Synthesis of daidzein 7-O-β-d-glucuronide-4′-O-sulfate

Article abstract of DOI:10.1016/j.steroids.2007.07.001

The first synthesis of daidzein 7-O-β-d-glucuronide-4′-O-sulfate, a mixed conjugate of an important dietary phytoestrogen is described.

Full text of DOI:10.1016/j.steroids.2007.07.001

steroids 7 2 ( 2 0 0 7 ) 851–854
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journal homepage: www.elsevier.com/locate/steroids
Synthesis of daidzein 7-O-␤-d-glucuronide-4ꢀ-O-sulfate
Otto Soidinsalo, Kristiina Wa¨ha¨la¨ ∗
Laboratory of Organic Chemistry, Department of Chemistry, P.O. Box 55, A.I. Virtasen aukio 1, University of Helsinki,
Helsinki 00014, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
The first synthesis of daidzein 7-O-␤-d-glucuronide-4ꢀ-O-sulfate, a mixed conjugate of an
important dietary phytoestrogen is described.
Received 5 October 2006
Received in revised form
15 March 2007
© 2007 Elsevier Inc. All rights reserved.
Accepted 6 July 2007
Published on line 13 July 2007
Keywords:
Daidzein
Isoflavone
Glucuronide
Sulfate
Phytoestrogen
Mixed conjugate
1.
Introduction
To the best of our knowledge there are no published synthe-
ses of the mixed sulfate/glucuronide isoflavones. In general,
analytical work on the naturally occurring isoflavone conju-
gates appears to suffer from the lack of authentic reference
samples [5]. To further study the analytics and biological
activity of daidzein 7-O-glucuronide-4ꢀ-O-sulfate (5), synthetic
reference is needed. We report here the first synthesis of
the sodium salt of daidzein 7-O-␤-d-glucuronide-4ꢀ-O-sulfate
(5) by way of selective 4ꢀ-O benzyl protection of daidzein
(Scheme 1).
Daidzein (1), one of the major dietary isoflavones, is
further metabolised in mammals to various glucuronide,
sulfate and sulfoglucuronide conjugates [1]. Daidzein 7-O-␤-
d-glucuronide-4ꢀ-O-sulfate (5), the major biliary metabolite
in rat, has been isolated and identified from rat urine [2].
Recently Tsuchihashi et al. isolated and identified various
isoflavone conjugates from human urine, including daidzein
7-O-␤-d-glucuronide-4ꢀ-O-sulfate (5) [3]. They also reported
that a daidzein 7-O-glucuronide-4ꢀ-O-sulfate isolate showed a
stimulatory effect on the growth of MCF-7 cells and exhibited
binding activity to human estrogen receptors (hERs) [4]. They
also studied the ER-dependent ␤-galactosidase induction of
several isoflavone conjugates, and daidzein 7-O-glucuronide-
4ꢀ-O-sulfate (5) showed potent hER ␣- and ␤-dependent
induction [4].
2.
Experimental
2.1.
General
Daidzein (1) was prepared according to our published pro-
cedure [6]. NMR spectra were measured on a Varian Inova
∗
Corresponding author. Fax: +358 9 19150357.
E-mail address: kristiina.wahala@helsinki.fi (K. Wa¨ha¨la¨).
0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2007.07.001

852
steroids 7 2 ( 2 0 0 7 ) 851–854
Scheme 1 – Synthetic route: (a) (i) KOBu-t (3 equiv.), DMF, rt, 2 h and (ii) BnCl (1.1 equiv.), rt, 16 h (72%); (b) (i) Bu4NBr
(1.2 equiv.), K2CO3 (5 equiv.), CHCl3, H2O, rt, 10 min, (ii) acetobromo-␣-d-glucuronic acid, methyl ester (1 equiv.), rt, 2 d and (iii)
acetobromo-␣-d-glucuronic acid, methyl ester (1 equiv.), rt, 5 d (33%); (c) thioanisole (50 equiv.), TFA, +40 ◦C, 3 h (70%); (d) (i)
ClSO3H (10 equiv.), pyridine, rt, 14 h and (ii) 5% aq. NaHCO3, pH 8 (74%); (e) (i) ClSO3H (10 equiv.), pyridine, rt, 14 h and (ii)
