Metabolic studies of four soy isoflavones in rats by HPLC-HR-MS

Article abstract of DOI:10.1080/10286020.2014.902939

In this paper, the metabolites of four soy isoflavones, daidzein, daidzin, genistein, and genistin, on perfused rat intestine-liver model were investigated by high-performance liquid chromatography coupled with high-resolution mass spectrometer/tandem mass spectrometer. Totally 16 metabolites were detected and identified based on accurate mass, fragmentation patterns, and multiple-stage mass data (MSⁿ). The metabolic site of dadzein-7-methyl ether (D-7-M) was further confirmed by nuclear magnetic resonance. Methylation, glucuronide conjugation, and sulfate conjugation were the primary metabolic processes. Among them, six metabolites, daidzin-4′,7-diglucoside, genistein-4′- glucoside, D-7-M, dadzein-4′,7-dimethyl ether, genistein-4′-methyl ether, and genistein-7-methyl ether were detected in rats for the first time and not reported in humans. The metabolic pathways of daidzein, daidzin genistein, and genistin in rats were postulated. The biological effects of these metabolites are worthy of further investigation.

Full text of DOI:10.1080/10286020.2014.902939

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Metabolic studies of four soy
isoflavones in rats by HPLC–HR-MS
Ying-Fei Lia, Qiang Rena, Ying Jina, Cai-Sheng Wua, Cai-Hong
Wanga, Zhi-Xin Jiaa & Jin-Lan Zhanga
a State Key Laboratory of Bioactive Substance and Function of
Natural Medicines, Institute of Materia Medica, Chinese Academy
of Medical Sciences & Peking Union Medical College, Beijing
100050, China
Published online: 03 Apr 2014.
Click for updates
To cite this article: Ying-Fei Li, Qiang Ren, Ying Jin, Cai-Sheng Wu, Cai-Hong Wang, Zhi-Xin Jia &
Jin-Lan Zhang (2014) Metabolic studies of four soy isoflavones in rats by HPLC–HR-MS, Journal of
Asian Natural Products Research, 16:5, 497-510, DOI: 10.1080/10286020.2014.902939
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Journal of Asian Natural Products Research, 2014
Vol. 16, No. 5, 497–510, http://dx.doi.org/10.1080/10286020.2014.902939
Metabolic studies of four soy isoflavones in rats by HPLC–HR-MS
Ying-Fei Li1, Qiang Ren1, Ying Jin, Cai-Sheng Wu, Cai-Hong Wang, Zhi-Xin Jia
and Jin-Lan Zhang*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College,
Beijing 100050, China
(Received 25 October 2013; final version received 6 March 2014)
In this paper, the metabolites of four soy isoflavones, daidzein, daidzin, genistein, and
genistin, on perfused rat intestine–liver model were investigated by high-performance
liquid chromatography coupled with high-resolution mass spectrometer/tandem mass
spectrometer. Totally 16 metabolites were detected and identified based on accurate
mass, fragmentation patterns, and multiple-stage mass data (MSn). The metabolic site
of dadzein-7-methyl ether (D-7-M) was further confirmed by nuclear magnetic
resonance. Methylation, glucuronide conjugation, and sulfate conjugation were the
primary metabolic processes. Among them, six metabolites, daidzin-40,7-diglucoside,
genistein-40-glucoside, D-7-M, dadzein-40,7-dimethyl ether, genistein-40-methyl ether,
and genistein-7-methyl ether were detected in rats for the first time and not reported in
humans. The metabolic pathways of daidzein, daidzin genistein, and genistin in rats
were postulated. The biological effects of these metabolites are worthy of further
investigation.
Keywords: soy isoflavones; metabolites; HPLC–HR-MS/MSn
1. Introduction
investigators have reported that the con-
jugates may be biologically active or may be
precursorsofbiologicallyactivecompounds
for specific target cells. For example, a
sulfoconjugate of daidzein, daidzein-40,7-
disulfate (D-40,7-diS), inhibits sterol sulfa-
tase in hamster liver microsomes [7].
Daidzein and genistein glucuronides have
weak estrogenic activity and can activate
human natural killer cells [8]. Hence the
possible biological activities of the metab-
olites that to evaluate the health effects of
soy isoflavones needs to completely under-
stand their in vivo processes including
intake, absorption, metabolism and
excretion. Among of them metabolites
identification is the first step. Soy isofla-
vones are known to be biotransformed to
their corresponding aglycones and sugar
moieties by enteral microorganisms [9].
Soy isoflavones in soybean and soybean-
derived products are the major source of
phytoestrogens with potent estrogenic
activity in the human diet. Genistein and
daidzein are the two most active isoflavones
in soybeans. Soy isoflavones have been the
subject of rigorous investigation during the
past two decades. A remarkable amount of
epidemiological studies and in vitro exper-
iments suggested that soy isoflavones
provide important health benefits, including
a reduced risk of coronary heart disease and
breast, endometrial and cancers and allevia-
tion ofmenopause-related hot flashes[1–6].
It is still ambiguous, however, what is the
whole material basis responsible for the
effects in these epidemiological studies?
Metabolites play an important role in
bioactive effects of soy isoflavones. Several
*Corresponding author. Email: zhjl@imm.ac.cn
q 2014 Taylor & Francis

