Development of a simple method for the determination of genistein, daidzein, biochanin A, and formononetin (Biochanin B) in human urine

Article abstract of DOI:10.1021/jf990159z

A simple method was developed for the determination of free and/or total isoflavones daidzein, genistein, and their respective 4'-methoxy derivatives biochanin A and formononetin (biochanin B) at low levels in human urine. A solid-phase extraction on octadecyl silica (C₁₈) columns was used for the isolation of the phytoestrogens from the matrix. An extraction on a ChemElut 1010 column connected on-line to a Florisil cartridge by a Teflon stopcock was used for effective eluate purification. A mixture of dichloromethane and ethyl acetate was used for elution of the isoflavones from the columns in tandem. The isoflavones were determined as trimethylsilyl (TMS) ethers using GC/MSSIM after separation on an HP-5MS fused silica column. TMS ethers were obtained by using BSTFA containing 1% of TMCS. For the determination of free isoflavones 6-hydroxyflavone was used as internal standard, whereas robigenin was used in the case of total isoflavone determination. Recoveries for free isoflavones under study varied from 63.5 to 89.6% at the 25 ng mL^-1 level and from 63.5 to 89.2% at the 5 ng mL^-1 level in urine. Analytical curves were linear between 5 and 25 ng mL^-1. Detection limits varied from 1 ng mL^-1 for formononetin to 2.3 ng mL^-1 for daidzein. Recoveries for total isoflavone determination after enzymatic hydrolysis with glucuronidase from Helix pomatia ranged from 56.5 to 77.1% at the 25 ng mL^-1 level.

Full text of DOI:10.1021/jf990159z

J. Agric. Food Chem. 1999, 47, 3489−3494
3489
ARTICLES
Develop m en t of a Sim p le Meth od for th e Deter m in a tion of
Gen istein , Da id zein , Bioch a n in A, a n d F or m on on etin (Bioch a n in B)
in Hu m a n Ur in e
J ozef Tekeł,† Els Daeseleire,‡ An Heeremans,‡ and Carlos van Peteghem*,‡
Faculty of Pharmacy, Comenius University, Odbojarov 10, SK 832 32 Bratislava, Slovak Republic, and
Laboratory of Food Analysis, University of Gent, Harelbekestraat 72, B-9000 Gent, Belgium
A simple method was developed for the determination of free and/or total isoflavones daidzein,
genistein, and their respective 4′-methoxy derivatives biochanin A and formononetin (biochanin B)
at low levels in human urine. A solid-phase extraction on octadecyl silica (C18) columns was used
for the isolation of the phytoestrogens from the matrix. An extraction on a ChemElut 1010 column
connected on-line to a Florisil cartridge by a Teflon stopcock was used for effective eluate purification.
A mixture of dichloromethane and ethyl acetate was used for elution of the isoflavones from the
columns in tandem. The isoflavones were determined as trimethylsilyl (TMS) ethers using GC/MS-
SIM after separation on an HP-5MS fused silica column. TMS ethers were obtained by using BSTFA
containing 1% of TMCS. For the determination of free isoflavones 6-hydroxyflavone was used as
internal standard, whereas robigenin was used in the case of total isoflavone determination.
Recoveries for free isoflavones under study varied from 63.5 to 89.6% at the 25 ng mL-1 level and
from 63.5 to 89.2% at the 5 ng mL-1 level in urine. Analytical curves were linear between 5 and 25
ng mL-1. Detection limits varied from 1 ng mL-1 for formononetin to 2.3 ng mL-1 for daidzein.
Recoveries for total isoflavone determination after enzymatic hydrolysis with glucuronidase from
Helix pomatia ranged from 56.5 to 77.1% at the 25 ng mL-1 level.
Keyw or d s: Urine; phytoestrogens; liquid/ liquid extraction column; gas chromatography/ mass
spectrometry
INTRODUCTION
Diphenolic phytoestrogens are plant substances that
show some structural similarity to estradiol-17â and to
the estradiol receptor producing estrogenic effects.
There are three main groups of nonsteroidal dietary
phytoestrogens, namely, the flavones, the isoflavones,
and the coumestans. The main known phytoestrogens
are the isoflavones genistein, daidzein, biochanin A, and
formononetin, the structures of which are shown in
Figure 1. Certain phytochemicals in fruits, vegetables,
and grains have possible cancer-preventive properties
that may inhibit tumor initiation, prevent oxidative
damage, or affect steroid hormones or prostaglandin
metabolism to block tumor promotion (Caragay, 1992).