0.5 M aq. Na2CO3, pH 10, rt, 24 h (41%).
500 spectrometer with TMS as an internal standard. Chemi-
cal shifts are in ı values (ppm) and coupling constants in Hz
(s, singlet; d, doublet; t, triplet; dd, double doublet; m, mul-
tiplet). Mass spectra were obtained with a JEOL JMS SX102
mass spectrometer at 70 eV or Mariner ESI-TOF on negative
or positive mode with TuneMix (Agilent Technologies) as the
internal standard. Melting points were determined in open
capillary tubes with a GWB melting point apparatus, and are
uncorrected. TLC was conducted on Merck silica gel 60 F254
plates or Merck RP-18 F254s plates. MPLC was performed with
Buchi Sepacore using 40 mm × 150 mm silica gel 60 packed
columns.
HRMS (EI) calculated for: C22H16O4 344.1049; found
344.1044.
2.3.
4ꢀ-O-Benzyldaidzein 7-O-triacetylglucuronide
methyl ester (3)
(2) (150 mg, 0.44 mmol), Bu4NBr (169 mg, 0.52 mmol), K2CO3
(301 mg, 2.18 mmol), CHCl3 (13 ml) and H2O (13 ml) were
placed in round bottomed flask and the mixture was
stirred at room temperature. After obtaining a clear solu-
tion (10 min), acetobromo-␣-d-glucuronic acid methyl ester
(173 mg, 0.44 mmol) was added. After 2 days another equiv-
alent (173 mg, 0.44 mmol) of acetobromo-␣-d-glucuronic acid
methyl ester was added. The reaction mixture was stirred
for a total of 7 days. CHCl3 (50 ml) was added and the sepa-
rated organic phase was washed with 10% aq. AcOH (2× 15 ml),
water (15 ml), 0.1 M Na2S2O3 (15 ml), water (15 ml), sat. NaHCO3
(2× 15 ml) and water (2× 15 ml), and dried with MgSO4. The sol-
vent was removed in vacuo to give a solid (490 mg), which was
purified by MPLC using CH2Cl2:MeOH (95:5) as an eluent. This
gave a white solid (368 mg) which was recrystallised from EtOH
to give (3) (96 mg, 33%); m.p. 223–225 ◦C; 1H NMR (500 MHz,
DMSO-d6): ı 2.01, 2.03 (2s, 9H, 3× COCH3), 3.64 (s, 3H, OCH3),
4.81 (d, 1H, J = 10.0 Hz, H-5ꢀꢀ), 5.12 (t, 1H, J = 10.0 Hz, H-4ꢀꢀ), 5.16
(s, 2H, H-7ꢀꢀꢀ), 5.19 (m, 1H, H-2ꢀꢀ), 5.48 (t, 1H, J = 10.0 Hz, H-3ꢀꢀ),
5.91 (d, 1H, J = 8.0 Hz, H-1ꢀꢀ), 7.08 (d, 2H, J = 9.0 Hz, H-3ꢀ, H-5ꢀ),
7.15 (dd, 1H, J = 9.0 Hz, 2.5 Hz, H-6), 7.29 (d, 1H, J = 2.0 Hz, H-8),
7.34 (d, 1H, J = 7.0 Hz, H-4ꢀꢀꢀ), 7.40 (t, 2H, J = 7.0 Hz, H-3ꢀꢀꢀ, H-5ꢀꢀꢀ),
7.47 (d, 2H, J = 7.5 Hz, H-2ꢀꢀꢀ, H-6ꢀꢀꢀ), 7.53 (d, 2H, J = ca. 9.0 Hz, H-
2ꢀ, H-6ꢀ), 8.10 (d, 1H, J = 9.0 Hz, H-5), 8.46 (s, 1H, H-2); 13C NMR
(125 MHz, DMSO-d6): 20.2, 20.2, 20.3 (3× COCH3), 52.6 (OCH3),
68.7 (C-4ꢀꢀ), 69.2 (C-7ꢀꢀꢀ), 70.3 (C-2ꢀꢀ), 71.0 (C-3ꢀꢀ), 71.1 (C-5ꢀꢀ), 96.3
(C-1ꢀꢀ), 103.7 (C-8), 114.5 (C-3ꢀ, C-5ꢀ), 115.2 (C-6), 119.3 (C-4a),
123.4 (C-1ꢀ), 124.1 (C-3), 127.5 (C-5), 127.6 (C-2ꢀꢀꢀ, C-6ꢀꢀꢀ), 127.8
(C-4ꢀꢀꢀ), 128.4 (C-3ꢀꢀꢀ, C-5ꢀꢀꢀ), 130.1 (C-2ꢀ, C-6ꢀ), 137.0 (C-1ꢀꢀꢀ), 153.8
2.2.