498
Y.-F. Li et al.
And then the isoflavones are metabolized as
glucuronide, sulfate, and sulfoglucuronide
conjugates [10,11]. But it is not the whole
metabolism fate of soy isoflavones. With the
development of hybride mass spectrometer
with high resolution and high sensitivity, the
metabolites in vivoare easilydiscoveredand
identified.
date knowledge of metabolic fate of
daidzein, genistein, daidzin, and genistin
after oral administration by rats.
2. Results
The reference substances and bio-samples
including processed perfusate, intestinal
contents, and bile were analyzed by
HPLC–LTQ-FTICR-MSn. Aglycones of
daidzin and genistin were found in bio-
samples after administration of daidzin
and genistin, respectively, by comparing
with the information of standards. Based
on multiple-stage mass data of standard
and possible metabolic pathway, metab-
olites were filtered out. Then the structures
of the metabolites were elucidated accord-
ing to the relative mass information,
including the proposed elemental compo-
sition based on accurate mass, fragmenta-
tion patterns, MS2 and MS3 spectra
collected with data-dependent MS/MS
analysis, and possible metabolic pathways.
Furthermore, the biotransformation
site of one metabolite of dadzein
was validated as dadzein-7-methyl ether
(D-7-M) by nuclear magnetic resonance
(NMR).
In this report, a high-performance
liquid chromatography coupled with a
hybrid linear ion-trap and fourier transform
ion cyclotron resonance mass spectrometry
(HPLC–LTQ-FTICR-MSn) method was
used to discover metabolites of daidzein,
genistein, daidzin, and genistin on perfused
rat intestine–liver model. The in situ
single-pass vascularly perfused rat intes-
tine–liver preparation [12] was utilized to
produce and collect metabolites of daid-
zein, genistein, and their respective glyco-
sides. The advantages of this model include
that composition of perfusion medium is
fully controlled; the effect of enterohepatic
circulation is eliminated; the native archi-
tecture of the small intestine is preserved
allowing the simultaneous examination of
intestinal metabolic, absorptive and
secretory processes; perfusate was col-
lected continuously beyond sampling time
limitation of blood, etc. Also bile duct
catheterized rats were used to get metab-
olites after hepatic metabolism. High-
resolution mass spectrometer (HR-MS)
and multiple-stage mass spectrometric
data of compounds in biological samples
were acquired automatically using HPLC–
LTQ-FTICR-MSn via data-dependent
scan. The structures of the metabolites
were identified on the basis of accurate
mass, fragmentation patterns, and MSn
data. As a result, a total of 16 metabolites as
shown in Figure 1 were detected and
identified in biological samples. Also the
metabolic pathways of metabolites were
proposed. HPLC–LTQ-FTICR-MSn had
been successfully utilized to discover and
identify metabolites of soy isoflavones in
rats and the combined method provided a
better tool for study and complement up-to-
Except aglycones metabolized from
daidzin and genistin, a total of 14 different
metabolites of daidzein, daidzin, genis-
tein, and genistin from three biological
samples were detected. Metabolites of
daidzein, daidzin, genistein, and genistin
detected in three biological samples and
related MS data are summarized in
Table 1. The representative extract ion
current (EIC) chromatograms of daidzein,
daidzin, genistein, genistin, and their
metabolites are presented in Figures 2
and 3, respectively.
2.1 Mass fragmentation of daidzein,
daidzin, genistein, and genistin
Daidzein, daidzin, genistein, and genistin
were analyzed by HPLC–LTQ-FTICR-
MSn. The combination of FTICR mass

Journal of Asian Natural Products Research
499
Figure 1. Structures of daidzein, daidzin, genistein, genistin and their metabolites considered in
this study. Abbreviations: D-7-M, dadzein-7-methyl ether; D-40,7-diM, dadzein-40,7-dimethyl ether;
D-7-Glu, daidzein-7-glucuronide; D-40-Glu, daidzein-40-glucuronide; D-7-Glu-40-S, daidzein-7-
glucuronide-40-sulfate; D-40-Glu-7-S, daidzein-40-glucuronide-7-sulfate; D-40,7-diGlc, daidzin-40,7-
diglucoside; G-7-M, genistein-7-methyl ether; G-40-M, genistein-40-methyl ether; G-7-Glu,
genistein-7-glucuronide; G-40-Glu, genistein-40-glucuronide; G-7-Glu-40-S, genistein-7-
glucuronide-40-sulfate; G-40-Glu-7-S, genistein-40-glucuronide-7-sulfate; D-40-Glc, genistein-40-
glucoside.
analyzer and tandem mass spectrometry is
capable of providing useful information
such as characteristic fragment ions and
fragmentation pattern. And the fragmenta-
tion patterns of reference compounds were
utilized in correlation to propose the
structures of metabolites.
with retention time of 18.16 min,
10.24 min, 24.81 min, and 13.38 min,
respectively (Table 1).
In the MS2 spectrum of daidzein, the
most abundant fragment ion at m/z 199 was
formed via loss of 2CO. A fragment ion
resulting from retro-Diels–Alder (RDA)
fragmentation was found at m/z 137 (62)
due to loss of C8H6O. The product ions at
m/z 227 (49), 237 (22), and 145 (11) were
generated by the loss of CO, H2O, and
C6H6O2, respectively. In the MS3 spec-
trum, the neutral loss of H2O from m/z 199
was observed and the corresponding base
peak at m/z 181 was generated [13].
In the full scan of high-resolution mass
spectrum, authentic daidzein, daidzin,
genistein, and genistin exhibited
[M þ H]þ ions at m/z of 255.06477
(C15H11O4, error 21.63 ppm), 417.11725
(C21H21O9, error 21.85 ppm), 271.05966
(C15H11O5, error 21.62 ppm), and
433.11221 (C21H21O10, error 21.65 ppm)

