Talanta 168 (2017) 329335

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Talanta
journal homepage: www.elsevier.com/locate/talanta

Evaluation of new natural deep eutectic solvents for the extraction of MARK
isoavones from soy products

Sylwia Bajkacza, , Jakub Adamekb
a
Department of Inorganic, Analytical Chemistry and Electrochemistry, Faculty of Chemistry, Silesian University of Technology, M. Strzody 7, 44-100
Gliwice, Poland
b
Department of Organic and Bioorganic Chemistry and Biotechnology, Faculty of Chemistry, Silesian University of Technology, Krzywoustego 4, 44-100
Gliwice, Poland

A R T I C L E I N F O A BS T RAC T

Keywords: Natural deep eutectic solvents (NADESs) are considered to be new, safe solvents in green chemistry that can be
Natural deep eutectic solvent widely used in many chemical processes such as extraction or synthesis.
Extraction In this study, a simple extraction method based on NADES was used for the isolation of isoavones (daidzin,
Green chemistry genistin, genistein, daidzein) from soy products. Seventeen dierent NADES systems each including two or
Isoavones
three components were tested. Multivariate data analysis revealed that NADES based on a 30% solution of
Soy products
choline chloride: citric acid (molar ratio of 1:1) are the most eective systems for the extraction of isoavones
from soy products. After extraction, the analytes were detected and quantied using ultra-high performance
liquid chromatography with ultraviolet detection (UHPLC-UV).
The proposed NADES extraction procedure achieved enrichment factors up to 598 for isoavones and the
recoveries of the analytes were in the range 64.799.2%. The developed NADES extraction procedure and
UHPLC-UV determination method was successfully applied for the analysis of isoavones in soy-containing
food samples. The obtained results indicated that new natural deep eutectic solvents could be an alternative to
traditional solvents for the extraction of isoavones and can be used as sustainable and safe extraction media for
another applications.

1. Introduction of information on practical issues related to their application as

One of the most important principles of Green Chemistry is the use
of safer solvents and auxiliaries [1]. There is no doubt that solvents
play an essential role in chemical processes of mass and heat transfer.
Selection of appropriate solvents plays a decisive role in extraction
process. From the pharmaceutical and biochemical research point of
view, the development of new, green methods for the extraction of
bioactive compounds from natural sources is crucial. It is not surpris-
ing that in recent years a wide range of solvents (including ionic
liquids) has been considered for this purpose. However, the discovery
and widespread utilisation of natural deep eutectic solvents (NADES)
represents a long-expected breakthrough in this area of research.
NADESs have been recognised as a novel class of sustainable solvents
because they are made from widely available, naturally occurring, non-
toxic and biodegradable components. The remarkable physicochemical
properties and low cost of synthesis and utilisation of NADES have
attracted a great deal of interest in analytical and engineering elds.
Although NADESs have attracted much interest, there is still a lack
extraction solvents, such as their eciency, their optimal concentra-
tion, and the recovery of compounds from NADES extracts [2]. To our
knowledge there are currently only three manuscripts in the literature
about the application of NADES in extraction processes [35]. Dai
et al. studied the extraction of avonols from saower, using dierent
NADESs lactic acid: glucose, glucose: choline chloride, and fructose:
glucose: sucrose. In those works, it was found that NADES have a
strong ability to extract phenolic compounds, related to the H-bond
interactions that are established between the phenolic compounds and
the NADES components.
Further information is need to better understanding of the inter-
actions and complexing actions of dierent NADES compositions, as
well as their applicability in the extraction of biologically active
compounds from the various materials. To this end, it is essential to
develop NADES synthesis methods and their applications in extraction
processes. Thus, this study aimed to develop new NADES and apply
them in the extraction of isoavones from soy product samples. To the
best of our knowledge, no extraction procedures based on these

Corresponding author.
E-mail address: sylwia.bajkacz@polsl.pl (S. Bajkacz).

