Analytica Chimica Acta 1103 (2020) 122e133

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Analytica Chimica Acta

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

Rapid determination of isoflavones and other bioactive compounds in

soybean using SWATH-MS
Han-Ju Chien a, Chang-Sheng Wang b, c, Yu-Hsun Chen a, Jie-Teng Toh a, Yi-Feng Zheng a,
Xiang-Gui Hong a, Hung-Yu Lin a, Chien-Chen Lai a, c, d, e, *
a
Institute of Molecular Biology, National Chung Hsing University, Taichung, 40227, Taiwan
b
Department of Agronomy, National Chung Hsing University, Taichung, 40227, Taiwan
c
Advanced Plant Biotechnology Center, National Chung Hsing University, Taichung, 40227, Taiwan
d
Graduate Institute of Chinese Medical Science, China Medical University, Taichung, 40402, Taiwan
e
Department of Pharmacology, National Defense Medical Center, Taipei City, 11490, Taiwan

h i g h l i g h t s g r a p h i c a l a b s t r a c t

High throughput SWATH-MS method

for the determination of 12 iso-
flavones in soybeans.

Amino acids, saponin Ba, Bb, and Bc
can be semi-quantified from the
SWATH-MS data.

All bioactive compounds were quan-
tified within 5 min in 10 min run
time.

This approach provides good quanti-
tative linearity and reproducibility.

7 varieties of soybeans were
compared with this robust screening
method.

a r t i c l e i n f o a b s t r a c t

Article history: Isoflavones are the major bioactive components in soybeans. Sequential window acquisition of all
Received 1 November 2019 theoretical fragment ions (SWATH) is a kind of data-independent acquisition (DIA), such that all frag-
Received in revised form ments of each precursor will be preserved in a SWATH-Mass Spectrometry (SWATH-MS) run. In this
15 December 2019
study, a high-throughput SWATH-MS method for the determination of 12 isoflavones in soybeans was
Accepted 18 December 2019
Available online 24 December 2019
established. Furthermore, amino acids, saponins can be semi-quantitated from the same SWATH-MS
data. Combination of targeted quantification and untargeted profiling with SWATH, all bioactive com-
pounds were analyzed within 5 min in 10 min run time, and the method had good linear regression with
Keywords:
Isoflavone
r2 > 0.99. The precisions (RSD %) of the intra-day and inter-day analyses ranged from 2.11% to 18.7%, and
Soybean the accuracies (RE%) ranged from 14.39% to 17.48%. The matrix effect ranged from 88.66% to 114.82%.
SWATH-MS Moreover, 7 varieties of soybeans were analyzed and compared with this robust screening method.
High-throughput © 2019 Elsevier B.V. All rights reserved.
Amino acid
Saponin

* Corresponding author. Institute of Molecular Biology, National Chung Hsing University, No. 250, Kuo-Kuang Road, Taichung, 40227, Taiwan.
E-mail addresses: ru0300618@hotmail.com (H.-J. Chien), wangchansen@nchu.edu.tw (C.-S. Wang), hinagiku83520@gmail.com (Y.-H. Chen), thoutought@gmail.com
(J.-T. Toh), yvonne19930307@hotmail.com (Y.-F. Zheng), xghong@smail.nchu.edu.tw (X.-G. Hong), noahlovealice@hotmail.com (H.-Y. Lin), lailai@dragon.nchu.edu.tw
(C.-C. Lai).

https://doi.org/10.1016/j.aca.2019.12.054
0003-2670/© 2019 Elsevier B.V. All rights reserved.
 H.-J. Chien et al. / Analytica Chimica Acta 1103 (2020) 122e133 123

Abbreviations HPLC-DAD/MSD high performance liquid chromatography-

diode array detector/mass spectrometry
ACN acetonitrile detection
CE collision energy LC-ESI-MS liquid chromatography-electron spray ionization-
DAI daidzein mass spectrometry
DAI-G daidzin LOD limit of detection
DAI-GA acetyldaidzin LOQ limit of quantification
DAI-GM malonyldaidzin LLOQ lower limit of quantification
DDA data-dependent acquisition MRM multiple reaction monitoring
DIA data-independent acquisition MS/MS tandem mass spectrometry
GEN genistein PVDF polyvinylidene fluoride
GEN-G genistin QC quality control
GEN-GA acetylgenistin RSD relative standard deviation
GEN-GM malonylgenistin SWATH-MS sequential window acquisition of all theoretical
GLY glycitein fragment ions-mass spectrometry
GLY-G glycitin UHPLC-PDA ultra-high performance liquid chromatography-
GLY-GA acetylglycitin photo diode array
GLY-GM malonylglycitin

1. Introduction malonylgenistin (GEN-GM), and malonylglycitin (GLY-GM) [4].

Soybean (Glycine max L.) is a major source of food in the world
[1]. Many breeders have sought to determine the best varieties of
the crop [2], and to that end, it is important to be able to quantify
the desired properties of a given variety within a larger set of va-
rieties within a short period of time. Isoflavones are polyphenolic
compounds that are the major bioactive components in soybeans
[3], with the isoflavones present in soybeans including daidzein
(DAI), genistein (GEN), glycitein (GLY), daidzin (DAI-G), genistin
(GEN-G), glycitin (GLY-G), acetyldaidzin (DAI-GA), acetylgenistin
(GEN-GA), acetylglycitin (GLY-GA), malonyldaidzin (DAI-GM),
Some of these isoflavones also act as phytoestrogens in mammals,
since their structures and functions are similar to those of 17-
estradiol, which is a kind of mammalian estrogen [5]. Isoflavones
have many benefits to human; for instance, they have antioxidant,
anti-inflammatory, and antiproliferative activities [6]. The purpose
of this study was to develop a rapid method for the determination
of 12 isoflavones in soybeans.
A number of analytical methods for quantifying isoflavones have
previously been established [7]. In 2011, Hong et al. build a 96-min
method for the quantification of 15 isoflavones that utilizes high
performance liquid chromatography-diode array detector/mass

Table 1
The transition MS/MS coordinates and collision energy values of isoflavones and amino acids.