Most significant sources of isoflavone and coumestan
phytoestrogens include soybean, soy flour, soy flakes,
isolated soy protein, traditional soy food such as tofu
and soy drinks, second-generation soy foods, sprouts
(Reinli and Block, 1996), and other leguminous plants
(Kaufman et al., 1997). The three isoflavones (genistein,
daidzein, glycitein) in soybean and soybean products
F igu r e 1. Chemical structures of daidzein, genistein, for-
occur in four possible forms: the free phenolic forms,
mononetin, biochanin A, 6-hydroxyflavone, and robigenin.
* Author to whom correspondence should be addressed
glucosides, malonyl glucosides, and acetyl glucosides
(telephone 32 9 264 81 34; fax 32 9 264 81 99; e-mail
(Wang and Murphy, 1994).
carlos.vanpeteghem@rug.ac.be).
† Comenius University.
‡ University of Gent.
Epidemiological data (Adlercreutz et al., 1991a,b,
1993a,b, 1995a,b; Franke and Custer, 1996; Lu et al.,
10.1021/jf990159z CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/31/1999

3490 J. Agric. Food Chem., Vol. 47, No. 9, 1999
Tekeł et al.
1995a,b) suggest that dietary legumes may be protective
against breast, prostate, and colon cancer development.
Populations with high isoflavone exposure through soy
consumption have low cancer rates. It is well-known
that the traditional J apanese diet is associated with a
very low breast cancer occurrence. The low mortality
from breast (Adlercreutz et al., 1991a) and prostate
(Adlercreutz et al., 1993b) cancer of J apanese women
and men may be due to high intake of soybean products.
Isoflavonoid compounds, especially genistein and daid-
zein, have been implicated in cancer prevention (Adler-
creutz, 1995; Messina and Barnes, 1991; Messina et al.,
1994). Genistein is a specific inhibitor of tyrosine protein
kinase, DNA topoisomerase II, and protein histidin
kinase. All of the main isoflavonoids (genistein, daid-
zein), their 4′-methyl ether precursors (biochanin A,
formononetin), and main isoflavonoid metabolites (equol,
O-desmethylangolensin) detected in human and animal
urine bind to the estrogen receptor (Adlercreutz et al.,
1995b). Still other metabolites have been detected
(J oannou et al., 1995) in human urine after soy intake.
Urinary lignan and isoflavonoid excretion changed in
response to alterations in vegetable, fruit, and legume
intake under controlled dietary conditions (Hutchins et
al., 1995; Seow et al., 1998).
retains water from aqueous samples. The high surface
area of cartridge filling allows efficient emulsion-free
interaction between the sample and the organic extrac-
tion solvent. Extraction did not require vacuum but was
carried out using gravity only. Ethyl acetate was used
for the elution of the isoflavones from the column. The
chromatographic methods require an extensive cleanup
procedure prior to analysis. Radioimmunoassay (RIA)
methods are a simple alternative and are used for
screening purposes. A radioimmunoassay for the analy-
sis of formononetin in blood plasma and rumen fluid of
wethers fed red clover (Wang et al., 1994) utilized
antibodies raised against a formononetin-7-O-(carboxy-
methyl) ether hapten (bovine serum albumin, BSA). The
3
tracer used was a H-labeled derivative of formononetin.
The radioimmunoassay for identification of daidzein and
genistein in human biological fluids (serum, urine) was
established on antibodies against daidzein-4′-O-CME-
BSA and genistein-4′-O-CME-BSA. Both methods
(Lapcik et al., 1997; Hampl et al., 1998) used 125I-labeled
tracer. RIA was used also for identification of the
isoflavonoids in beer (Lapcik et al., 1998).
In this paper a fast, simple, and effective isolation and
cleanup procedure for the determination of low isofla-
vone levels in human urine by GC/MS is reported.
Free urinary isoflavones were often analyzed by gas
chromatography/mass spectrometry in single ion moni-
toring mode (GC/MS-SIM) after derivatization as tri-
methylsilyl (TMS) ethers. Different silanization agents
were used, most frequently N,O-bis(trimethylsilyl)tri-
fluoroacetamide (BSTFA). Enzymatic hydrolysis of the
urinary isoflavone glucuronides was carried out with
â-glucuronidase/arylsulfatase from Helix pomatia. This
procedure was used by different authors for the analysis
of isoflavones and/or their metabolites in biological
samples. GC/MS was used for the analysis of isoflavones
in human urine (Adlercreutz et al., 1991a,b; J oannou
et al., 1995; Kelly et al., 1993; Lu et al., 1995a,b), human
milk (Franke and Custer, 1996), human plasma (Adler-
creutz et al., 1993a,b, Morton et al., 1994), human feces
(Adlercreutz et al., 1993b), beer (Rosenblum et al., 1992),
and food samples (Mazur et al., 1996). An anhydrous
methanolic hydrogen chloride was used for the hydroly-
sis (methanolysis) of steroid conjugates from male horse
urine sample and was tested as an alternative (Tang
and Crone, 1989).