http://dx.doi.org/10.1016/j.talanta.2017.02.065
Received 11 August 2016; Received in revised form 22 February 2017; Accepted 27 February 2017
Available online 01 March 2017
0039-9140/ 2017 Elsevier B.V. All rights reserved.
S. Bajkacz, J. Adamek
solvents have been reported for extraction of this class of avonoids.
Isoavones are a group of chemical compounds that are widespread
in the plant world. Recently there has been interest in isoavones has
been increasing, especially concerning genistein and daidzein. Their
biological activity has been exploited in pharmaceutical formulations
and dietetics. Isoavones form a group of nonsteroidal natural
chemicals that demonstrate oestrogenic, antioxidant and anti-allergic
agent properties. Isoavones are also used in the cosmetics industry, to
delay the start of the aging of the skin, stimulate collagen biosynthesis
in broblasts and accelerate the regeneration of skin cells [6].
Although these compounds are not steroids, they have hydroxyl
groups in the 7 and 4 positions in the same conguration as those
found in oestradiol. Isoavones occur as inactive glycosides and as free
aglycones. They can accumulate in the body. After absorption of
isoavone aglycones they are metabolized via UDP-glucuronosyltrans-
ferase and sulphotransferase [7].
Maintenance of the proper level of isoavones in the diet is
important in the prevention of many diseases such as cardiovascular
diseases, or cancer. Soy products have been suggested to provide a
protective eect against breast, intestine, liver, bladder, prostate, skin
and stomach cancer. Isoavones also help maintain a healthy heart,
strong bones and support the immune system. In soy beans, isoa-
vones are present both as aglycones and as glycosides (-glycosides, 6-
O-acetyl- and 6-O-malonyl-glycosides). Glycosides are the predomi-
nant forms found in soy beans. The content and distribution of
isoavones in soy products depend on the type of seed, growth, and
harvesting, as well as on the type of processing [811].
For soy and soy products, classical extraction methods such as
solid-liquid extraction (SLE) with dierent organic solvents [1214],
solid-phase extraction (SPE) [15] and supercritical uid extraction
[16,17] are used. These methods utilise large amounts of solvent as
well as sample and are considerably time consuming. Alternative
separation protocols are based on pressurised liquid extraction (PLE)
[1820], microwave assisted extraction (MAE) [2123] or ultrasound-
assisted extraction [2426]. Current methods tend to minimise the
consumption of solvents, sample amounts, and extraction times by
applying additional energy and/or pressure to the mixture of sample
and solvent.
More research on the preparation of NADES is required to make
tailor-made NADES for specic applications and specic anaytes (it has
been studied only for the extraction of quercetin and rutin). Therefore
in this study, we aimed to evaluate and maximise the potential and
eectiveness of NADESs as green extraction solvents to selectively
isolate isoavones, such as genistein (GT), daidzein (DA), genistin
(GTGL) and daidzin (DAGL) from soy-containing food samples (Fig.
S1).
2. Experimental
2.1. Chemicals and reagents
Standards of genistein (GT), daidzein (DA), genistin (GTGL),
daidzin (DAGL) and biochanin A (BIO) were obtained from Sigma
Aldrich (St. Louis, MO, USA). Acetonitrile, methanol, water, triuor-
oacetic acid, acetic acid, and formic acid (all HPLC grade) were
purchased from Merck (Darmstadt, Germany). Methanol was obtained
from Stanlab (Lublin, Poland). Choline chloride, saccharose, D(+)-
glucose, D(+)-xylose, L(+)-tartaric acid, citric acid and urea were
purchased from Alfa Aesar (Lancashire, United Kingdom). Glycerine
was obtained from Chempur (Piekary lskie, Poland).
2.2. Preparation of standard solutions, calibration standards and
quality control (QC) samples
Standard stock solutions of analytes (GT, DA, GTGL, DAGL) and an
internal standard (BIO) were prepared by dissolving weighted amounts
330
Talanta 168 (2017) 329335
in methanol to give a nal concentration of 1 mg mL1. Working stock
solutions of analytes at concentration 100 g mL1 and 10 g mL1
were prepared by appropriate dilution of the standard solutions with
methanol and were stored at 4 C. A working IS solution (50 g mL1)
was prepared in methanol.
Calibration curve (CC) standards and quality control (QC) samples
were prepared by spiking breakfast cereals that had been dried to
constant weight (blank sample) with the working solutions of the
analytes in methanol. Calibration standards were prepared at 0.5, 1.25,
2.5, 5.0, 10.0, 17.5 and 25.0 g g1 concentrations. Quality control
samples were prepared at three dierent levels: low 1.0 g g1 (LQC),
medium 7.5 g g1 (MQC) and high 20.0 g g1 (HQC) concentrations.
Spiked samples were left until the solvent had totally evaporated and
stored in closed vessels at room temperature for 24 h before the
extraction.