Classification Analyte Q1 (m/z) Q3 (m/z) Collision energy Retention time (min) No. of SWATH isolation window

Isoflavones
Daidzein (DAI) 255.06 137.03 45 3.8 20
Genistein (GEN) 271.06 153.02 46 4.2 21
Glycitein (GLY) 285.08 242.06 45 3.8 22
Daidzin (DAI-G) 417.12 255.07 17 2.7 26
Genistin (GEN-G) 433.11 271.07 17 3.1 27
Glycitin (GLY-G) 447.13 285.08 14 2.8 27
Acetyldaidzin (DAI-GA) 459.12 255.06 18 3.5 29
Acetylgenistin (GEN-GA) 475.12 271.06 19 3.9 29
Acetylglycitin (GLY-GA) 489.14 285.08 15 3.5 29
Malonyldaidzin (DAI-GM) 503.12 255.06 21 3.2 30
Malonylgenistin (GEN-GM) 519.11 271.06 22 3.5 30
Malonylglycitin (GLY-GM) 533.13 285.08 18 3.2 30

Amino acids
Serine 106.05 60.05 14 0.3 1
Proline 116.07 70.07 18 0.3 3
Valine 118.09 72.08 13 0.4 3
Threonine 120.07 74.06 15 0.3 3
Isoleucine/Leucine 132.1 86.1 13 0.5 5
Asparagine 133.06 74.03 18 0.3 5
Aspartic acid 133.96 74.03 17 0.3 5
Lysine 147.11 130.09 15 0.3 7
Glutamic acid 148.06 84.05 16 0.3 7
Methionine 150.06 104.06 17 0.4 8
Histidine 156.08 110.07 18 0.3 8
Phenylalanine 166.09 120.08 14 0.8 10
Arginine 175.12 70.07 21 0.3 11
Tyrosine 182.08 136.08 16 0.5 12
Tryptophan 205.1 188.07 13 1.4 15
124 H.-J. Chien et al. / Analytica Chimica Acta 1103 (2020) 122e133

Scheme 1. The workflow of the development of a rapid SWATH-MS method for the determination of isoflavones, and its application to real samples.

spectrometry detection (HPLC-DAD/MSD) [8]. In 2015, Shim et al.
quantified 14 isoflavones in 25 min by using ultra-high perfor-
mance liquid chromatography-photo diode array (UHPLC-PDA) [9].
In 2018, Akitha Devi et al. developed a 35 min liquid
chromatography-electron spray ionization-mass spectrometry (LC-
ESI-MS) procedure for the measurement of 11 isoflavones [10].
Even with that relatively rapid procedure, however, the required
analytical time still had room for improvement.
Sequential window acquisition of all theoretical fragment ions
(SWATH) is a kind of data-independent acquisition (DIA), such that
all fragments of every precursor in each SWATH mass isolation
window will be collected in a SWATH-MS run, which is the reason
why DIA has more MS/MS information than data-dependent
acquisition (DDA) [11]. Also, SWATH can provide a sufficient num-
ber of data points [12]. DDA usually entails the use of exclusion time
for precursor ions in order to detect more compounds, but SWATH
dose not, therefore the number of data points per peak of SWATH
can be made greater than one of DDA. Furthermore, the sensitivity

Fig. 1. (A) The peak shapes and peak points in the SWATH0, SWATH1, and SWATH2 methods. (B) The pie chart of peak points distribution of calibration curves of 12 isoflavones.
 H.-J. Chien et al. / Analytica Chimica Acta 1103 (2020) 122e133 125

2. Materials and methods

2.1. Materials and reagents

DAI, GEN, GLY, DAI-G, GEN-G, GLY-G, DAI-GA, GEN-GA, GLY-GA,

DAI-GM, GEN-GM, GLY-GM (HPLC level, purity 98%) were obtained
from Sigma Aldrich. Arginine, aspartic acid, asparagine, glutamic
acid, histidine, isoleucine, leucine, lysine, methionine, phenylala-
nine, proline, serine, threonine, tryptophan, tyrosine, and valine
were also purchased from Sigma-Aldrich. One mg L1 of each iso-
flavone and amino acid were prepared in 80% ACN and ddH2O,
respectively. Deionized water was purified using a Milli-Q system
(Millipore, Milford, MA, USA). All of the analytes were analyzed
with infusion experiments for the obtainment of the m/z of pre-
cursor and fragment ions, as well as the best collision energy.

Fig. 2. The relationship of total ion intensities and window width in the SWATH2 2.2. Soybean materials
method.