High-performance liquid chromatography (HPLC)
with a reversed phase (C18) column can be directly used
for the analysis of these compounds in free and conju-
gated form in samples without a derivatization step.
This method has been used for the analysis of the
isoflavone content in human urine (Franke et al., 1998),
human milk (Franke and Custer, 1996), soybean foods
(Wang and Murphy, 1994), legumes (Franke et al.,
1994), and soybean infant formula (Murphy et al., 1997).
EXPERIMENTAL PROCEDURES
Ch em ica ls a n d Sta n d a r d s. Methanol and water of HPLC
grade were obtained from BDH (Poole, Dorset, U.K.). Dichlo-
romethane (stabilized with 20 ppm amylene) p.a. and ethyl
acetate were obtained from Acros Organics (Geel, Belgium).
The standards of daidzein (7,4′-dihydroxyisoflavone), genistein
(5,7,4′-trihydroxyisoflavone), and biochanin A (5,7-dihydroxy-
4′-methoxyisoflavone) were obtained from ICN Biomedical
(Aurora, OH). The standard of formononetin (7-hydroxy-4′-
methoxyisoflavone) was obtained from Extrasynthese (Genay,
France). The standard of 6-hydroxyflavone (external standard
1, ES-1) was purchased from Sigma Chemical Co. (St. Louis,
MO), whereas the standard of robigenin (3,5,7,4′-tetrahydroxy-
flavone; kaempferol) (external standard 2, ES-2) was from
Fluka Chemie AG (Buchs, Switzerland). BSTFA containing 1%
of trimethylchlorosilane (TMCS) was obtained from Alltech
Associates, Inc. (Deerfield, IL). Helix pomatia juice [(â-glucu-
ronidase (5.5 units/mL) and arylsulfatase (2.6 units/mL)] were
obtained from Boehringer (Mannheim, Germany).
Special indicator paper pH 4.0-7.0, acetic acid, and sodium
acetate trihydrate were from Merck (Darmstadt, Germany).
Octadecyl (C18) disposable extraction columns (500 mg) were
from J . T. Baker (Phillipsburg, NJ ). ChemElut columns CE-
1010 (part 1219-8007) for liquid/liquid extraction were from
Varian Sample Preparation Products (Harbor City, CA), and
the Florisil Sep-Pak cartridges (part 51960) were from Milli-
pore Corp. (Milford, MA). The extraction system Baker-10 for
SPE was from J . T. Baker Chemical Co.
The derivatization vials were silanized with a solution of
5% dimethyldichlorosilane (Merck) in toluene (Merck) before
use.
Urine samples were supplied by people working in the
laboratory. Rubber gloves were worn when handling the urine
samples.
For the determination of free isoflavones 6-hydroxyflavone
(ES-1) was used as external standard, whereas robigenin (ES-
2) was used for the total isoflavone determination.
Sa m p le Clea n u p P r oced u r e. Twenty milliliters of urine
sample was centrifuged for 10 min at 3600 rpm. After
centrifugation, 3 mL of acetate buffer (3 mol L-1, 9.9 g of acetic
acid and 18.4 g of sodium acetate trihydrate in 100 mL of
water) was added to the decanted supernatant of the urine.
The content of the glass tube was premixed and pH was
controlled using an indicator strip and adjusted if necessary
with acetic acid to a value between 4.7 and 5.2. The sample
Investigators have used ion-exchange chromatogra-
phy (Adlercreutz et al., 1991a,b, 1993a,b, 1995a; Hutch-
ins et al., 1995; J oannou et al., 1995; Kelly et al., 1993;
Mazur et al., 1996; Morton et al., 1994) or solid-phase
extraction (SPE) on octadecyl silica (C18) phase (Franke
and Custer, 1996; Franke et al., 1998) prior to GC/MS
or liquid chromatography diode array detection (LC-
DAD) analysis of isoflavones in urine or plasma. The
procedure developed by Lu et al. (1995a,b) for the
extraction of daidzein and genistein from urine samples
used liquid/liquid extraction columns. These columns
were filled with diatomaceous material that absorbs and

Detection of Phytoestrogens in Human Urine
J. Agric. Food Chem., Vol. 47, No. 9, 1999 3491
Ta ble 1. GC Reten tion Tim es of Sta n d a r d s of Isofla von es
a n d Selected Ion s for th e Selected Ion Mon itor in g
temperature being held for 20 min. The temperature of the
ion source was 200 °C, and the electron voltage was 70 eV.