2.3. NADES preparation
Natural deep eutectic solvents were prepared according to the
previously published procedure [3]. The two- or three-component
mixtures with calculated amounts of deionised water were added to a
glass vials sealed with a screw-caps and equipped with a magnetic
stirring bars. The mixtures were vigorously stirred (600 rpm) and
heated to 70 C until a homogeneous liquid was formed (1530 min).
Seventeen dierent NADES systems each including two or three
components were obtained (Table S1, in Supplementary material).
2.4. Optimisation of NADES extraction procedure
A breakfast cereals sample (with low amount of isoavones, blank
sample) spiked with a known amount of isoavones was used to
optimise the extraction procedure. Breakfast cereals samples were
dried to constant weight using a WPS 30 S moisture analyser
(RADWAG, Radom, Poland). The samples used for optimisation of
NADES extraction procedure were spiked with isoavones (standard
solution in methanol, nal concentration of isoavones 4.0 g g1).
Fortied samples were left to stand at room temperature for 24 h
before analysis. All optimisation procedures were carried out in
triplicate. Selection of the optimal conditions was based on the
recoveries obtained for isoavones and the enrichment factor (Ef).
The enrichment factor was calculated as follows:
Cf
Ef =
C0
where Cf is the concentration found/predicted in NADES and C0 is the
original concentration in the spiked our.
The following extraction parameters were studied:
NADES type: mixture of dierent compounds (Table S1, in
Supplementary material)
water content in NADES: 10, 15, 20, 25, 30, 40, 50, 75%
ratio of NADES volume (L) to sample amount (mg): 200/200=1:1,
400/200=2:1, 600/200=3:1, 800/200=4:1, 1000/200=500:1, 1500/
200=7.5:1
extraction time: 40120 min
extraction temperature: 3080 C central composite design(CCD)
ultrasonic power: 264616 W.
Central composite design (CCD) was used to nd the optimal values
for three independent variables: extraction time (A), extraction tem-
perature (B) and ultrasonic power (C) at ve levels (, 1, 0, +1, and
+). When applying experimental design methodologies, it is advisable
to keep the number of variables as low as possible in order to avoid very
complex response models and high variability. This design confounds
some of the main eects with interactions or interactions among
S. Bajkacz, J. Adamek
themselves, resulting in a smaller set of experiments but nevertheless it
is able to identify the inuence of each parameter as well as the rst-
order interactions between the factors. The whole experiments con-
sisted of 20 experimental points that included six replicates of the
centre points. The centre values were 80 min for extraction time, 55 C
for extraction temperature and 440 W for ultrasonic power. Statistical
comparisons were performed using Statistica 12 software (StatSoft,
Poland) using a two-tailed t-test and a one-way analysis of variance
(ANOVA). The p-values < 0.05 were considered signicant.
2.5. NADES microextraction procedure
Samples belonging to dierent soy-containing food categories
(soybeans, our, pasta, breakfast cereals, cutlets, tripe, soy drink, soy
nuts, soy cubes and three dierent dietary supplements) were pur-
chased from a local grocery store and pharmacies. Immediately after
reception the material was ground in a home style coee grinder. In the
case of the dietary supplements, the contents of 1020 capsules were
pooled in order to minimise the eect of variation between single
capsules. The ground material was then dried to constant weight using
a WPS 30 S moisture analyser (RADWAG, Radom, Poland). All
samples were stored in a closed vessels at room temperature (approxi-
mately 21 C, protected from moisture) until the time of extraction.
From each commercial samples 200 mg was weighed out and 50 L
of the BIO internal standard solution (50 g mL1) was added. 600 L
of the NADES was added into each sample (a ratio of NADES volume to
sample equal to 3:1). Further UAE extraction was performed by for
60 min at 60 C and 440 W. The UAE extraction was carried out in an
ultrasonic device (Sonorex Digital 10 P, Bandelin Electronic), equipped
with a digital timer and a temperature controller. All sample solutions
were then centrifuged for 5 min at 2000g in an IKA mini G centrifuge
(IKA, Staufen, Germany) and 300 L of the liquid supernatant was
moved to another tube. The solutions were then diluted with 300 L of
methanol and subsequently centrifuged for 5 min at 6000 rpm. Finally
the supernatant was transferred into a sample tube for UHPLC-UV
analysis.
2.6. Instrumentation and analytical conditions
The UHPLC system (Merck Hitachi, Germany) used in this study
consisted of a pump (Model L-2160U), a UV detector (Model L-
2400U), an autosampler (Model L-2200), a thermostated column
compartment (Model L-2350U) and a degasser module. The column
used was a Poroshell 120 EC-C18 analytical column (100 mm3.0 mm;