4ꢀ-O-Benzyldaidzein (2)
A suspension of daidzein (300 mg, 1.18 mmol) and dry potas-
sium tert-butoxide (397 mg, 3.5 mmol) in freshly distilled DMF
(40 ml) was stirred at room temperature under Ar for 2 h.
Freshly distilled BnCl (0.15 ml, 1.3 mmol) was added and the
reaction mixture was stirred overnight and then poured into
water and neutralised with 10% HCl. The precipitated prod-
uct was filtered off, washed with water, dried in vacuo and
purified with MPLC using CH2Cl2:EtOAc (8:2) as an eluent.
Recrystallisation from EtOH gave white crystals (290 mg, 72%);
m.p. 243–245 ◦C; 1H NMR (500 MHz, DMSO-d6): ı 5.16 (s, 2H, H-
7ꢀꢀꢀ), 6.87 (d, 1H, J = 2.5 Hz, H-8), 6.94 (dd, 1H, J = 10.0, 2.5 Hz, H-6),
7.06 (d, 2H, J = 7.0 Hz, H-3ꢀ, H-5ꢀ), 7.34 (d, 1H, J = 7.0 Hz, H-4ꢀꢀꢀ),
7.40 (t, 2H, J = 7.0 Hz, H-3ꢀꢀꢀ, H-5ꢀꢀꢀ), 7.47 (d, 2H, J = 7.0 Hz, H-2ꢀꢀꢀ,
H-6ꢀꢀꢀ), 7.52 (d, 2H, J = 7.0 Hz, H-2ꢀ, H-6ꢀ), 7.97 (d, 1H, J = 9.0 Hz,
H-5), 8.34 (s, 1H, H-2), 10.79 (s, 1H, –OH); 13C NMR (125 MHz,
DMSO-d6): 69.2 (C-7ꢀꢀꢀ), 102.1 (C-8), 114.5 (C-3ꢀ, C-5ꢀ), 115.1 (C-6),
116.6 (C-4a), 123.1 (C-1ꢀ), 124.6 (C-3), 127.3 (C-5), 127.6 (C-2ꢀꢀꢀ,
C-6ꢀꢀꢀ), 127.8 (C-4ꢀꢀꢀ), 128.4 (C-3ꢀꢀꢀ, C-5ꢀꢀꢀ), 130.0 (C-2ꢀ, C-6ꢀ), 137.1
(C-1ꢀꢀꢀ), 153.1 (C-2), 157.4 (C-8a), 158.0 (C-4ꢀ), 162.5 (C-7), 174.5
(C-4).

steroids 7 2 ( 2 0 0 7 ) 851–854
853
(C-2), 156.8 (C-8a), 158.1 (C-4ꢀ), 159.9 (C-7), 166.9 (C-6ꢀꢀ), 169.0
(C O), 169.3 (C O), 169.5 (C O), 174.5 (C-4).
HRMS (M + H)+ calculated for: C35H33O13 661.19157; found
661.19348.