500
Y.-F. Li et al.
d

Journal of Asian Natural Products Research
501
Figure 2. Representative EIC chromatograms of daidzein, daidzin, and their metabolites. See
abbreviations in the caption of Figure 1.
In the MS2 of genistein, the most
abundant fragment ion at m/z 153 was
formed via RDA fragmentation by loss of
C8H6O. The product ions at m/z 215 (80),
243 (72), 253 (37), 149 (20), and 145 (14)
were generated via loss of 2CO, CO, H2O,
C7H6O2, and C6H6O3, respectively. In the
MS3 spectrum, the neutral losses of H2O
from m/z 215 were observed and the base
peak at m/z 197 was generated [13].
sidic moiety in the MS2 spectrum. The
MS3 ions at m/z 199 (100), 227 (75), 137
(65), 237 (31), 145 (11) of daidzin were
same as the MS2 ions of daidzein due to
the loss of 2CO, CO, C8H6O, H2O, and
C6H6O2. The MS3 ions at m/z 153 (100),
215 (50), 243 (49), 149 (30), 253 (29),
and 145 (15) of genistin were same as the
MS2 ions of genistein due to the loss of
C8H6O, 2CO, CO, C7H6O2, H2O, and
C6H6O3. The fragmentation pathways of
daidzein and genistein are proposed in
Figure 4 [13].
Both daidzin and genistin produced
quasi-molecular ion at m/z of 255 and
271, respectively, via loss of the glyco-

502
Y.-F. Li et al.
Figure 3. Representative EIC chromatograms of genistein, genistin, and their metabolites. See
abbreviations in the caption of Figure 1.
2.2 Identification of metabolites of
daidzein and daidzin
metabolite of daidzein. In the MS2
spectrum, the product ions at m/z 213
(100), 151 (62), and 241 (33) were
generated by the loss of 2CO, C8H6O, and
CO, respectively. The fragment ions at m/z
213 and 151 showing 14 Da higher than
those of daidzein at m/z of 199 and 137
indicated a methyl group attached to the
C-7 hydroxyl group. To further verify the
methylated position on the metabolite
formed from daidzein, an authentic com-
pound with same retention time, MS2, and
MS3 as D-7-M was synthesized, and the
NMR spectral data of daidzein and the
authentic compound were obtained
(Table 2). The 1H NMR data of the
authentic compound exhibited three pro-
tons of methyl at d 3.89 (3H, s, MeO-7), not
Totally eight different metabolites of
daidzein and daidzin were detected in
perfusate, intestine, and bile samples
(Figure 1). The structures of metabolites
were identified and tentatively character-
ized on the basis of the proposed formulas
of the precursor ions, the fragmentation
rules, and MSn data (Table 1).
D-7-M (tR ¼ 27.1 min) exhibited the
quasi-molecular ion at m/z 269.08075
(C16H13O4, error 20.32 ppm), 14 Da
higher than that of daidzein, in intestinal
contents of perfused rat intestine–liver
preparation after administration of daidzein
in duodenum, suggesting a mono-methyl

Journal of Asian Natural Products Research
503
Figure 4. Fragmentation of daidzein, daidzin, genistein, and genistin.
found in the 1H NMR spectrum of daidzein,
one proton of hydroxyl group at d 9.52 (1H,
s, 40-OH), and eight protons at d 8.35 (1H, s,
2-H), 8.01 (1H, d, 5-H), 7.12 (1H, d, 6-H),
7.05 (1H, dd, 8-H), 7.39 (2H, d, 20-H and 60-
H), and 6.80 (2H, d, 30-H and 50-H). While
two signals for hydroxyl groups at d 10.74
(1H, s) and 9.49 (1H, s) were found in the
1H NMR spectrum of daidzein. These
results confirmed that a methyl group was
transferred to the C-7 hydroxyl group of
daidzein to get D-7-M.
Dadzein-40,7-dimethyl ether (D-40,7-
diM, tR ¼ 32.35 min) gave an [M þ H]þ
ion at m/z 283.09660 (C17H15O4, error
21.15 ppm), 28 Da higher than that of
daidzein, in perfusate and intestinal con-
tents of perfused rat intestine–liver
preparation after administration of daid-
zein in duodenum which indicated two
Table 2. 1H NMR spectral data for daidzein and D-7-M.a
Proton
Daidzein
D-7-M
CH3
7-OH
40-OH
2
–
10.74 (1H, s)
9.49 (1H, s)
8.34 (1H, s)
3.89 (3H, s)
–
9.52 (1H, s)
8.35 (1H, s)
5
6
8
20,60
30,50
7.95 (1H, d, J ¼ 9.0 Hz)
6.92 (1H, dd, J ¼ 9.0, 2.0 Hz)
6.85 (1H, d, J ¼ 2.0 Hz)
7.36 (2H, d, J ¼ 8.0 Hz)
6.77 (2H, d, J ¼ 8.0 Hz)
8.01 (1H, d, J ¼ 8.5 Hz)
7.05 (1H, dd, J ¼ 8.5, 2.5 Hz)
7.12 (1H, d, J ¼ 2.5 Hz)
7.39 (2H, d, J ¼ 8.5 Hz)
6.80 (2H, d, J ¼ 8.5 Hz)
Note: d, doublet; dd, doublet of doublets; s, singlet.
a The reported data were obtained in DMSO-d6.