2.7 m, Agilent Technologies, USA) and kept at 40 C. The binary
mobile phase consisted of solvent A (0.1% formic acid in water) and
solvent B (acetonitrile) combined in the following gradient: 80% A
(0 min), 60% A (3.0 min), 20% A (3.5 min) and 10% A (6.0 min). The
ow rate was set between 0.4 and 0.6 mL min1 (0.4 mL min1 0 min,
0.4 mL min1 3 min, 0.4 mL min1 3.5 min and 0.6 mL min1 6 min).
Finally, the system was re-equilibrated using the initial solvent
composition for 2 min between injections. The volumes loaded in the
UHPLC system were 2 L for each sample. Monitoring and quantita-
tion were performed at 254 nm for all analytes and for the IS
(biochanin A). The whole UHPLC conguration was driven by EZ
Chrom Elite System Manager software.
2.7. Method validation
The NADES-UHPLC-UV methodology proposed was properly vali-
dated in terms of linearity, limit of detection (LOD), limit of quantica-
tion (LOQ), precision, accuracy, and recovery, according to our
previous work [27,28]. The description was placed in Supplementary
material.
331
Talanta 168 (2017) 329335
3. Results and discussion
3.1. Chromatographic conditions
The chromatographic conditions were optimised to obtain better
separation in shorter times. Dierent types of columns (Zorbax RRHD
SB-C18 (50 mm2.1 mm, 1.8 m), Poroshell 120 EC-C18
(100 mm3.0 mm, 2.7 m), Hypersil GOLD (100 mm2.1 mm,
1.9 m)), mobile phases (acetonitrile-water, methanol-water), types
of acid (triuoroacetic acid, formic acid and acetic acid), concentrations
of additive (0.05%, 0.1%, and 0.2%), column temperatures (20, 30 and
40 C) and ow rates (0.2, 0.3, 0.4 and 0.6 mL min1) were optimised.
It was found that the peaks resolution and symmetry decreased
when using the Zorbax RRHD SB-C18 and Hypersil GOLD columns
when compared to the Poroshell 120 EC-C18 column. Therefore, the
Poroshell 120 EC-C18 column was selected as the analytical column.
Analyte retention times were shorter using acetonitrilewater com-
pared to methanolwater, so acetonitrilewater was chosen as the
mobile phase. The addition of 0.1% of each acid in the mobile phase
decreased peak tailing signicantly, and formic acid yielded the best
results. Then several gradient programs were compared and the
percentage of acetonitrile was optimised to obtain a proper separation
of isoavones and IS. The gradient elution program was optimised as
described in the Instrumentation and analytical conditions section.
The column temperature was kept at 40 C and the injection volume
was 2 L. Based on the ultraviolet (UV) spectra of the analysed
compounds, a detection wavelength of 254 nm was chosen. UHPLC
chromatograms of a standard mixture under the optimised UHPLC
conditions are shown in Fig. 1A. The total chromatography analysis run
time per sample using the optimised method was 6.0 min.
3.2. Optimisation of the NADES extraction parameters
3.2.1. Eect of NADES composition
Seventeen dierent NADES systems each including two or three
components were tested. Because of the very high viscosity of NADES,
30% (w/w) water in NADES solution was used for the extraction. The
eect of the NADES composition on the extraction eciency is shown
in Fig. 2. The most eective extraction systems were NADESs based on
choline chloride: citric acid (molar ratio of 1:1 and 2:1) and choline
chloride: glycerine (molar ratio of 2:1). The use of three-component
NADES including choline chloride: citric acid: glycerine (molar ratio of
2:2:1) improved the extraction eciency of genistein and daidzein but
decreased the yield of genistin and daidzin. The extraction yield seems
to be closely related to the types of interactions that may occur between
solutes and solvents. In this context, not only the polarity of solvent,
but especially the possibility of hydrogen bond formation between
solvent molecules and isoavone compounds is likely of great impor-
tance.
Based on the above described results NADES 3 (a choline chloride:
citric acid; molar ratio of 1:1) was selected as the best solvent type,
critical for extraction eciency, and applied in further tests.
3.2.2. Eect of water content in NADES
NADES 3 (choline chloride: citric acid (1:1)) with dierent con-
centrations of water (10, 15, 20, 25, 30, 40, 50, 75%, w/w) was
evaluated for the extraction of isoavones. As can be seen in Fig. S2 (in
Supplementary material), the extraction eciency strongly depends on
the water content. In the case of low water concentrations (10, 15%),
extraction eciencies are also low, probably because the viscosity of
NADES is very high, which is benecial for the mass transport. The
addition of 2530% water signicantly improved the extraction results.
A further increase in water content (4075%) led to a decrease in
extraction eciency. High concentrations of water can limit interac-
tions between isoavones and NADES components.
Based on these results a concentration of 30% (w/w) water in
S. Bajkacz, J. Adamek Talanta 168 (2017) 329335