Seven varieties of soybean (Glycine max) were collected from the

Taiwan Agricultural Research Institute, Council of Agriculture, Ex-
ecutive Yuan, in Spring of 2018, including the No. 1, 11, 63, 65, 77, 92,
of SWATH is better than that of DDA, because the reduced total
and 103 varieties. They were provided by Prof. Chang-Sheng Wang
number of MS/MS experiments could be used to increase accu-
(Department of Agronomy, National Chung Hsing University, Tai-
mulation time without increasing cycle time [13]. SWATH may have
chung, Taiwan). After being collected, the soybeans were sealed in
quantitative performance similar to that of multiple reaction
bags with deoxidizer and stored at 4  C before undergoing analysis.
monitoring (MRM), the quantification gold scan mode, and SWATH
have the possibility of retrospectively selecting precursors and
2.3. Extraction of soybean
fragments for data processing [14]. The MRM channel should be set
for each specific analytes prior to LC/MS/MS analysis, whereas
Extraction solvent (80% ACN) was added to a homogeneous tube
SWATH, in which every fragment ion of each precursor ion can be
with the ratio of 0.2 g soybean: 1 mL of 80% ACN, then the mixture
conserved, allows for retrospectively selecting precursors and its
was homogenized four times with Homogenizer (Precellys® evo-
fragments for qualitative and quantitative data processing. More-
lution, Bertin, France). The details of this process were as follows:
over, SWATH-MS shows good reproducibility by acquiring of large-
the tube size was 2 mL, the speed was set at 6800 rpm, the cycle
scale data in multiple labs [15], and it has been applied to small
time was 2  20s, the pause time was 30 s, and the samples were
molecules, such as identification and quantitation of pesticides
put on ice between each run time. Then, the extracts were centri-
residues in food [11].
fuged at 8000 rpm for 15 min, and the supernatants were filtered
Free amino acids and saponins are important components in
with a 0.22 mm PVDF syringe filter. The filtered extraction solutions
soybean. The accumulation of free amino acids can increase the
were stored at 20  C and diluted with 5% ACN before undergoing
abundance of seed proteins in soybean [16], and soybean proteins
LC-MS/MS analysis.
have many beneficial effects, such as the ability to reduce blood
pressure [17] and a hypocholesterolemic effect [18]. Meanwhile, the
2.4. LC gradient optimization
saponins also provide many benefits in humans, including protec-
tive effects against some cancers, such as colon cancer [19] and
Each soybean extract was analyzed by UHPLC-QTOF MS. One
prostate cancer [20], as well several beneficial physiological activ-
microliter of extract was separated into a UHPLC Ultimate 3000
ities, including hypolipidemic, anti-oxidative, and hemolytic ac-
RSLC system (Thermo-Dionex, Sunnyvale, CA, USA) using a COR-
tivities, amongst others [21]. There are many types of saponins,
TECS UPLC T3 column (2.1  50 mm, 1.6 mm, Waters) at a flow rate
including Aa (A4), Ab (A1), Ac, Ad, Ae (A5), Af (A2), Ag (A6), Ah (A3),
of 450 mL min1. The mobile phase A and B were water with 0.1%
Ba (V), Bb (I), Bb’ (III), Bc (II), Bc’ (IV), ag, bg, ba, gg, and ga [22]. In
formic acid and acetonitrile with 0.1% formic acid, respectively.
this study, only the Ba, Bb, and Bc saponins were further investi-
There were 3 LC gradients compared in this study, and the total run
gated in the experiments due to the fact that the exact mass and
times were 10, 15, and 20 min. For the 10 min gradient run, a
MS/MS spectrum of each of those saponins has been reported in
gradient of 5%e10% B within 1 min, 10% B to 30% B within 2 min,
previous studies [23,24]. Therefore, amino acids and the Ba, Bb, and
30% B to 50% B within 1 min, 50% B to 85% B within 1 min, and 85% B
Bc saponins can be detected by retrospectively selecting precursors
to 95% B within 2 min was followed by re-equilibration with 5% B
and fragments from existing SWATH data which reserve all pre-
for 3 min. For the 15 min gradient run, a gradient of 5%e10% B
cursors and its related fragments.
within 2 min, 10% B to 30% B within 2 min, 30% B to 50% B within
More specifically, after the use of SWATH-MS in this study for
1 min, 50% B to 80% B within 5 min, and 80% B to 90% B within 2 min
the rapid determination of isoflavones in soybean, validation ex-
was followed by re-equilibration with 5% B for 3 min. For the 20 min
periments were also performed. Furthermore, untargeted profiling
gradient run, a gradient of 5% B was held for 2 min, followed by 5% B
of 16 amino acids and the Ba, Bb, and Bc saponins in soybeans were
to 50% B within 3 min, 50% B to 80% B within 7 min, 80% B to 90% B
executed from the existing SWATH data. Combination of targeted
within 3 min, and re-equilibration with 5% B for 5 min. The me-
quantification and untargeted profiling analysis, the results of this
tabolites eluted were analyzed with a Q-TOF MS (TripleTOF 6600,
work should not only help breeders to filter out the varieties of
Sciex, Framingham, MA) which was operated with top 20 data-
soybean with high isoflavone contents more quickly, but should
dependent acquisition (DDA) using the positive ion mode. Duo-
also provide more information regarding other analytes through
Spray Source (Sciex) was used for ionization by applying a 5500 V
the use of the same SWATH data.
voltage. The accumulation times of MS and MS/MS were 250 and
Table 2

126
The linear range, calibration curve, r2, and LLOQ values of 12 isoflavones in solvent, and the precision and accuracy of LQC, MQC, and HQC in interday and intraday analyses.

Analyte Linear range Calibration curves Correlation LLOQ LLOQ precision LLOQ Nominal cons. Interday Intraday
(ng/mL) coefficient (r2) (ng/mL) (RSD, %) accuracy (RE, (ng/mL)
Observed Precision Accuracy Observed Precision Accuracy
%)
concentration (ng/ (RSD, %) (RE, %) concentration (ng/ (RSD, %) (RE, %)
mL) mL)

DAI 20e2000 y ¼ 9.93189x - 38.82292 0.99805 20 12.21 3.96 50 51.17 12.66 2.34 56.66 6.63 13.32
800 803.98 9.40 0.50 823.51 7.40 2.94
2000 2000.02 8.32 0.00 2029.71 5.91 1.49

GEN 20e2000 y ¼ 8.59515x - 102.22368 0.99487 20 3.37 13.94 50 44.84 10.79 10.33 46.63 10.06 6.74
800 819.49 10.56 2.44 828.24 7.52 3.53
2000 2187.86 9.96 9.39 2143.33 7.69 7.17

GLY 20e2000 y ¼ 9.27289x - 32.79540 0.99803 20 13.75 1.21 50 54.15 11.95 8.29 51.72 8.10 3.45
800 773.41 7.89 3.32 822.35 2.76 2.79
2000 1861.86 8.11 6.91 2028.80 4.57 1.44

DAI-G 20e2000 y ¼ 35.67289x þ 65.10133 0.99667 20 5.16 2.55 50 52.54 14.93 5.08 48.66 9.92 2.69
800 766.38 12.82 4.20 814.87 3.63 1.86

H.-J. Chien et al. / Analytica Chimica Acta 1103 (2020) 122e133

2000 1727.80 14.04 13.61 1940.43 3.99 2.98

GEN-G 10e2000 y ¼ 65.99061x þ 337.51439 0.99843 10 14.25 2.13 10 9.47 14.55 5.27 11.06 6.50 10.57
600 666.69 10.08 11.12 599.88 7.40 0.02
2000 2087.97 11.33 4.40 599.88 7.40 0.02