504
Y.-F. Li et al.
methyl groups substituted on daidzein.
In the MS2, the loss of 2CH3 formed the
characteristic ion at m/z 255. Then the MS3
of m/z 255 presented the specific fragment
ions at m/z 199 (100), 227 (78), 137 (75),
and 145 (16) via loss of 2CO, CO, C8H6O,
and C6H6O2. According to the possible
metabolic path, methyl groups were
attached to 7-OH and 40-OH of daidzein,
respectively, to get D-40,7-diM.
error 20.57 ppm) and 511.05405
(C21H19O13S, error 20.08 ppm), respect-
ively. The MS2 of both quasi-molecular
ions at m/z 511 presented the base peak at
m/z 335 via loss of 176 Da. The subsequent
MS3 of m/z 335 generated the fragment ion
at m/z 255 by loss of 80 Da. Based on the
above data, both compounds were sulfo-
glucuronides of daidzein. According to the
report [14], the daidzein sulfates whose
conjugation site was assigned to the
40-hydroxyl group were eluted earlier
than the metabolite whose conjugation
group was assigned to the 7-hydroxyl
group. So we proposed that the earlier
metabolite should be D-7-Glu-40-S
(tR ¼ 7.91 min) and the later one should
be D-40-Glu-7-S (tR ¼ 8.87 min).
Two glucuronide conjugates of daid-
zein, daidzein-7-glucuronide (D-7-Glu)
and daidzein-40-glucuronide (D-40-Glu),
were detected both in perfusate of perfused
rat intestine–liver preparation and bile
samples of rat after duodenum or oral
administration of daidzein and daidzin,
respectively. D-7-Glu and D-40-Glu
exhibited protonated ions at m/z
431.09683 (C21H19O10, error 21.03 ppm)
and 431.09650 (C21H19O10, error
21.79 ppm), respectively, 176 Da higher
than that of daidzein. Both MS2 gave the
base peak of m/z 255 via loss of a
glucuronide moiety (176 Da) from m/z
431. In the MS3 spectrum of D-7-Glu, the
m/z 255 ion produced specific ions at m/z
199 (100) and 137 (89). In the MS3 of D-
40-Glu, the m/z 255 ion produced specific
ions at m/z 199 (100), 237 (75), 227 (55),
137 (13), and 145 (10) due to the loss of
2CO, H2O, CO, C8H6O, and C6H6O2.
According to the report [14], the glucur-
onide of daidzein whose conjugation site
was assigned to the 7-hydroxyl group was
eluted earlier than the metabolite whose
conjugation group was assigned to the
40-hydroxyl group. We proposed that the
metabolite with earlier retention time
should be D-7-Glu (tR ¼ 10.68 min) and
the later one should be D-40-Glu
(tR ¼ 11.15 min).
In the full scan of FTICR mass
spectrum, a trace glycoside conjugate of
daidzin, daidzin-40,7-diglucoside (D-40,7-
diGlc), was found in perfusate of perfused
rat intestine–liver preparation at 9.48 min
and gave a protonated ion at m/z
579.17029 (C27H31O14, error 20.94 ppm)
after administration of daidzin in duode-
num. In the MS2, two fragment ions were
observed. The higher product ion at m/z
255 was formed by the loss of two glucose
moieties from the precursor ion. The lower
fragment ion at m/z 417 was generated by
cleaving one glycoside. In the MS3, the
neutral losses of CO, H2O, and 2CO from
m/z 255 were observed and the corre-
sponding fragment ions at m/z 227 (100),
237 (41), and 199 (27) were generated,
respectively. Based on the possible meta-
bolic path, we postulated that one glyco-
side was attached to 7-OH and 40-OH of
daidzein, respectively, to get D-40,7-diGlc.
Daidzein was found in perfusate,
intestinal contents, and bile samples after
administration of daidzin which was ident-
ified by comparison with standard daidzein.
The proposed metabolic pathways of
daidzein and daidzin in rats are shown in
Figure 5.
Also two isomers, daidzein-7-glucur-
onide-40-sulfate (D-7-Glu-40-S) and daid-
zein-40-glucuronide-7-sulfate (D-40-Glu-7-
S), were detected in bile samples of rat
after oral administration of daidzein and
daidzin. These two compounds showed
[M þ H]þ at m/z 511.05380 (C21H19O13S,