Fig. 1. Representative UHPLC-UV chromatograms of (A) standards mixture containing the isoavones and IS (concentration 2 g mL1), (B) dietary supplement and (C) soy cubes
samples using the proposed NADES-UHPLC-UV method. Compounds: daidzin (DAGL, 1.81 min), genistin (GTGL, 2.73 min), daidzein (DA, 4.63 min), genistein (GT, 5.01 min),
biochanin A (BIO, 5.50 min). Chromatographic conditions: Poroshell 120 EC-C18 analytical column (100 mm3.0 mm; 2.7 m), mobile phase: (A) 0.1% formic acid in water, (B)
acetonitrile, gradient elution, column temperature: 40 C, detection: UV =254 nm.

NADES was chosen for subsequent experiments.

3.2.3. Eect of the ratio of NADES volume to sample amount

Extraction was carried out at dierent liquid (extractant, NADES
volume)solid (sample mass) ratio (1:17.5:1) conditions while the
other extraction parameters were the same as described in Section 2.5.
The extraction recoveries of the isoavones signicantly increased as
the ratio of liquid to solid increased from 1:1 to 3:1 (Fig. S3, in
Supplementary material), due to the increase of the driving force for
the mass transfer of the analytes. These phenomena are related to the
extraction capacity of the solvent. When the volume of the extraction
solvent is large the extraction capacity of solvent is high and analytes
are extracted into the solvent quickly, while at lower volumes, more
time is required for analyte extraction. However, when the ratio
continued to increase above three, extraction recoveries did not
increase further. Consequently, a ratio of NADES volume (600 L) to
sample amount of three (200 mg) was used as the optimum, because it
Fig. 2. Eect of NADES composition (Table 1) on extraction eciency. Extraction
provided quantitative recoveries and high enrichment factors for the
conditions: water content: 30%; ratio of NADES volume to sample amount: 3:1;
extraction time: 60 min; extraction temperature: 60 C; ultrasonic power: 616 W.
analysed compounds.

