ANALYTICAL BIOCHEMISTRY 264, 1–7 (1998)
ARTICLE NO. AB982825
Extraction and Quantification of Daidzein
and Genistein in Food
Jason Liggins,1 Leslie J. C. Bluck, W. Andy Coward, and Sheila A. Bingham
Dunn Nutrition Centre, Medical Research Council, University of Cambridge,
Hills Road, Cambridge CB2 2DH, United Kingdom
Received April 2, 1998
A simple analytical method has been developed for
routine quantification of a broad range of concentra-
tions of the isoflavones daidzein and genistein in food.
The synthetic glucosides daidzin and genistin were
used as internal standards, combined with each food
prior to extraction. The recovery of the aglycones
daidzein and genistein from these internal standards
were used to ensure the completeness of the extrac-
tion and aid quantification of isoflavones from the
food. Hydrolytic enzymes from Aspergillus niger were
used, in aqueous buffer, to liberate daidzein and
genistein from their respective glycosides. The agly-
cone isoflavones were partitioned from the aqueous
buffer into ethyl acetate. After evaporation of the
ethyl acetate under nitrogen, the isoflavones were de-
rivatized with N-tert-(butyldimethylsilyl)-N-methyltri-
fluoroacetamide and quantified by comparison with
authentic synthetic standards using gas chromatog-
raphy–mass spectrometry in selected ion mode. The
isoflavone content of a stock soy flour was determined,
using 36 separate assays, to be 1.05 mg daidzein and
1.11 mg genistein per gram of freeze-dried food, and
the interassay coefficient of variation was 2.7 and 4.7,
respectively. © 1998 Academic Press
The impact of dietary phytoestrogens such as the
isoflavones daidzein and genistein upon the health of
both adults and developing infants is a growing con-
cern of the medical profession, food regulatory bodies,
and the food production industry. These naturally oc-
curring compounds have been reported to have wide-
ranging beneficial effects upon health, notably upon
breast, bowel, prostate, and other cancers, cardiovas-
cular disease, brain function, alcohol abuse, osteoporo-
sis, and menopausal symptoms (1– 4). On the other
1
To whom correspondence should be addressed. Fax: 144 1223
413763.
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Copyright © 1998 by Academic Press
All rights of reproduction in any form reserved.
hand, phytoestrogens could be potentially harmful to
the development of infants. Children fed exclusively on
soy infant formulas have particularly high intakes and
although the effect of this remains unknown, current
advice is that soy formulas should only be used follow-
ing medical advice (5–7).
The scientific literature concerning the health effects
of daidzein and genistein in humans has been gener-
ated from evidence using relatively rich sources of the
phytoestrogens, such as soy. Effects shown include an
increase in the length of the menstrual cycle by dietary
isoflavones, a reduction of menopausal symptomology,
and cardiovascular risk factors (1, 8, 9). However, foods
containing lower amounts of the isoflavones may have
more subtle effects upon long-term health, especially if
different sources of the compounds are eaten together,
generating a cumulative dose. The overall health im-
plications of dietary isoflavones cannot be completely
assessed until their distribution in foods has been de-
termined.
The majority of published protocols for the assay of
isoflavones were developed for their application to hu-
man biological fluids and soy-based foods (5, 10 –26).
The aim of the work contained in this article was to
develop a simple assay that could be used routinely to
determine a wide range of isoflavone concentrations,
much lower than those found in soy, in a variety of
foods.
Isoflavones occur naturally within plants mainly as
glycosides. They are possibly used by the plant to pro-
mote symbiosis with rhizobacteria in combination with
other roles, such as the defence of the plant against
pests and pathogens (22, 27, 28). Although only three
carbohydrate conjugates of daidzein and genistein
have so far been identified in soy (Fig. 1), it is likely
that in plants in general a larger variety of glycosides
exist (22, 26). Other possible conjugates include those
linked at one or more of the other positions on the
isoflavone (e.g., R1 or R2 in Fig. 1), or like the lignans,
1
2 LIGGINS ET AL.
FIG. 1. Molecular structures of isoflavones.
carbohydrate moieties may be composed of more than a
single monosaccharide (29). Upon ingestion, the carbo-
hydrate component of isoflavone conjugates is removed
by gut microflora, releasing the aglycone isoflavone for
either further modification by the bacteria or absorp-
tion.