Journal of Asian Natural Products Research
505
Figure 5. Proposed metabolic pathways of daidzein, daidzin, genistein, and genistin in rats.
2.3 Identification of metabolites of
genistein and genistin
253 (28), 257 (21), 271 (16), and 149 (11)
via loss of CH4, 2CO, CH4O, CO, CH2,
and C8H8O2, respectively. In the MS3, the
isomer with earlier retention time yielded
the fragment ions at m/z 167 (100), 241
(24), and 225 (22) due to the loss of
C7H2O, CO, and CO2 from m/z 269,
respectively. The MS3 spectrum of later
isomer eluted displayed fragment ions at
m/z 151 (100) and 253 (46) derived from
loss of C7H2O2 and CH4 from m/z 269,
respectively. According to the above data,
both the compounds were mono-methyl
metabolite of genistein. For the later
isomer, the characteristic ion at m/z 229
(46) in MS2 which was 14 Da higher than
the MS2 ion of protonated genistein at m/z
215 indicated that the methyl was attached
to 7-OH or 5-OH. In consideration of the
chemical inertia of the 5-hydroxyl group
which formed a hydrogen bond with the
ketone carbonyl group in the structure of
flavone, the glycosidation on the
5-hydroxyl group is impossible. So we
proposed that the metabolites with reten-
Totally eight metabolites of genistein and
genistin were detected in perfusate,
intestine, and bile samples (Figure 1).
The structures of metabolites were ident-
ified and tentatively characterized on the
basis of the proposed formulas of the
precursor ions, the fragmentation rules,
and the MSn data (Table 1).
Genistein-40-methyl ether (G-40-M,
tR ¼ 33.00 min) and genistein-7-methyl
ether (G-7-M, tR ¼ 33.38 min) were also
isomers detected in rat intestinal contents
of perfused rat intestine–liver preparation
after administration of genistein in duode-
num. The isomers showed [M þ H]þ at
m/z 285.07559 (C16H13O5, error
20.56 ppm) and 285.07553 (C16H13O5,
error 20.77 ppm), respectively. The MS2
of G-40-M presented the fragment ions at
m/z 269 (100) and 271 (11) via loss of CH4
and CH2. The MS2 of G-7-M displayed
product ions at m/z 269 (100), 229 (46),

506
Y.-F. Li et al.
tion times of 33.00 and 33.38 min were
G-40-M and G-7-M, respectively.
Two metabolites with quasi-molecular
ions at m/z 527.04907 (C21H19O14S, error
0.07 ppm) and 527.04895 (C21H19O14S,
error 20.10 ppm) were found at 10.25 and
10.93 min in the chromatogram of rat bile
after oral administration of genistein or
genistin. Both peaks produced the base
peak at m/z 351 in the MS2 via loss of a
glucuronide moiety (176 Da). Then sub-
sequent MS3 of m/z 351 generated the base
peak at m/z 255 by loss of SO3 (80 Da).
Therefore, these two metabolites were
tentatively identified as sulfoglucuronides
of genistein. Based on the report [14],
genistein sulfate whose sulfate conju-
gation site was assigned to the 40-hydroxyl
group was eluted earlier than the metab-
olite whose sulfate conjugation group was
assigned to the 7-hydroxyl group. So we
proposed that the earlier metabolite should
be genistein-7-glucuronide-40-sulfate
(G-7-Glu-40-S) and the later one should
be genistein-40-glucuronide-7-sulfate0.
Aglycone of genistin was found in
perfusate, intestinal contents, and bile (0–
36 h) after duodenum and oral adminis-
tration of genistin and was identified by
comparison with standard daidzein.
The metabolite with the quasi-molecu-
lar ion at m/z 433.11212 (C21H21O10, error
21.85 ppm) was discovered at 14.08 min
in the chromatogram of intestinal contents
of perfused rat intestine–liver preparation
after administration of genistein in duode-
num. In the MS2, the base peak of m/z 271
was generated by loss of a glycoside from
the quasi-molecular ion at m/z 433. In the
MS3, the characteristic fragment ions at
m/z 243 (100), 153 (94), 215 (89), 253
(34), and 149 (25) were observed due to
the neutral losses of CO, C8H6O, 2CO,
H2O, and C7H6O2 from the fragment ion at
m/z 271, respectively. Based on the
possible metabolic path and earlier reten-
tion time than genistin, we speculated that
the glycosylation occurred at 40-OH to get
genistein-40-glucoside (G-40-Glc).
Genistein-7-glucuronide (G-7-Glu,
tR ¼ 13.84 min) and Genistein-40-glucuro-
nide (G-40-Glu, tR ¼ 14.29 min) were
screened both in perfusate of perfused rat
intestine–liver preparation and bile samples
after duodenum or oral administration of
genistein and genistin. The isomers with
[M þ H]þ ions at m/z of 447.09213
(C21H19O11, error 20.13 ppm) and
447.09207 (C21H19O11, error 20.26ppm)
showed similar fragmentation rules. Both
the MS2 fragmentations yielded the base
peak ions at m/z 271 from the quasi-
molecular ions at m/z 447 due to the loss of
a glucuronide moiety (176Da). The typical
fragment ions at m/z 153 (100), 215 (90),
243 (80), 253 (59), 149 (37), 145 (29), and
159 (18) in MS3 of G-7-Glu from the ion at
m/z 271 were observed. Similar typical
fragment ions at m/z 153 (100), 215 (90),
243 (74), 253 (29), 149 (25), 159 (22), and
145 (14) in MS3 spectrum of G-40-Glu from
ion at m/z 271 were observed. Due to
chemical inertia of the 5-hydroxyl group,
the metabolites were tentatively identified
as G-7-Glu and G-40-Glu, glucuronide of
genistein, according to the retention
time [14].
The proposed metabolic pathways of
genistein and genistin in rats are shown in
Figure 5.
3. Discussion
The aim of this study was to identify the
metabolites of daidzein, daidzin genistein,
and genistin in rats based on HPLC–LTQ-
FTICR-MSn analysis incorporating spec-
tral tree data mining method. Although
previous reports investigated metabolites
and conversion pathway of soy isoflavones
(daidzin, daidzein, genistein, etc.) in
biological samples of humans [15,16],
rodents, or their intestine bacteria [17,18],
the application of HPLC combined with
high resolution and multiple stage mass
spectrometer for investigation of metab-
olites of soy isoflavones in multiple bio-
samples (rat perfusate, intestinal contents,