freshly distilled pyridine (4 ml) at −16 ◦C under argon atmo-
sphere. The reaction was allowed to warm by stirring at room
temperature. After 14 h at room temperature 5% aq. NaHCO3
was added until the pH was 8. After evaporation of the sol-
vents, the crude product was purified on Sephadex LH-20
eluting with water. White solid (34 mg, 74%). 1H NMR (500 MHz,
DMSO-d6): ı 2.01, 2.03, 2.03 (3s, 9H, COCH3), 3.65 (s, 3H, OCH3),
4.81 (d, 1H, J = 10.5 Hz, H-5ꢀꢀ), 5.12 (t, 1H, J = 9.0 Hz, H-4ꢀꢀ), 5.17
(m, 1H, H-2ꢀꢀ), 5.47 (t, 1H, J = 10.0 Hz, H-3ꢀꢀ), 5.92 (d, 1H, J = 8.0 Hz,
H-1ꢀꢀ), 7.14 (dd, 1H, J = 9.0 Hz, 2.5 Hz, H-6), 7.23 (d, 2H, J = 9.0 Hz,
H-3ꢀ, H-5ꢀ), 7.29 (d, 1H, J = 2.5 Hz, H-8), 7.49 (d, 2H, J = ca. 9.0 Hz,
H-2ꢀ, H-6ꢀ), 8.11 (d, 1H, J = 9.0 Hz, H-5), 8.48 (s, 1H, H-2); 13C NMR
(125 MHz, DMSO-d6): 20.2, 20.2, 20.3 (3× COCH3), 52.6 (OCH3),
68.8 (C-4ꢀꢀ), 70.3 (C-2ꢀꢀ), 71.0 (C-3ꢀꢀ), 71.1 (C-5ꢀꢀ), 96.3 (C-1ꢀꢀ), 103.7
(C-8), 115.2 (C-6), 119.4 (C-4a), 120.0 (C-3ꢀ, C-5ꢀ), 123.6 (C-3), 126.2
(C-1ꢀ), 127.6 (C-5), 129.3 (C-2ꢀ, C-6ꢀ), 153.5 (C-2), 154.0 (C-4ꢀ), 156.9
(C-8a), 159.9 (C-7), 166.9 (C-6ꢀꢀ), 169.0 (C O), 169.3 (C O), 169.5
2.4.
Daidzein 7-O-triacetylglucuronide methyl ester (4)
(3) (85 mg, 0.13 mmol) was dissolved in thioanisole (0.76 ml,
6.43 mmol) and TFA (4 ml). The reaction mixture was stirred
at 40 ◦C for 3 h. After completion of the reaction, TFA was
evaporated in vacuo and the product was precipitated from
Et2O/hexane. The solvents were decanted and the residue
washed with Et2O. Recrystallization from EtOH/H2O gave a
white solid (51 mg, 70%); m.p. 218–220 ◦C; 1H NMR (500 MHz,
DMSO-d6): ı 2.01, 2.03 (2s, 9H, 3× COCH3), 3.64 (s, 3H, OCH3),
4.80 (d, 1H, J = 10.5 Hz, H-5ꢀꢀ), 5.12 (t, 1H, J = 9.5 Hz, H-4ꢀꢀ), 5.18 (m,
1H, H-2ꢀꢀ), 5.47 (t, 1H, J = 10.0 Hz, H-3ꢀꢀ), 5.91 (d, 1H, J = 8.0 Hz, H-
1ꢀꢀ), 6.82 (d, 2H, J = 8.5 Hz, H-3ꢀ, H-5ꢀ), 7.13 (dd, 1H, J = 9.0, 2.5 Hz,
H-6), 7.28 (d, 1H, J = 2.5 Hz, H-8), 7.41 (d, 2H, J = 8.5 Hz, H-2ꢀ, H-6ꢀ),
8.09 (d, 1H, J = 8.5 Hz, H-5), 8.41 (s, 1H, H-2), 9.55 (s, 1H, 4ꢀ-OH);
13C NMR (125 MHz, DMSO-d6): 20.2, 20.2, 20.3 (3× COCH3), 52.6
(OCH3), 68.8 (C-4ꢀꢀ), 70.2 (C-2ꢀꢀ), 71.0 (C-3ꢀꢀ), 71.1 (C-5ꢀꢀ), 96.3 (C-
1ꢀꢀ), 103.7 (C-8), 115.0 (C-3ꢀ, C-5ꢀ), 115.1 (C-6), 119.4 (C-4a), 122.1
(C-1ꢀ), 123.8 (C-3), 127.6 (C-5), 130.0 (C-2ꢀ, C-6ꢀ), 153.5 (C-2), 156.8
(C-8a), 157.3 (C-4ꢀ), 159.8 (C-7), 166.9 (C-6ꢀꢀ), 169.0 (C O), 169.3
(C O), 169.5 (C O), 174.6 (C-4).
(C O), 174.5 (C-4). HRMS (M–Na)− calculated for: C28H25O16
S
649.08578; found 649.08688.
3.