GLY-G 10e2000 y ¼ 71.81841x þ 444.70071 0.99724 10 14.04 5.71 20 17.01 14.84 9.82 17.81 11.72 10.96
600 555.03 10.13 3.23 555.34 6.10 7.44
2000 1852.38 11.42 7.38 1915.04 5.66 4.25

DAI-GA 10e2000 y ¼ 164.20538x þ 947.57586 0.99741 10 11.77 0.8 10 9.54 18.70 4.61 9.33 2.11 6.70
600 672.83 11.67 8.50 628.29 10.78 4.71
2000 2018.24 10.11 0.91 2163.34 4.86 8.17

GEN- 10e2000 y ¼ 119.29177x - 761.50839 0.99827 10 7.13 9.5 10 11.75 9.64 17.48 10.54 5.12 5.44
GA
600 661.53 8.82 10.26 658.28 8.62 6.91
2000 2107.22 11.82 5.36 2001.99 4.20 0.10

GLY- 10e2000 y ¼ 170.73311x - 122.83629 0.99696 10 10.96 5.29 10 10.33 9.81 3.30 10.29 9.64 2.91
GA
600 614.50 11.17 2.42 599.12 5.39 0.15
2000 1804.05 8.60 9.80 1884.71 8.33 5.76

DAI- 10e2000 y ¼ 121.05656x þ 780.23752 0.99824 10 12.42 0.78 10 8.39 16.17 0.31 8.56 10.08 14.39
GM
600 551.65 7.85 8.06 598.10 3.57 0.32
2000 1844.82 11.80 7.41 2037.23 5.05 1.86

GEN- 10e2000 y ¼ 86.93616x þ 1.52490 0.9966 10 6.5 2.81 10 10.81 14.38 8.10 10.71 6.64 7.09
GM
600 678.51 7.14 13.08 660.70 6.76 10.12
2000 2154.81 9.59 7.74 2140.63 8.29 7.03

GLY- 10e2000 y ¼ 141.27575x þ 638.57120 0.99709 10 10.34 0.73 10 9.07 16.38 9.28 10.05 9.96 0.49
GM
600 595.06 12.53 0.82 580.02 10.91 3.33
2000 1735.79 10.13 13.21 1911.59 7.55 4.42
 H.-J. Chien et al. / Analytica Chimica Acta 1103 (2020) 122e133
Fig. 3. The calibration curves of 12 isoflavones.
100 ms, respectively. The mass range of MS was 100 to 1000 m/z,
and that of MS/MS was 50 to 1000 m/z. The exclusion time for each
analyzed precursors was 6 s, and only single charged precursor ions
were analyzed. Dynamic background substitution was used. Peak-
View v2.0 was used for the acquisition of the number of MS/MS
spectra in each experiment.
2.5. SWATH method optimization
Three SWATH methods (Table S1) were compared in this study,
including one fixed window SWATH method (SWATH0) and two
variable window SWATH methods (SWATH1 and SWATH2). The
fixed window SWATH0 method consisted of the acquisition of
36 MS/MS scans of overlapping sequential precursor isolation
windows covering the 100 to 1000 m/z mass range (25 m/z isolation
window, 1 m/z overlap, high sensitivity mode) combined with a
previous TOF-MS scan (100e1000 m/z) for each cycle. For the var-
iable window methods, all the peaks and intensities of DDA data for
10 min of total run time were obtained from PeakView v2.1 and put
into SWATH Variable Window Calculator V1.0, after which the list
of variable windows was exported into Analyst TF v1.7.1. The
collision energy (CE) values of some isolation windows were
adjusted for the isoflavones (SWATH1). Moreover, the number of
SWATH windows and the window width were adjusted in the
127
SWATH2 method. The TOF-MS accumulation times were 25 ms for
SWATH0, 50 ms for SWATH1, and 25 ms for SWATH2. The SWATH
window numbers were 36 for SWATH0, 35 for SWATH1, and 30 for
SWATH2. The MS/MS accumulation times were 26.4 ms for
SWATH0 and 25 ms for SWATH1 and SWATH2. The cycle times
were 977 ms, 926 ms, and 806 ms for SWATH0, SWATH1, and
SWATH2, respectively.
2.6. Data processing and statistical analysis
The SWATH data were processed using MultiQuant v2.0 soft-
ware (Sciex), and the results were exported as Excel files. The
transition parameter settings and retention times for the 12 iso-
flavones, 16 amino acids, and saponin Ba, saponin Bb, and saponin
Bc are listed in Table 1. All mass extraction windows was set at
0.02 m/z.
2.7. Method validation
The validation was performed according to the bioanalytical
method validation guidance for industry of Food and Drug
Administration (FDA) [25]. The calibration curve, lower limit of
quantification (LLOQ), sensitivity, accuracy, precision and matrix
effect were evaluated.
128 H.-J. Chien et al. / Analytica Chimica Acta 1103 (2020) 122e133

Table 3
The matrix effect values of LQC, MQC, and HQC of 6 isoflavones.

Analytes Nominal concentration (ng/mL) Matrix effect (%)

Daidzein (DAI) 20 111.67

800 113.51
2000 93.03
Genistein (GEN) 20 107.45
800 112.42
2000 100.37
Glycitein (GLY) 20 96.12
800 114.28
2000 114.82
Daidzin (DAI-G) 20 102.60
800 112.08
2000 98.17
Genistin (GEN-G) 20 97.86
800 110.95
2000 113.58
Glycitin (GLY-G) 20 105.09
800 90.22
2000 90.63
Acetyldaidzin (DAI-GA) 20 109.95
800 113.85
2000 103.59
Acetylgenistin (GEN-GA) 20 93.13
800 112.09
2000 100.78
Acetylglycitin (GLY-GA) 20 98.73
800 99.56
2000 88.66
Malonyldaidzin (DAI-GM) 20 97.89
800 106.52
2000 114.14
Malonylgenistin (GEN-GM) 20 94.63
800 92.61
2000 105.92
Malonylglycitin (GLY-GM) 20 104.16
800 97.97
2000 112.60