3.2.4. Eect of extraction time, temperature and ultrasonic power

To study the eect of extraction time, temperature and ultrasonic

332
S. Bajkacz, J. Adamek
Table 1
Summary of calibration curve, linearity range, LOD, LOQ and Ef (n=6).
Analyte Regression equation Linear rang (g g1) Coecient of determination r2
DAGL y=0.4509x+0.0346 0.525
GTGL y=0.5822x+0.0464 0.525
DA y=0.5352x+0.0253 0.525
GT y=0.5998x+0.04459 0.525
power a circumscribed central composite design (CCD) with ve levels
was used. Statistics-based optimisation of variables using RSM can be
advantageous over the classical one-variable-at-a-time (OVAT) ap-
proach because it allows for the evaluation of interacting eects
between variables and variable optimisation with overall scope from
fewer experiments. In order to evaluate the eciency of the proposed
procedure, the extraction recovery of isoavones was considered as the
response. The experiments were performed in random order to avoid
systematic error. The design matrix including the experiments (un-
coded variables) and the related responses is shown in Table S2 (in
Supplementary material).
The variables and the response were processed to build a dierent
quadratic multiple regression model for each isoavone:
n n n 1 n
Y = 0 + iXi + iiXi2 + ijXiXj
i =1 i =1 i =1 j =2
j>i
The model quality was evaluated in terms of the square of the
correlation coecient (R2) and the lack-of-t was evaluated by analysis
of variance (ANOVA) at the 95% condence level. The resulting R2
values were 0.9123 or higher in the three models, indicating that the
experimental data were in relatively good agreement with predicted
extraction yields for each model. All of the F-values for the lack-of-t of
these models were insignicant, which supported the hypothesis that
these models were sucient to accurately represent the experimental
data. The models were expressed as second order polynomial quadratic
equations for the extraction recovery (Y) and the factors (A, B, and C)
as follows:
YDAGL=91.8+7.20A11.4A21.82B4.39B21.03C+1.73C2
5.25AB0.34AC+1.22BC
YGTGL=95.9+7.25A18.3A2+2.47B5.04B20.23C2.19C2
2.56AB0.54AC+0.78BC
YDA=78.4+8.43A8.31A23.97B+6.02B2+0.19C0.07C29.86
AB1.19AC0.04BC
YGT=87.11.48A22.7A2+6.57B3.13B2+3.78C2.74C230.3
AB0.95AC+2.88BC
For graphical interpretation of the signicant interactions between
the variables, a three-dimensional (3D) response surface plot of the
model was considered. Fig. S4A (in Supplementary material) shows the
response surface plots for extraction time and extraction temperature
at a constant ultrasonic power of 440 W. As can be seen, when the time
of the extraction increases, the response also increases. This might be
due to a requirement for a longer time of exposure of the isoavones to
the release medium where the NADES penetrated into the dried
powdered material, dissolved the isoavones and subsequently diused
out from the material. Recoveries reached a maximum at 60 min, and
did not increase further as the extraction proceeded. This indicated that
60 min was sucient to obtain a satisfactory extraction eciency.
The extraction eciency for the analysed compounds increased
when the extraction temperature increased from 10 C to 60 C. As
shown in Fig. S4A (in Supplementary material), the maximum
recoveries of isoavones were observed when extraction temperature
was 60 C. Above 60 C the extraction eciency remained constant.
Therefore, an extraction temperature of 60 C was used for all
subsequent experiments.
Talanta 168 (2017) 329335
LOD (g g1) LOQ (g g1) Ef