Results and discussion
Glucuronation at position 7-OH required prior selective pro-
tection at the 4ꢀ-hydroxyl. We have shown that selective 4ꢀ-O-
or 7-O-alkylation of daidzein is possible by careful adjust-
ment of the reaction conditions [7]. Thus, 4ꢀ-O-benzyldaidzein
(2) was readily prepared by reaction of daidzein dipotas-
sium salt with slightly more than 1 equiv. of benzyl chloride.
In our work, glucuronidation at 7-OH using acetobromoglu-
curonic acid methyl ester was performed by a phase transfer
procedure described earlier for phenols [8,9]. An alterna-
tive technique employs the Koenigs–Knorr reaction used for
the glucuronidation of daidzein [10]. Trichloroacetimidates
have also been used for glucuronidation purposes but mod-
est yields or a complete failure have been reported in some
cases [8,11]. Attempted debenzylation of 4ꢀ-O-benzyldaidzein
7-O-triacetylglucuronide methyl ester (3) with Pd/C and H2
resulted in partial reduction of the ring C double bond.
However, debenzylation with thioanisole/TFA [12] proceeded
smoothly, with retention of the acetyl groups, the methyl ester
and the ring C enone functionality.
Sulfation of the resulting daidzein 7-O-triacetylglucuronide
methyl ester (4) was successfully performed by our earlier pub-
lished procedure [13]. In situ hydrolysis of the acetyl groups and
methyl ester gave daidzein 7-O-␤-d-glucuronide-4ꢀ-O-sulfate
sodium salt (5) in 41% yield. If required, the glucuronide triac-
etate methyl ester sulfate sodium salt (6) may also be prepared
by omitting the hydrolysis step.
Products were analyzed by 1H and 13C NMR. 2D (HMQC,
HMBC) measurements were run to confirm the identities of
1H and 13C signals in NMR spectra. The presence of the sul-
fate group is clearly seen by the shifts of the protons 3ꢀ and
5ꢀ and carbons 3ꢀ, 4ꢀ and 5ꢀ as reported earlier [2,3,13]. The
NMR spectra of (5) were virtually identical with the previously
published 1H and 13C NMR spectra of natural daidzein 7-O-␤-
d-glucuronide-4ꢀ-O-sulfate [2,3].
HRMS (M – H)− calculated for: C28H25O13 569.12897; found
569.13112.
2.5.
Daidzein 7-O-ˇ-d-glucuronide-4ꢀ-O-sulfate
disodium salt (5)
Chlorosulfonic acid (58 ␮l, 0.88 mmol) was added dropwise
(Caution) into a stirred solution of (4) (50 mg, 0.088 mmol) in
freshly distilled pyridine (5 ml) at −16 ◦C under Ar. The reac-
tion was allowed to warm by stirring at room temperature.
After 14 h at room temperature, 0.5 M aq. Na2CO3 (20 ml)
was added until the pH was 10. The reaction mixture was
stirred 24 h at room temperature. After completion of the
reaction solvents were removed in vacuo and the remaining
solid was purified with Sephadex LH-20 eluting with water.
Further purification was performed with preparative RP-18
PLC plate using MeOH:H2O (50:50) as an eluent. Recystallisa-
tion from H2O/EtOH gave a white solid (20 mg, 41%). 1H NMR
(500 MHz, DMSO-d6): ı 3.32–3.40 (m, 3H, H-2ꢀꢀ, H-3ꢀꢀ, H-4ꢀꢀ), 3.98
(d, 1H, J = 9.0 Hz, H-5ꢀꢀ), 5.28 (d, 1H, J = 7.0 Hz, H-1ꢀꢀ), 7.16 (dd, 1H,
J = 9.0 Hz, 2.5 Hz, H-6), 7.22 (d, 2H, J = 9.0 Hz, H-3ꢀ, H-5ꢀ), 7.28 (d,
1H, J = 2.5 Hz, H-8), 7.49 (d, 2H, J = 9.0 Hz, H-2ꢀ, H-6ꢀ), 8.08 (d, 1H,
J = 9.0 Hz, H-5), 8.45 (s, 1H, H-2); 13C NMR (125 MHz, DMSO-d6):
71.4 (C-4ꢀꢀ), 72.8 (C-2ꢀꢀ), 75.0 (C-5ꢀꢀ), 75.8 (C-3ꢀꢀ), 99.5 (C-1ꢀꢀ), 103.4
(C-8), 115.5 (C-6), 118.6 (C-4a), 120.0 (C-3ꢀ, C-5ꢀ), 123.5 (C-3), 126.4
(C-1ꢀ), 127.2 (C-5), 129.4 (C-2ꢀ, C-6ꢀ), 153.4 (C-2), 153.9 (C-4ꢀ), 157.0
(C-8a), 161.1 (C-7), 170.4 (C-6ꢀꢀ), 174.5 (C-4). HRMS (M–2Na)2−
calculated for: C21H16O13S 254.01504; found 254.01576.