For the preparation of calibration and quality control (QC)
samples, 1 mg L1 of each isoflavones and 0.1% FA were used to
produce a 2000 ng mL1 mixed standard stock. For calibration
samples, the mixed standard stock was diluted into the concen-
trations of 10, 20, 50, 400, 800, 1600 and 2000 ng mL1. For QC
samples of DAI, GEN, GLY and DAI-G, they were diluted into the
concentrations of 50, 800 and 2000 ng mL1; for the others, they
were diluted into the concentrations of 10, 600 and 2000 ng mL1,
except the concentration of low QC of GLY-G, which was
20 ng mL1.
The calibration curves of the 12 isoflavones were acquired for
five consecutive runs. The peak areas of the analytes were calcu-
lated and plotted against the nominal concentrations. The sensi-
tivity was measured by quantifying the LLOQ, its accuracy and
precision should be within 20% of nominal concentration and
relative standard deviations (RSD). The precision and accuracy
were also evaluated over intra-day and inter-day periods; five
repeated QC samples were analyzed on the same day for the intra-
day analysis and once per day for five consecutive days for the inter-
day analysis. The % RSD of the QC samples, which were required to
less than 15% (20% for the LLOQ), were used to estimate the
precision of the method, and the relative errors (%RE), which were
required to be within ±15%, were used to estimate the accuracy.
The matrix effect was determined by using 20, 800 and
2000 ng mL1 of each analyte for 3 replicates, which should be
within ±15%. The equation was calculated as follows: matrix effect
(%) ¼ [(the peak area of post-extracted spiked sample) e (the peak
area of the analyte in a sample)]/(the peak area of standard
solution) x 100.
3. Results and discussion
The workflow is shown in Scheme 1. First, the transition MS/MS
coordinates of the analytes were obtained by infusion, then the LC
gradient and SWATH-MS method were optimized. Next, the line-
arity, LLOQs, precision, accuracy, and matrix effect were measured
in the method validation. Finally, this method was applied to real
samples for the determination of their isoflavone contents.
Furthermore, amino acids, saponin Ba, saponin Bb, and saponin Bc
can be semi-quantitated using untargeted profiling with the same
SWATH-MS raw data.
3.1. Method development
In the beginning, 10-, 15-, and 20-min LC gradients were
compared by using the number of MS/MS spectra; 0.02x diluted No.
1 real sample extract was used and analyzed with the DDA acqui-
sition method. There were 890, 1011, and 1153 MS/MS spectra in the
10-, 15-, and 20-min gradients, respectively. Although the number
of MS/MS spectra was the least in the 10-min LC method, this
approach saved considerable time for the rapid determination of
isoflavones in soybeans, and the spectra of the 10-min method
were not missing a lot compared with those for the 15- and 20-min
methods. Then, different dilution folds of real sample were tested.
Specifically, No. 1 sample extract was diluted by 10 fold, 50 fold, or
2000 fold and then analyzed with the DDA acquisition method; the
 H.-J. Chien et al. / Analytica Chimica Acta 1103 (2020) 122e133
Fig. 4. The absolute quantification of (A) isoflavones and relative quantification of (B)
amino acids and (C) saponin Ba, Bb and Bc in 7 real samples.
number of MS/MS spectra were 1147, 890, and 813, respectively.
The 0.1x diluted extract had the most spectra. Therefore, the 10-
min LC gradient method and the 10-fold dilution of real sample
extract were used in the following experiments.
Next, the SWATH acquisition method was optimized by
comparing the fixed and variable isolation windows, with the 0.1x
diluted No.1 real sample extract spiked with 1 mg L1 of the iso-
flavone standards being used. The SWATH0 method was the fixed
25 Da isolation window SWATH method, and SWATH1 and
SWATH2 methods were the variable isolation window methods. In
the SWATH0 method, the CE values of DAI, GEN, and GLY were not
adequately intense. They needed to be 45, 46, and 45, respectively,
for fragmentation, but their respective values were only 23.604,
23.604, and 25.044 in the SWATH0 method. In the SWATH1
method, this problem of the SWATH0 methods was resolved, as the
CE values of the DAI, GEN, and GLY isolation windows were
adequate for fragmentation. However, the isolation windows
developed from the SWATH window calculator still needed some
correction. The closed m/z values of the precursor ions were put in
the same SWATH isolation window easily, and the precursor ions of
GEN-GA, GLY-GA, DAI-GM, and GEN-GM were in the same window.
This was okay for GLY-GA and DAI-GM, which had different frag-
ment ion values (285.08 m/z for GLY-GA and 255.06 m/z for DAI-
GM), but it was not okay for GEN-GA and GEN-GM, as they had
the same 271.06 m/z value of fragment ions and their retention
times of 3.9 min and 3.5 min, respectively, were close. Therefore,
the windows of GEN-GA and GEN-GM needed to be separated. In
129
the SWATH2 method, the precursor ions with the same fragment
ion values and close retention times, such as DAI-GA (255.06 m/z,
3.5 min) and DAI-GM (255.06 m/z, 3.2 min), GEN-GA (271.06 m/z,
3.9 min) and GEN-GM (271.06 m/z, 3.5 min), and GLY-GA (285.08 m/
z, 3.5 min) and GLY-GM (285.08 m/z, 3.2 min), were separated into
different isolation windows (Table 1), and the number of isolation
windows and cycle time were decreased in order to increase the
number of peak points. For instance, for GLY-G, there were 8 peak
points in both the SWATH0 and SWATH1 methods but 10 peak
points in the SWATH2 method, and the peak shapes of the SWATH2
method were better. Moreover, the peak intensity of GLY-G in the
SWATH2 method was better than the peak intensity in the SWATH0
method (Fig. 1A). Besides, the peak points of the calibration curves
of 12 isoflavones were measured and summarized in Fig. 1B, the
percentage of peak points more than 10 was 88%, and the 8 to 9
points and less than 8 points were 7% and 5%, respectively. It
demonstrated that most of peak points were good for quantifica-
tion. The number of SWATH windows were 36, 35, and 30 in the
SWATH0, SWATH1, and SWATH2 methods, respectively. The accu-
mulation times of TOF-MS and MS/MS were 25 ms and 36 ms,
respectively, for the SWATH0 method; 50 ms and 25 ms, respec-
tively, for the SWATH1 method; and 25 ms and 25 ms, respectively,
for the SWATH2 method. The cycle timed were 977, 926, and
806 ms for the SWATH0, SWATH1, and SWATH2 methods, respec-
tively (Table S2). These results indicated that the variable SWATH
window methods were better than the fixed SWATH window
method. Above all, we considered the SWATH2 method to be the
best MS method investigated in this study. The relationship of total
ion intensity and window width is shown in Fig. 2, and the basic
peak chromatography of the No. 1 soybean variety is shown in
Fig. S1.
3.2. Method validation
The Linearity, LLOQs, precision, accuracy, and matrix effect were
measured in this study. With respect to linearity and LLOQs, as
shown in Table 2, the calibration curves of the 12 isoflavones had
good linear regression (r2 > 0.99) (Fig. 3). The linear ranges of DAI,
GEN, GLY, and DAI-G ranged from 20 to 2000 ng mL1, and those of
the others ranged from 10 to 2000 ng mL1. Meanwhile, the LLOQs
of DAI, GEN, GLY, and DAI-G were determined to be 20 ng mL1, and
those of the others were determined to be 10 ng mL1, the precision
and accuracy at LLOQ of 12 isoflavones were ranged from 3.37% to
14.25% and 5.71%e13.94%, respectively (Table 2). The concentra-
tions of analytes in soybean samples can be measured by using the
linearity equation. With respect to precision and accuracy, the
precisions (RSD %) of the intra-day and inter-day analyses of the 12
isoflavones in the low, medium, and high QC samples ranged from
7.14% to 18.7% and 2.11%e11.72%, respectively, whereas the accu-
racies (RE%) ranged from 13.6% to 17.48% and 14.39%e13.32%,
respectively (Table 2). All of the precision and accuracy results were
within the criteria of ±15% (±20% for LLOQ). Twelve isoflavones
were selected to evaluated the matrix effect. The matrix effect
ranged from 88.66% to 114.82% (Table 3), revealing that there was
no matrix interference in the analyses of the soybeans.
It was reported in the literature that a drawback of SWATH-MS
approach compared to the targeted analysis (PRM or SRM) method
in terms of 3-10-fold less sensitive [26]. The comparison experi-
ments between SWATH and MRM were also performed in this
study. In MRM results, the LOQ of 12 isoflavones ranged from 1.5 to
6.25 ng mL1(data not shown); in SWATH results, the LLOQ ranged
from 10 to 20 ng/mL1. Indeed, the sensitivity of MRM was better
than SWATH. However, the measurements of 12 isoflavones in real
samples by using SWATH were performed in next section, it shown
that the ability of quantification was enough for real samples.
130 H.-J. Chien et al. / Analytica Chimica Acta 1103 (2020) 122e133