compound
tR (min)
selection of ions (m/z)
RESULTS AND DISCUSSION
6-hydroxyflavone (ES-1)
formononetin
biochanin A
daidzein
genistein
12:41
15:01
15:38
16:10
16:31
18:42
310, 295, 236, 165
340, 325, 269, 226
413, 370, 341, 269
398, 383, 355, 253
471, 399, 355, 327
559, 487, 458, 415
A new isolation and cleaning procedure based on SPE
(C18) for the isolation of isoflavones from urine samples
was developed. The elution of the compounds under
study from the C18 phase is sufficiently effective with
pure methanol. The columns in tandem (ChemElut 1010
and Florisil cartridge) were efficient for a rapid purifica-
tion of the urine matrix. An advantage of the new
purification procedure was an easy regulation of the flow
of the elution mixture across the Florisil cartridge by a
Teflon stopcock. Other authors (Lu et al., 1995a,b) used
ethyl acetate for the elution of isoflavones from the
ChemElut column. Our experiments, however, showed
that a mixture of dichloromethane and ethyl acetate (1:
1, v/v) used on the combined columns gave extracts that
were significantly cleaner (results not shown). The exact
time between application of sample onto the ChemElut
1010 column and elution thereof was very crucial for
the reproducibility of the isolation process. An optimal
waiting time of 10 min is recommended. Longer (at most
15 min) waiting times had no further positive influence
on the elution. Centrifugation of the urine samples
before application on the SPE columns is highly recom-
mended to avoid obstruction.
The GC temperature program was optimized so that
separation between the compunds of interest was
achieved. The retention times for genistein, daidzein,
biochanin A, formononetin (biochanin B), and external
standards are summarized in Table 1. The external
standards were chosen according to their retention time
and the specificity of the mass spectrum of the TMS
derivative. A derivatization temperature of 60 °C and
a reaction time of 60 min were used for the TMS ether
preparation of all isoflavones and external standards.
Derivatization with BSTFA containing 1% of TMCS was
fast, simple, and reproducible. As an example, the
spectra of daidzein and genistein are shown in Figure
2.
robigenin (ES-2)
was preconcentrated on a C18 column that had previously been
conditioned with 2 × 5 mL of methanol and 2 × 5 mL of water.
After application of the urine, the cartridge was washed with
2 × 5 mL of water and the free isoflavones were eluted with
2 × 2 mL of methanol into a glass tube. The eluate was dried
in a water bath at 40 °C under a stream of nitrogen. The
residues were dissolved in 0.2 mL of methanol, and 5 mL of
water was added followed by vortexing.
This mixture was cleaned on a ChemElut 1010 column on-
line connected with a Teflon stopcock to a florisil cartridge.
The sample was applied onto the extraction column, and after
10 min, the isoflavones were eluted from the two superimposed
columns with 50 mL of a mixture of dichloromethane and ethyl
acetate (1:1, v/v) using gravity. The eluate was collected in a
rotavapor flask and evaporated under vacuum at 40 °C to
dryness. The residue was dissolved in 1 mL of methanol and
put into a silanized derivatization vial. The methanol was
evaporated to dryness at 60 °C under nitrogen.
En zym a tic Hyd r olysis of Isofla von e Glu cu r on id es.
An enzymatic hydrolysis of the isoflavone glucuronides was
used for the total isoflavone content determination in the urine
samples. The eluate from the C18 column (see Sample Cleanup
Procedure) was evaporated to dryness at 40 °C under a stream
of nitrogen. The residue was dissolved in 0.2 mL of methanol,
and 5 mL of a 0.2 mol L-1 acetate buffer (pH 4.6) and 50 µL of
H. pomatia digestive juice was added. The sample was
incubated for 90 min at 60 °C. After cooling to room temper-
ature, the hydrolyzed urine was cleaned up as described above
(Chem Elut plus Florisil). However, the elution was done with
70 mL of a mixture of dichloromethane and ethyl acetate (2:
1, v/v).
Der iva tiza tion . After the external standard was added,
100 µL of BSTFA containing 1% of TMCS was put into the
vial. After vortexing, the solution was heated for 60 min at 60
°C. After cooling, 2 µL of this solution was injected into the
GC/MS instrument.