2.6.
Daidzein 7-O-triacetylglucuronide-4ꢀ-O-sulfate
methyl ester sodium salt (6)
This compound will be useful as an internal standard in
LC/MS analysis of the biological samples, as well as in the
studies on the metabolism of isoflavones in man.
Chlorosulfonic acid (46 ␮l, 0.69 mmol) was added dropwise
(Caution) into a stirred solution of (4) (39 mg, 0.069 mmol) in

854
steroids 7 2 ( 2 0 0 7 ) 851–854
with [13C3]isoflavone internal standards. Anal Biochem
2002;309:158–72.
Acknowledgements
[6] Wa¨ha¨la¨ K, Hase T. Expedient synthesis of
polyhydroxyisoflavones. J Chem Soc, Perkin Trans 1
1991:3005–8.
[7] Al-Maharik NI, Kaltia SA, Wa¨ha¨la¨ K. Regioselective
mono-O-carboxymethylation of polyhydroxyisoflavones.
Molecular 1999;3:20–4. Online.
[8] Hongu M, Saito K, Tsujihara K. Solid–liquid phase transfer
catalyzed novel glycosylation reaction of phenols. Synth
Commun 1999;29:2775–81.
We thank Helsinki University Research Funds and Bioorganic
and Medicinal Chemistry Graduate School for financial sup-
port.
r e f e r e n c e s
[9] Li M, Han X, Yu B. Synthesis of quercetin
3-O-(2ꢀꢀ-galloyl)-␣-l-arabinopyranoside. Tetrahedron Lett
2002;43:9467–70.
[10] Needs PW, Williamson G. Syntheses of daidzein-7-yl
␤-d-glucopyranosiduronic acid and daidzein-4ꢀ-7-yl
di-␤-d-glucopyranosiduronic acid. Carbohyd Res
2001;330:511–5.
[11] Needs P, Kroon P. Convenient synthesis of metabolically
important quercetin glucuronides and sulfates. Tetrahedron
2006;62:6862–8.
[12] Kiso Y, Ukawa K, Nakamura S, Ito K, Akita T. Efficient
removal of protecting groups by a “push-pull” mechanism II.
Deprotection of O-benzyltyrosine with
[1] Adlercreutz H, van der Wildt J, Kinzel J, Attalla H, Wa¨ha¨la¨ K,
Ma¨kela¨ T, et al. Lignan and isoflavonoid conjugates in
human urine. J Steroid Mol Biol 1995;52:97–103.
[2] Yasuda T, Kano Y, Saito K, Ohsawa K. Urinary and biliary
metabolites of daidzin and daidzein in rats. Biol Pharm Bull
1994;17:1369–74.
[3] Tsuchihashi R, Okawa M, Nohara T, Okabe H, Kinjo J. Soy
isoflavone metabolites isolated from human urine. Nat Med
2004;58:71–5.
[4] Kinjo J, Tsuchihashi R, Morito K, Hirose T, Aomori T, Nagao T,
et al. Interactions of phytoestrogens with estrogen receptors
␣ and ␤ (III). Estrogenic activities of soy isoflavone aglycones
and their metabolites isolated from human urine. Biol
Pharm Bull 2004;27:185–8.
thioanisole–trifluoroacetic acid system without O-to-C
rearrangements. Chem Pharm Bull 1980;28:673–6.
[13] Soidinsalo O, Wa¨ha¨la¨ K. Synthesis of phytoestrogenic
isoflavonoid disulfates. Steroids 2004;69:613–6.
[5] Clarke DB, Lloyd AS, Botting NP, Oldfield MF, Needs PW,
Wiseman H. Measurement of intact sulfate and glucuronide
phytoestrogen conjugates in human urine using isotope
dilution liquid chromatography–tandem mass spectrometry