Fig. 5. The XIC of the precursor ions of isoflavone standards in (A) solvent and (B) the No. 1 soybean variety, as well as (C) the transition MS/MS coordinates of analytes in the No. 1
soybean variety.

Moreover, it also provided information about other compounds,
such as amino acid and saponin Ba, Bb and Bc, whereas the MRM
couldn’t do that.
3.3. The content of 12 isoflavones in real samples
After the establishment of the platform for the rapid deter-
mination of the 12 isoflavones, 7 varieties of soybeans were
analyzed using this SWATH-MS approach, including the No. 1, 11,
63, 65, 77, 92, and 103 varieties (Table S3). The No. 1 variety was
the parent of the other varieties, which were derived from it by
the mutation of sodium azide. The results are displayed in Fig. 4.
The total amounts of isoflavones in the No. 1, 11, 63, 65, 77, 92, and
103 soybeans were 3.74, 4.02, 8.43, 2.57, 2.65, 6, and 6.78 mg g1,
respectively (Table S3). We found that the No. 63 variety had the
highest contents of isoflavones, at 2.25 times the amount in the
No. 1 parent variety, whereas the No. 65 variety had the lowest
amount, at only 0.69 times the amount in the parent variety. The
extracted ion chromatography (XIC) of the isoflavone standards
and the analytes in the real samples are shown in Fig. 5. The
 H.-J. Chien et al. / Analytica Chimica Acta 1103 (2020) 122e133 131

Fig. 6. The XIC of the precursor ions of amino acid standards in (A) solvent and (B) the No. 77 soybean variety, as well as (C) the transition MS/MS coordinates of analytes in the No.
77 soybean variety.