Recovery studies were performed on urine samples
spiked at the 5 and 25 ng mL-1 level (n ) 5 for each
concentration). External standard was added just before
derivatization and the ratios of the area of the sum of
the selected ions of the compound of interest to the area
of the sum of the selected ions of the external standard
were calculated. These values were compared to the
ratios obtained for purified urine extracts that were
spiked at the 5 or 25 ng mL-1 level just before deriva-
tization. The obtained results varied from 63.5 to 89.2%
for the 5 ng mL-1 level and from 61.3 to 89.6% for the
25 ng mL-1 level. Recoveries for the determination of
Calibration curves were constructed in blank human urine
samples.
GC/MS An a lysis. The analyses were carried out on a GCQ
Finnigan ion trap detector (San J ose, CA), linked to an HP
5890 Series II gas chromatograph Hewlett-Packard (Palo Alto,
CA) equipped with an HP-5MS fused silica capillary column
(30 m × 0.25 mm i.d., film thickness ) 0.25 µm). Injections
were carried out in the splitless mode. The carrier gas was
high-purity helium (L’Air Liquide, Liege, Belgium) at a flow
rate of 1 mL min-1. The injector and interface temperatures
were at 280 and 275 °C, respectively. The oven temperature
was programmed from 70 °C (held for 1 min) to 200 °C at 40
°C min-1 and then to 280 °C at 10 °C min-1, the final
Ta ble 2. Recover y for F r ee (5 a n d 25 n g m L-1 Levels) a n d Tota l Isofla von es (25 n g m L-1 Level) in Ur in e a n d Detection
Lim it for F r ee Isofla von esa
recovery ( RSD (%), n ) 5
detection limit
free
total
25 ng mL-1
(ng mL-1
)
compound
5 ng mL-1
25 ng mL-1
free isoflavones
formononetin
biochanin A
daidzein
89.2 ( 30.0
63.5 ( 16.6
89.1 ( 17.2
78.6 ( 21.5
88.5 ( 9.5
61.3 ( 4.2
85.6 ( 5.6
89.6 ( 13.9
70.6 ( 11.2
56.5 ( 9.7
77.1 ( 6.2
75.9 ( 7.8
1.05
1.5
2.3
genistein
2.2
a
External standard was 6-hydroxyflavone for free isoflavone and robigenin for total isoflavone determination. Recoveries were based
on comparison of the ratios of the area of the sum of the selected ions of the isoflavone to the area of the sum of the selected ions of the
external standard obtained for a spiked sample to the ratio obtained for a blank matrix spiked just before derivatization.

3492 J. Agric. Food Chem., Vol. 47, No. 9, 1999
Tekeł et al.
F igu r e 2. Mass spectra of the TMS derivatives of daidzein and genistein.

Detection of Phytoestrogens in Human Urine
J. Agric. Food Chem., Vol. 47, No. 9, 1999 3493
F igu r e 3. Total ion chromatograms for daidzein, genistein, formononetin, and biochanin A spiked in human urine at a
concentration of 10 ng mL-1
.
total isoflavones (n ) 5) after enzymatic hydrolysis (H.
pomatia) varied from 56.5 to 75.9% for the 25 ng mL-1
level. All results are presented in Table 2. The total ion
chromatogram obtained in urine at a concentration of
10 ng mL-1 is shown in Figure 3.
For the determination of the total isoflavone content
a hydrolysis was required. Methanolysis (Tang and
Crone, 1989) of isoflavone glucuronides with anhydrous
hydrogen chloride did not provide extracts that were
pure enough for the GC/MS detection, even after
cleanup on the combined columns. Therefore, hydrolysis
was carried out enzymatically making use of H. poma-
tia.
free isoflavones. For the determination of total isofla-
vones problems with the stability of the standards
during the hydrolysis procedure at low concentration
were encountered. From the experiments it was con-
cluded that for daidzein and genistein a detection limit
of 10 ppb is real. For formononetin and biochanin A this
is not possible. Further studies concerning the hydroly-
sis conditions are necessary to solve this problem.
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Received for review February 16, 1999. Revised manuscript
received J une 23, 1999. Accepted J uly 8, 1999. This work was
supported by The National Fund for Scientific Research (Grant
G0019.97) and the Belgian Office for Scientific, Technical and
Cultural Affairs (DWTC).
Lu, L.-J . W.; Grady, J . J .; Marshall, M. V.; Ramanujam, S. V.
M. Anderson, K. E. Altered time course of urinary daidzein
J F990159Z