Journal of Asian Natural Products Research
507
and bile) presented in this manuscript
provided a better tool for study and
complement up-to-date knowledge in bio-
transformation of daidzein, genistein,
daidzin, and genistin after oral adminis-
tration by rats.
However, whether the newly detected
metabolites, D-40,7-diGlc, G-40-Glc, D-7-
M, D-40,7-diM, G-40-M, and G-7-M, are
present in humans after oral administration
of soy isoflavones needs to be investigated
further. Based on the corresponding
pharmacodynamic effects and pharmaco-
kinetic data of the newly detected
metabolites of soy isoflavones, the phar-
macological effects of soy isoflavones
could be further investigated.
Most of the isoflavone glycosides are
hydrolyzed to their aglycones by enteral
microorganisms [17,18]. After adminis-
tration of daidzin or genistin in duodenum,
daidzein or genistein, the aglycone of
daidzin or genistin, was found in intestinal
contents and perfusate. And intact agly-
cones were still detected in bile at relative
low level after oral administration of
daidzein and genistein. Several studies
have shown that the main metabolites of
isoflavone are 7-glucuronides followed by
40-glucuronides and that there are also
small proportions of sulfate esters.
In perfusate and bile samples, two glucur-
onide conjugates of daidzein and genistein
were found. In addition, two sulfoglucur-
onides of daidzein and genistein in bile
were detected after oral administration of
daidzein, daidzin, genistein, and genistin to
rats, respectively. However, there was no
mono- or di-sulfate conjugates detected in
perfusate and bile for both daidzein and
genistein. In addition, only one mono
glycoside of daidzin, D-40,7-diGlc, was
filtered in perfusate after administration of
daidzin in duodenum. And one mono
glycoside of genistein, G-40-Glc, was
detected in perfusate after administration
of genistein in duodenum. In addition,
except phase II metabolites, methylation
metabolites of daidzein (D-7-M and D-40,7-
diM) and genistein (G-40-M and G-7-M)
were found in perfusate or intestinal
contents after administration of daidzein
in duodenum. To the best of our knowl-
edge, D-40,7-diGlc, G-40-Glc, D-7-M, D-
40,7-diM, G-40-M, and G-7-M were
detected as metabolites of isoflavone in
rats for the first time.
4. Experimental
4.1 Materials and reagents
Daidzein, daidzin, genistein, and genistin
with purity more than 98.0% were supplied
by Shanghai Xinran Biotechnology Co.,
Ltd (Shanghai, China). Dextran T40,
bovine serum albumin, glucose, dexa-
methasone injection (2 mg/ml), and norepi-
nephrine injection (2 mg/ml) were supplied
by Beijing Double-Crane Pharmaceutical
Co., Ltd (Beijing, China), Bovogen Biolo-
gicals Pty Ltd (East Keilor, VIC, Australia),
Sinopharm Chemical Reagent Beijing Co.,
Ltd (Beijing, China), Tianjin Pharmaceu-
tical Group Jiaozuo Co., Ltd (Tianjin,
China), and Shanghai Harvest Pharmaceu-
tical Co., Ltd (Shanghai, China), respect-
ively. Urethane was purchased from Sigma-
Aldrich (St. Louis, MO, USA).
Acetonitrile of mass grade was pro-
vided by Mallinckrodt Baker, Inc. (Phil-
lipsburg, NJ, USA). Acetic acid of HPLC
grade was purchased from Mallinckrodt
Baker, Inc., and the other chemicals used
were all analytical reagent grade. Deio-
nized water was purified using a water
purification system (Millipore, Billerica,
MA, USA).
4.2 Synthesis of D-7-M
A total of 100 mgof daidzein (0.39 mmol) in
4 ml of N-dimethyl formamide were added
to 50mg of anhydrous K2CO3 (0.36 mmol)
and 20ml dimethyl sulfate (0.21 mmol). The
mixture was stirred for 5 h [19]. After
Conjugated metabolites of soy isofla-
vones were identified and their pharmaco-
kinetic data have been estimated.