precursor ions of the isoflavone standards in solvent (Fig. 5A) were
extracted, and these analytes can be distinguished easily by LC
separation and high-resolution MS. In addition, the XIC of the real
samples (Fig. 5B) were extracted, and the results revealed that the
isoflavones did indeed exist in the soybeans, with the RT and m/z
values of the analytes being matched. Moreover, the transition
MS/MS coordinates in the real samples (Fig. 5C) were also
extracted, and the peak areas of the analytes were used for
quantification. We found that there were some peaks (not quan-
tification ions) in the DAI, GEN, and GLY results (Fig. 5C). Because
DAI-G had a fragment ion value of 255.07 m/z, which was the same
m/z value of the precursor ion of DAI, and because its best CE value
was only 17 (Table 1), the CE of the TOF-MS was set at 10. The
precursor ion of it could thus be fragmented by in-source collision
induced dissociation (CID), resulting in the peak appearing at
2.8 min. Similar situations occurred with both GEN-G and GEN
and GLY-G and GLY. Specifically, GEN-G and GLY-G had fragment
ion values of 271.07 m/z and 285.08 m/z, respectively, which were
the same m/z values as the precursor ions of GEN and GLY,
respectively, and the best CE values of GEN-G and GLY-G were 17
and 14 (Table 1), respectively, while their peaks appeared at
3.2 min and 2.89 min, respectively. These results demonstrated
that in-source CID occurred, and according to the results of stan-
dard curves (Table 2), the performance of the quantification of
these analytes was not influenced.
132 H.-J. Chien et al. / Analytica Chimica Acta 1103 (2020) 122e133
Fig. 7. (A) The XIC of the precursor ions, as well as (B) the transition MS/MS coordinates and (C) the MS/MS spectra of saponin Ba, Bb and Bc in the No. 103 soybean variety.
3.4. The relative quantification of 16 amino acids and saponin Ba,
saponin Bb, and saponin Bc from past SWATH-MS data
Before quantification, the transition MS/MS coordinates, colli-
sion energy, retention time, and number of SWATH isolation win-
dow for each of the 16 amino acids were obtained by infusion and
LC-MS (Table 1). Among the 16 amino acids, because asparagine
and aspartic acid were in the same No. 5 SWATH isolation window
and had the same fragment ion value of 74.03 m/z and same
retention time of 0.3 min, they could not be distinguished. There-
fore, the contents of the two amino acids were combined together.
As for the contents of isoleucine and leucine, they were isomers.
The spectrum of the 16 amino acids are shown in Fig. 6. The peak
areas of the 16 amino acids in the No. 1, 11, 63, 65, 77, 92, and 103
soybean varieties were 27405.4, 86257.9, 76327.3, 20841.5, 50021.6,
141918.5, and 31251.8, respectively (Table S3). The No. 92 variety
had the highest contents of amino acids, at 1.825 times the amount
of the No. 1 parent variety, while the No. 65 variety had the lowest
amount, at 0.76 times the amount of the parent variety. The XIC of
the precursor ions of the amino acids standards in solvent and the
No. 77 soybean variety, and the transition MS/MS coordinates of
analytes in the No. 77 variety are shown in Fig. 6. The precursor ions
(Fig. 6B) and transition MS/MS coordinates (Fig. 6C) of the analytes
in real samples must appear in the same RT of those in solvent
(Fig. 6A) in order to have confidence in their identification. For
instance, serine, threonine, lysine, and histidine were not detected
in the No. 77 soybean variety. Meanwhile, the peak areas of saponin
B were 178254, 125126.3, 192411, 180550.7, 83473.3, 164343.3, and
236753.8 in No. 1, 11, 63, 65, 77, 92, and 103 soybean varieties,
respectively (Table S3). The No. 103 soybean variety had the highest
content of saponin B, which was 1.33 times that in the No. 1 soy-
bean variety, while the No. 77 soybean variety had the lowest
content, which was 0.47 times that in the No. 1 soybean variety.
Although the standards of saponin B were not used in this study,
there were precise monoisotopic mass and tandem mass spec-
trometry values reported in previous studies, Omizu et al. illus-
trated that with respect to the TOF-MS and MS/MS spectra of
saponin Ba and saponin Bb, the fragments of them were similar in
the MS/MS spectra, with the largest difference being a difference in
the intensity of 797.4 m/z for [M e Rha þ H]þ, which was higher in
saponin Bb than saponin Ba [23]. As such, it may be possible to use
this difference to distinguish between them, if only in terms of MS/
MS spectra. Meanwhile, Jervis et al. reported the exact mass of
saponin B observed by high-resolution MS [24]. These pieces of
information were used in this study for the confirmation of the
identification of saponin B. For instance, in No. 103 soybean, the XIC
of the precursor ions of saponin Ba, saponin Bb, and saponin Bc are
shown in Fig. 7A, and the XIC of their transition MS/MS coordinates
(Table 1) are shown in Fig. 7B, and even though they were in the
same SWATH isolation window, they can be distinguished by the
different RT of 4.82, 4.87, and 4.94 min for saponin Ba, Bb, and Bc,
respectively, as well as by their MS/MS spectra shown in Fig. 7C,
which were similar to those of a previous study. Relatedly the in-
tensity % of 797.47 m/z for saponin Ba was indeed lower than that
for saponin Bb. The MS/MS spectra of saponin Bc were not reported
in a previous study, but their structure was similar to those for
saponin Ba and Bb, indicating that the fragments of saponin Bc
would be similar to those of saponin Ba and Bb (Fig. 7C).
The SWATH spectrum-extracted results for amino acids and
saponin B provided herein not only indicated the contents of those
components in real samples for soybean breeders, but also proved
that we can extract other analytes from past SWATH data, some-
thing which cannot be done with MRM. In other words, SWATH
data can contain various forms of valuable information that can be
extracted in future.
4. Conclusions
In this study, a SWATH-MS method for the rapid quantification
of isoflavones was established, and the associated linearity, LLOQs,
precision, accuracy, and matrix effect values were determined for
the purpose of validation. The results demonstrated that the
method had good performance. Next, this method was successfully
applied in the measurement of isoflavones in real samples. More-
over, this study proved that the peak areas of amino acids and
saponin Ba, saponin Bb, and saponin Bc could be extracted from
past SWATH data without re-acquirement.
 H.-J. Chien et al. / Analytica Chimica Acta 1103 (2020) 122e133
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgment
This work was financially supported by the Ministry of Science
and Technology (MOST) and the Advanced Plant Biotechnology
Center from The Featured Areas Research Center Program within
the framework of the Higher Education Sprout Project by the
Ministry of Education (MOE) in Taiwan, R.O.C
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.aca.2019.12.054.
References
[1] S. Sharma, M. Kaur, R. Goyal, B. Gill, Physical characteristics and nutritional
composition of some new soybean (Glycine max (L.) Merrill) genotypes,
J. Food Sci. Technol. 51 (2014) 551e557.
[2] T. Saputro, K. Purwani, V. Fatimah, E. Stevia, N. Jadid, The tolerance
improvement of local soybean in waterlogging condition through the com-
bination of irradiation and in vivo selection, in: Journal of Physics: Conference
Series, IOP Publishing, 2018, 012001.
[3] D.A. Schreihofer, Neuroprotection by Dietary Isoflavones and Their Role in
Cerebral Ischemia, Bioactive Nutraceuticals and Dietary Supplements in
Neurological and Brain Disease, Elsevier, 2015, pp. 385e394.
[4] H.-j. Wang, P.A. Murphy, Isoflavone content in commercial soybean foods,
J. Agric. Food Chem. 42 (1994) 1666e1673.
[5] P.B. Kaufman, J.A. Duke, H. Brielmann, J. Boik, J.E. Hoyt, A comparative survey
of leguminous plants as sources of the isoflavones, genistein and daidzein:
implications for human nutrition and health, J. Altern. Complement. Med. 3
(1997) 7e12.
[6] C.-C. Hsieh, S. Ferna
ndez-Tome
, B. Herna
ndez-Ledesma, Functionality of
Soybean Compounds in the Oxidative Stress-Related Disorders, Gastrointes-
tinal Tissue, Elsevier, 2017, pp. 339e353.
[7] M. Bustamante-Rangel, M.M. Delgado-Zamarren ~ o, L. Pe
rez-Martín,


