294

100 15 30.5 99.67 1.994
125 20 36.26 101.87 1.567
Diadzin 6.8 75 5 12.1 102.54 1.652
100 7 13.53 98.06 1.128
125 9 15.6 98.73 1.695
Glycetin 2.4 75 2 4.44 100.83 1.821
100 2.5 4.83 98.63 1.061
125 3 5.33 98.82 0.943
Soyasaponin I 124.4 75 93 216.13 99.41 0.737
100 125 248.1 99.47 1.068
125 155 282.83 101.22 0.883
Soysaponin III 29.6 75 22 51.3 99.41 1.522
100 30 59.8 100.33 1.761
125 37 66.2 99.39 0.918

Table IV. Assay for Isoflavones and Soyasaponins in Soy Samples 4. Sakthivelu, G., Akita Devi, M.K., Giridhar, P., Rajasekaran, T.,
Ravishankar, G.A., Nikolova, M.T., et al.; Isoflavone composition, phe-
Soybean sample Soy milk infant formula nol content, and antioxidant activity of soybean seeds from India and
Compounds Amount found RSD Amount found RSD Bulgaria; Journal of Agriculture and Food Chemistry, (2008); 56:
in mg/g (n = 3) % in mg/g (n = 3) % 2090–2095.
5. Hu, J., Lee, S.O., Hendrich, S., Murphy, P.A.; Quantification of the group
Genistin 0.39 1.41 0.28 1.93 B soyasaponins by high performance liquid chromatography; Journal of
Diadzin 0.17 1.49 0.092 1.65 Agriculture and Food Chemistry, (2002); 50: 2587–2594.
Glycetin 0.06 2.2 0.01 2.4 6. Berhow, M.A., Kong, S.B., Vermillion, K.E., Duval, S.M.; Complete
Soyasaponin I 3.11 1.03 1.3 0.6 quantification of group A and group B soyasaponins in soybeans; Journal
Soysaponin III 0.74 1.43 0.26 1.99 of Agriculture and Food Chemistry, (2006); 54: 2035–2044.
7. Gu, L., Tao, G., Gu, W., Prior, R.L.; Determination of soyasaponins in
soy with LC-MS following structural unification by partial alkaline degra-
quantification of all five compounds by HPTLC was successfully dation; Journal of Agriculture and Food Chemistry, (2002); 50:
achieved for the first time. 6951–6959.
Although different TLC methods were published about the deter- 8. Rickert, D.A., Johnson, L.A., Murphy, P.A.; Improved fractionation of
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9. Rickert, D.A., Meyer, M.A., Hu, J., Murphy, P.A.; Effect of extraction
all five compounds simultaneously on a simple silica gel plate using
pH and temperature on isoflavone and saponin partitioning and profile
common reagents. The study was mainly targeted to the development during soy protein isolate production; Journal of Food Science, (2004);
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and precision. The validated method is straightforward being faster 11. MacDonald, R.S., Guo, J., Copeland, J., Jimmy, D., Browning, J., Sleper, D.,
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