508
Y.-F. Li et al.
cooling the reaction mixture, the mixture
was purified on a Sephadex LH-20 (Phar-
macia Fine Chemicals, Inc., Piscataway, NJ,
USA) column with methanol. The structure
ofthe synthesized compoundwas confirmed
by MS and 1H NMR techniques. The purity
of chemically synthesized D-7-M was more
than 98.5%, determined by HPLC–UV.
378C. Upon verification of the loss of pain
reflex, the abdomen was opened by a
3–4 cm midline longitudinal incision. The
bile duct, gastric artery, gastro duodenal
artery, and splenic blood vessels perfusing
the stomach and spleen were ligated in this
order. The celiac artery and the pyloric vein
were ligated. Immediately after the aorta
was tied off near the superior mesenteric
artery, a cut was made in the superior
mesenteric artery. A teflon catheter with
diameter of 1 mm was inserted into the
artery. The catheter was secured with 3-0
surgical silk (Shanghai Medical Suture
Needle Factory Co., Ltd, Shanghai, China).
Perfusion into the superior mesenteric
artery with blank perfusate was immedi-
ately initiated at a flow rate of 4 to 5 ml/min.
The thoracic cavity was opened and a cut
was made in the right atrium of the heart.
A teflon catheter from an i.v. placement
unit was inserted into the opening of the
right atrium till the hepatic vein and
secured in place. The catheter was con-
nected to tygon tubing allowing the exit of
perfusate from the liver. Then the proximal
part of the colon was ligated and an open
ligature was positioned around the duode-
num. Henceforth, the perfusion flow rate
was then adjusted to approximately 7 ml/
min. The experiment started after a pre-
perfusion to wash out the residual blood in
tissues using 30 ml of perfusate.
4.3 Animals
Male Wistar rats (220 ^ 20 g) were used
and supplied by Vital River Laboratories
(Beijing, China). The rats were housed
under standard conditions of temperature,
humidity, and light, and had free access to
standard rodent diet and water before the
experiment. Animal experiments were
conducted according to the Animal Care
and Welfare Committee of the Institute of
Materia Medica, Chinese Academy of
Medical Sciences and Peking Union
Medical College (Beijing, China).
4.4 Perfused rat intestine–liver
preparation
A perfuser consisting of a peristaltic pump,
oxygen, temperature chamber, and recir-
culating pump was used. Perfusate con-
sisted of 3% dextran T40, 1% bovine
serum albumin, 3 mg/ml glucose in Krebs-
Henseleit Bicarbonate solution buffered to
pH 7.4. Perfusate was oxygenated in
rotating reservoirs with 95% O2 and 5%
CO2. Before perfusion, 40 ml of dexa-
methasone injection and 20 ml of norepi-
nephrine injection were added for each
100 ml perfusate.
4.5 Bile duct catheterization
preparation
Also two rats for each compound were
used to collect bile samples. The rats were
anesthetized with i.p. injection of 20%
urethane (0.6 ml/100 g) and placed under a
tunable heating light to maintain a body
temperature of 378C. Upon verification of
the absence of a pain reflex, a midline
abdominal incision of 3–4 cm was made.
Then the intestine was moved away to
expose the hepatic artery and bile duct.
Following isolation, a polyethylene cath-
eter (0.2 mm i.d.) was inserted about 5 mm
A previously described surgical pro-
cedure [20,21] was modified in this study.
To get enough metabolites, two male
Wistar rats were used for each compound.
The rats were fasted overnight with free
access to tap water before the perfusion
experiment. The rats were anesthetized
with i.p. injection of 20% urethane (0.6 ml/
100 g) and placed under a tunable heating
light to maintain a body temperature of

Journal of Asian Natural Products Research
509
inside and firmly ligated. The abdominal
cavity was closed using surgical wire and
the catheter was passed under the skin of
the lower abdomen and out through a
puncture made in the tail. The skin of the
abdomen was then closed using surgical
wire. The bile duct cannula was secured
with a harness from the tail of the rat to the
roof of the metabolism cage. During the
whole period of the experiment, rats were
kept on a homoeothermic board with light
anaesthesia and were supplemented with
water containing 2% glucose and 0.9%
NaCl.
was pooled and evaporated to dryness
under N2 flow at 408C. The residues were
reconstituted in 2 ml of methanol and
1.5 ml supernatant was transferred after
5 min of vortex and centrifugation at
13,000 g for 10 min at 48C. Finally, a 3 ml
aliquot of supernatant was injected to
HPLC–HR-MS/MSn for analysis.
4.8 Instrumentation and analytical
conditions
HPLC–HR-MS/MSn was performed using
a Surveyor LC system coupled to a hybrid
LTQ-FTICR mass spectrometer (Thermo
Fisher Scientific, Bremen, Germany). LC
separation was performed on Kinetex C18
reversed phase column (4.6 £ 100 mm,
2.6 mm, Phenomenex (Macclesfield, UK))
and the column temperature was main-
tained at 358C. Purified water with the
addition of 0.1% formic acid was used as
solvent A and acetonitrile containing 0.1%
formic acid as solvent B. The gradient
profile was as follows: 0–5 min 10% B, 5–
38 min, a linear increase to 50% B, by a
linear increase to 95% B at 50 min to wash
the column. The column was then returned
to 10% B at 52 min and allowed to re-
equilibrate (18 min) before the next sample
was analyzed. Analyses were performed at
a flow rate of 0.6 ml/min with a splitter
after the analytical column to introduce
200 ml/min eluent to the ESI source.
The positive ion ESI-MS and tandem
MS were used for the detection of analytes
under the following mass spectrometric
conditions. The spray voltage was set at
3.5 kV, the temperature of the heated
capillary was set at 2508C, and the
capillary voltage was set at 30 V. The
sheath gas (N2) flow rate was 35 (arbitrary
units), the auxiliary gas flow rate was set at
10 (arbitrary units), the sweep gas flow rate
was set at 3 (arbitrary units), and tube lens
offset voltage was set at 120 V.
4.6 Drug administration and sample
collection
For rats accepting intestine–liver perfused
preparation, in order to collect enough
metabolite, a dosage of 10 mg suspended
daidzein, daidzin, genistein, and genistin
solved in 1 ml saline, respectively, was
administrated by an injector at the proximal
part of the duodenum after exsanguinations
of tissues, and then the open ligature
positioned around the duodenum was
ligated. Perfusions were continued at a
constant flow rate (7.0 ml/min). About 25
and 50 ml samples were taken before (blank
sample) and after (metabolite sample)
intra-duodenum administration of com-
pound at 120 min, respectively. Intestinal
content between the ligated duodenum and
ligated colon was collected.
For rats accepting bile duct catheter-
ization preparation, the same dosage of
daidzein, daidzin, genistein, and genistin
was administrated by oral gavage, respect-
ively. Bile was collected 2 h and 36 h,
respectively, before and after dosing.
4.7 Preparation of samples
Perfusate, intestinal contents, and bile
samples were concentrated to dryness
under reduced pressure at 2208C, and
then the residues were extracted by 5 ml
methanol for three times. The methanol
Full-scan experiments were performed
in the range of m/z 100–1500 at a
resolving power of 50,000 with data-