E. Rodríguez-Gonzalo, J. Domínguez-Alvarez, Analysis of isoflavones in foods,
Compr. Rev. Food Sci. Food Saf. 17 (2018) 391e411.
[8] J.-L. Hong, X.-Y. Qin, P. Shu, Q. Wang, Z.-F. Zhou, G.-K. Wang, B.-B. Lin,
Q. Wang, M.-J. Qin, Comparative study of isoflavones in wild and cultivated
soybeans as well as bean products by high-performance liquid chromatog-
raphy coupled with mass spectrometry and chemometric techniques, Eur.
Food Res. Technol. 233 (2011) 869.
[9] Y.-S. Shim, W.-J. Yoon, J.-B. Hwang, H.-J. Park, D. Seo, J. Ha, Rapid method for
the determination of 14 isoflavones in food using UHPLC coupled to photo
diode array detection, Food Chem. 187 (2015) 391e397.
133
[10] M.A. Devi, S.S. Kumar, P. Giridhar, LCeESIeMS based characterisation of iso-
flavones in soybean (Glycine max (L.) Merr.) from India, J. Food Sci. Technol.
55 (2018) 5045e5054.
[11] P.P. Va
zquez, A. Lozano, C. Ferrer, M.M. Bueno, A. Ferna
ndez-Alba, Improve-
ments in identification and quantitation of pesticide residues in food by LC-
QTOF using sequential mass window acquisition (SWATH®), Anal. Methods
10 (2018) 2821e2833.
[12] C. Ludwig, L. Gillet, G. Rosenberger, S. Amon, B.C. Collins, R. Aebersold, Data-
independent acquisition-based SWATH-MS for quantitative proteomics: a
tutorial, Mol. Syst. Biol. 14 (2018).
[13] B. Drotleff, M. Hallschmid, M. L€ ammerhofer, Quantification of steroid hor-
mones in plasma using a surrogate calibrant approach and UHPLC-ESI-QTOF-
MS/MS with SWATH-acquisition combined with untargeted profiling, Anal.
Chim. Acta 1022 (2018) 70e80.
[14] W. Wu, R. Dai, E. Bendixen, Comparing SRM and SWATH methods for quan-
titation of bovine muscle proteomes, J. Agric. Food Chem. 67 (5) (2019)
1608e1618.
[15] B.C. Collins, C.L. Hunter, Y. Liu, B. Schilling, G. Rosenberger, S.L. Bader,
D.W. Chan, B.W. Gibson, A.-C. Gingras, J.M. Held, Multi-laboratory assessment
of reproducibility, qualitative and quantitative performance of SWATH-mass
spectrometry, Nat. Commun. 8 (2017) 291.
[16] M. Takahashi, Y. Uematsu, K. Kashiwaba, K. Yagasaki, M. Hajika, R. Matsunaga,
K. Komatsu, M. Ishimoto, Accumulation of high levels of free amino acids in
soybean seeds through integration of mutations conferring seed protein
deficiency, Planta 217 (2003) 577e586.
[17] T. Kou, Q. Wang, J. Cai, J. Song, B. Du, K. Zhao, Y. Ma, B. Geng, Y. Zhang, X. Han,
Effect of soybean protein on blood pressure in postmenopausal women: a
meta-analysis of randomized controlled trials, Food Funct. 8 (2017)
2663e2671.
[18] D. Ramdath, E. Padhi, S. Sarfaraz, S. Renwick, A. Duncan, Beyond the
cholesterol-lowering effect of soy protein: a review of the effects of dietary
soy and its constituents on risk factors for cardiovascular disease, Nutrients 9
(2017) 324.
[19] C.-Y. Tsai, Y.-H. Chen, Y.-W. Chien, W.-H. Huang, S.-H. Lin, Effect of soy
saponin on the growth of human colon cancer cells, World J. Gastroenterol.:
WJG 16 (2010) 3371.
[20] B.F. Wang, J.S. Wang, J.F. Lu, T.H. Kao, B.H. Chen, Antiproliferation effect and
mechanism of prostate cancer cell lines as affected by isoflavones from soy-
bean cake, J. Agric. Food Chem. 57 (2009) 2221e2232.
[21] M. Shiraiwa, K. Harada, K. Okubo, Composition and structure of “group B
saponin” in soybean seed, Agric. Biol. Chem. 55 (1991) 911e917.
[22] S. Kamo, S. Suzuki, T. Sato, The content of soyasaponin and soyasapogenol in
soy foods and their estimated intake in the Japanese, Food Sci. Nutr. 2 (2014)
289e297.
[23] Y. Omizu, C. Tsukaoto, R. Chettri, J.P. Tamang, Determination of Saponin
Contents in Raw Soybean and Fermented Soybean Foods of India, 2011.
[24] J. Jervis, C. Kastl, S.B. Hildreth, R. Biyashev, E.A. Grabau, M.A. Saghai-Maroof,
R.F. Helm, Metabolite profiling of soybean seed extracts from near-isogenic
low and normal phytate lines using orthogonal separation strategies,
J. Agric. Food Chem. 63 (2015) 9879e9887.
[25] U.F.D. Administration, Bioanalytical Method Validation, Guidance for Industry,
US Department of Health and Human Services Food and Drug Administration,
Rockville, MD, 2018.
[26] L.C. Gillet, P. Navarro, S. Tate, H. Ro € st, N. Selevsek, L. Reiter, R. Bonner,
R. Aebersold, Targeted data extraction of the MS/MS spectra generated by
data-independent acquisition: a new concept for consistent and accurate
proteome analysis, Mol. Cell. Proteom. 11 (2012), 016717. O111.