0.9994 0.12 0.40 598
0.9997 0.14 0.45 571
0.9992 0.08 0.25 495
0.9992 0.06 0.20 516
Fig. S4B (in Supplementary material) shows the eect of the
interaction of extraction time and ultrasonic power at a constant time
of 80 min on the response. It is noticeable that the mixing intensity is a
mass transfer limiting factor. This nding suggests, that increasing the
ultrasonic power to 616 W would lead to a signicant improvement in
yield. Decreasing the power from 616 W to 264 W caused a decrease in
the recovery of Glu-DA from 94.8% to 85.7%. At low ultrasonic power,
the distribution of the sample was not as uniform as at higher power.
Based on the central composite design experiment the following
optimum NADES extraction conditions were selected: extraction time,
60 min; extraction temperature, 60 C; ultrasonic power, 616 W.
3.3. Method validation
The dependence of the chromatographic signal on analyte concen-
tration was veried under optimum conditions of NADES and UHPLC-
UV.
A linear calibration graph was obtained over the range 0.5
25 g g1 of analytes by the proposed method of extraction and
determination. The calculated regression lines showed good linearity
with determination coecients (r2) higher than 0.9992 for each analyte
(Table 1). The limit of detection (S/N=3) and limit of quantication (S/
N=10) ranged between 0.06 and 0.14 g g1 and 0.20 and 0.45 g g1,
respectively. Intra-day and inter-day precision (expressed as %RSD)
and accuracy (expressed as %RE) were less than 8.5% at the three
concentration levels assayed for all isoavones and for IS. Table 2
shows the results obtained in the precision and accuracy studies. The
average enrichment factor was in the range 495598.
The results for the recovery studies are shown in Table 2. The
recovery achieved by the present method ranged from 64.7% to 99.2%
with a %RSD lower than 5.4%.
3.4. Sample analysis
The optimised procedure was applied to analyse the isoavones in
soy beverages made by dierent manufacturers and available on the
market. Representative UHPLC-UV chromatograms of extracts from
the soy products are shown in Fig. 1B and C. The content of each
isoavone in the dierent samples was determined using the corre-
sponding regression equation and the mean contents are summarised
in Table S3 (in Supplementary material). Dierences were observed in
isoavone concentration, with an almost ten-fold dierences in some
cases, and most of the extracts were diluted before analysis.
As can be seen in Table S3 (in Supplementary material), the
contents of the four investigated isoavones were highly variable. The
total content of isoavone glycones and aglycones varied among soy
material types from 5.14 to 1200 g g1 and from 2.15 to 511 g g1 of
dry weight, respectively. All selected isoavones were detected in all
tested soy food samples. Daidzein was the main isoavone aglycoside
and these results are in good agreement with those obtained in another
study [24,26]. The total isoavone aglycoside content found in the soy-
containing food products was also in agreement with reports in the
literature [24,25,27]. The supplement samples with lowest soy content
(Supplement 3) presented the lowest isoavone concentration (from
68.9 to 182 g g1). Supplement 3 is a source of bioavonoids (the
macromolecules). Daidzein and genistein are aglycones, so they are not
bioavonoids, hence their low amount in the Supplement 3. The
333
S. Bajkacz, J. Adamek Talanta 168 (2017) 329335

Table 2
Method validation data: precision, accuracy and recovery of isoflavones (n=6).