510
Y.-F. Li et al.
dependent MS2 analysis triggered by the
two most abundant ions from full-scan
mass analysis, followed by MS3 analysis
of the most abundant product ions. Upon
data-dependent analyses, high accuracy
mass-to-charge and multiple-stage mass
spectrometry of compounds in biological
samples could be acquired automatically.
Data acquisition was performed with
Xcalibur version 2.0 software (Thermo
Fisher Scientific, San Jose, CA, USA).
[4] M. Messina and B. Lane, Future Lipidol.
2, 55 (2007).
[5] A.H. Wu, M.C. Yu, C.C. Tseng, and M.C.
Pike, Br. J. Cancer 98, 9 (2008).
[6] M. Messina and A.H. Wu, Am. J. Clin.
Nutr. 89, 1673S (2009).
[7] C.K. Wong and W.M. Keung, Biochem.
Biophys. Res. Commun. 233, 579 (1997).
[8] Y. Zhang, T.T. Song, J.E. Cunnick, P.A.
Murphy, and S. Hendrich, J. Nutr. 129,
399 (1999).
[9] K. Nemeth, G.W. Plumb, J.G. Berrin,
N. Juge, R. Jacob, H.Y. Naim, G.
Williamson, D.M. Swallow, and P.A.
Kroon, Eur. J. Nutr. 42, 29 (2003).
[10] D.R. Doerge, H.C. Chang, M.I. Church-
well, and C.L. Holder, Drug Metab.
Dispos. 28, 298 (2000).
[11] S.R. Shelnutt, C.O. Cimino, P.A. Wig-
gins, M.J. Ronis, and T.M. Badger, Am.
J. Clin. Nutr. 76, 588 (2002).
[12] K.S. Pang, W.F. Cherry, and E.H. Ulm,
J. Pharmacol. Exp. Ther. 233, 788 (1985).
[13] P.J. Madeira, C.M. Borges, and M.H.
Florencio, Rapid Commun. Mass Spec-
trom. 24, 3432 (2010).
4.9 NMR spectrometry
1H NMR spectra were measured on a
Bruker AV-500 instrument (500 MHz,
11.74 T) (Karlsruhe, Germany). Samples
were recorded in DMSO-d6. Chemical
shifts are given in d (ppm) scale relative to
solvent peaks as references.
Acknowledgments
This work was financially supported by the
Ministry of Science and Technology of China
(grant number 2012ZX09301002-001) and the
Fundamental Research Funds for the Central
Universities from the Institute of Materia
Medica (grant number 2012RC01), Chinese
Academy of Medical Sciences & Peking Union
Medical College.
[14] K. Hosoda, T. Furuta, and K. Ishii, Drug
Metab. Dispos. 39, 1762 (2011).
[15] K. Hosoda, T. Furuta, A. Yokokawa, and
K. Ishii, Anal. Bioanal. Chem. 397, 1563
(2010).
[16] K. Hosoda, T. Furuta, A. Yokokawa,
K. Ogura, A. Hiratsuka, and K. Ishii, Drug
Metab. Dispos. 36, 1485 (2008).
[17] I. Marotti, A. Bonetti, B. Biavati,
P. Catizone, and G. Dinelli, J. Agric.
Food Chem. 55, 3913 (2007).
[18] A. Matthies, T. Clavel, M. Gutschow,
W. Engst, D. Haller, M. Blaut, and A.
Braune, Appl. Environ. Microbiol. 74,
4847 (2008).
Note
1. These authors contributed equally to this
work.
References
[1] L. Yan and E.L. Spitznagel, Am. J. Clin.
Nutr. 89, 1155 (2009).
[2] A. Cassidy and L. Hooper, J. Br. Meno-
pause Soc. 12, 49 (2006).
[3] S.H. Li, X.X. Liu, Y.Y. Bai, X.J. Wang,
K. Sun, J.Z. Chen, and R.T. Hui, Am.
J. Clin. Nutr. 91, 480 (2010).
[19] G.Y. Gao, D.J. Li, and W.M. Keung,
Bioorg Med. Chem. 11, 4069 (2003).
[20] H. Hirayama, X. Xu, and K.S. Pang, Am.
J. Physiol. 257, G249 (1989).
[21] X. Xu, H. Hirayama, and K.S. Pang, Drug
Metab. Dispos. 17, 556 (1989).