Analyte Spiking level (g g1) Precisiona (%RSD) Accuracyb (%RE) Recovery (%)

Intra-day Inter-day Intra-day Inter-day

DAGL 0.5 4.1 5.0 5.7 6.0 95.5

7.5 3.4 3.7 2.9 5.0 96.4
20 2.9 3.2 2.6 3.8 97.2
GTGL 0.5 5.5 5.9 7.6 8.5 99.2
7.5 3.3 4.6 3.9 5.9 97.8
20 2.4 2.7 3.7 4.2 98.1
DA 0.5 3.8 4.4 5.2 6.6 81.3
7.5 3.5 4.2 4.0 3.3 80.5
20 3.1 4.1 1.5 2.8 83.3
GT 0.5 3.6 3.9 4.7 5.0 81.9
7.5 2.4 3.4 1.2 4.9 82.1
20 1.0 3.0 1.0 2.7 83.7
BIO (IS) 0.5 4.8 5.2 3.8 6.2 64.7
7.5 4.0 4.5 2.5 4.8 65.6
20 2.1 2.9 2.3 3.5 66.4

a
Expressed as relative standard deviation (RSD (%)).
b
Expressed as relative error (RE (%)).

supplements with the highest soy content (Supplement 1 and 2)
presented the highest isoavone concentration in the range from
15,000 to 90,000 g g1. Supplements 1 and 2 contain soybean extract
and they are used to relieve the menopause symptoms. This explains
the high concentration all detected compounds in Supplements 1 and
2.
Based on the obtained results, it can be stated that the proposed
NADES-UHPLC-UV method presented high accuracy, sensitivity and
high ecient matrix clean-up for the simultaneous analysis of four
isoavones in soy-containing food samples.
3.5. Comparison of the proposed method with other reported methods
Table S4 (in Supplementary material) compares dierent studies
considering several parameters, as follows: technique, sample volume,
extraction time and chromatographic time, LOD, water sample,
recovery, RSD% and total analytical eco-scale score.
According to the literature, this is the rst study that combines
NADES and UHPLC-UV for the extraction and determination of
isoavones in soy and its products. The total analysis time of the
UHPLC protocol was within 6 min, in contrast to the previous HPLC
procedures [15,19,30,31] that involved analysis times of 3045 min.
Other non-negligible advantages to our method are the low solvent
consumption and minimal sample requirement (ca. 600 L per sam-
ple). In addition, the NADES-UHPLC-UV procedure provided higher
recoveries with lower RSDs in comparison with the Soxhlet extraction-
HPLC-DAD, UAE-HPLC-DAD and MAE-HPLC-DAD methods
[19,21,29].
4. Conclusions
A green, novel and simple extraction method based on natural deep
eutectic solvents (NADES) was developed for the extraction of iso-
avones from soy product samples prior to their determination. The
choline chloride:citric acid (molar ratio of 1:1) as natural deep eutectic
solvent is relatively non-toxic and easily prepared using accessible,
renewable, non-ammable, non-volatile and biodegradable compo-
nents. Optimisation of the operational conditions improved the extrac-
tion eciency and the nal optimised conditions were as follows:
NADES composition choline chloride:citric acid (1:1); water content
in NADES: 30%; ratio of NADES volume to sample amount: 3;
extraction time: 60 min; extraction temperature: 60 C; ultrasonic
power: 616 W.
The proposed method was successfully applied for the extraction of
isoavones from soy-containing food samples with satisfactory recov-
eries indicating that the developed method is a promising tool for the
enrichment and purication of selected compounds in complex sam-
ples.
Compliance with ethical standards
Funding
This project was supported by funds from the National Science
Centre in the frame of the project SONATA No. 2014/13/D/ST4/01863
for 20152018 period, Cracow, Poland.
Conict of interest
Sylwia Bajkacz declares that she has no conict of interest. Jakub
Adamek declares that he has no conict of interest.
Ethical approval
This article does not contain any studies with human or animal
subjects.
Informed consent
Informed consent was not applicable.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.talanta.2017.02.065.
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