Quantification of all the different possible glycosides
of daidzein and genistein is impractical for a large
number of samples, especially in the absence of refer-
ence standards. This problem can be overcome by hy-
drolytically removing the carbohydrate component, by
chemicals or enzymes, and quantifying just the agly-
cones. Polyphenols, however, are degraded upon alkali
hydrolysis and the stability of isoflavones upon acid
hydrolysis is questionable (11, 23–25, 30, 31). Enzyme
hydrolysis using glucosidases has been described in the
literature, but most investigations have been limited to
the analysis of human biological fluids. The isoflavones
are excreted in urine as b-glucuronide and sulfate con-
jugates, which may be hydrolyzed with a crude prepa-
ration of Helix pomatia, containing b-glucuronidase
and sulfatase enzymes (32). Although the preparation
of H. pomatia has also been successfully applied to the
hydrolysis of glucosidic isoflavones from foods, it is
unliklely to be the best choice of enzyme for this pur-
pose and in addition it has been reported to contain the
phytoestrogens of interest (5, 10 –14, 16, 18, 19, 23–26,
31, 33).
Fermented soy products are produced by culturing
roasted soy beans with a variety of fungal molds in-
cluding some from the Aspergillus family. The effect of
fermentation is to change the isoflavone glycosides to
the unconjugated aglycones (34). The enzymes present
in these fungal cultures could thus be more suited to
the hydrolysis of isoflavone glycosides in foods than
those from H. pomatia. The effectiveness of acid hydro-
lysis compared with enzyme hydrolysis with b-gluc-
uronidase, from H. pomatia, b-glucosidase from al-
monds, and the novel use of enzymes from Aspergillus
niger is reported in this article.
To ensure good quality assurance of a routine extrac-
tion procedure, internal standards must be added to
the foods at the beginning of the extraction, because
addition part way through the procedure ignores any
losses in prior steps. The internal standard must also
be extracted with the same relative efficiency as the
target compounds. Isotopically labeled isoflavone stan-
dards are difficult to obtain and they have not been
widely used (10). The majority of analytical methods
for quantification of isoflavones have been validated
with synthetic daidzein and genistein, but the routine
use of such aglycones does not give any indication of
hydrolytic efficiency or differences in the solubility of
the glycosides and aglycones in a solvent (11–14, 17–
19, 23, 25, 31, 35). The extraction procedure described
in this article uses the synthetic glycosides daidzin and
genistin as internal standards; both are added to each
food sample assayed to simulate losses of the native
glycosides prior to hydrolysis and also the unconju-
gated isoflavones following hydrolysis.
For quantification gas chromatography–mass spec-
trometry, in the selected ion mode (GC–MS-SIM),2 was
used because it offers high sensitivity combined with a
high degree of selectivity; it is capable of separating
isomers of the isoflavones, thus necessitating only ru-
dimentary extraction of the isoflavones prior to load-
ing. The combination of the isoflavone extraction pro-
cedure and quantification by GC–MS-SIM is a simple
sensitive assay suitable for routine analysis of foods for
daidzein and genistein.
METHODS
All enzymes, reagents and chemicals were pur-
chased from Sigma/Aldrich (Poole, Dorset, UK) unless
otherwise stated. The soy flour used in this investiga-
tion was full fat produced by first toasting and then
milling soy beans (Arjuna Healthfoods, Cambridge,
UK). In order to inhibit losses of target compounds by
adsorption to glassware, all glassware was silanized in
2
Abbreviations used: GC–MS-SIM, gas chromatography–mass
spectroscopy in the selected ion mode; TBDMS, N-(tert-
butyldimethylsilyl)-N-methyltrifluoroacetamide; TMS, N-trimethyl-
silylimidazole; SPE, solid-phase extraction.
 EXTRACTION AND QUANTIFICATION OF DAIDZEIN AND GENISTEIN
TABLE 1
Mass Ions Used for Identification of Isoflavones
Compound m/z No. 1 m/z No. 2
Daidzein 425 482
Genistein 555 249
6,7-Dihydroxyflavone 425
a 10% solution of dimethyldichlorosilane in toluene,
followed by deactivation of excess reagent in methyl-
ated spirits and oven drying (120°C).
Extraction and Quantification of Daidzein and
Genistein in Food
Isoflavone extraction protocol. To two and a half
grams of freeze-dried food was added at least 5 ml of
80% aqueous methanol; the isoflavone glycosides were
dissolved into the methanol using 10 min of sonication
to break up cellular material, followed by a further
hour of soaking in the solvent. Insoluble material was
removed by filtration through a double layer of filter
paper (Whatman No. 4 and then No. 1), and any ad-
sorbed isoflavones washed through with fresh 80%
aqueous methanol (.5 ml). The alcohol in the filtrate
was evaporated and 100 Fishman units of cellulase,
from A. niger, added in 5 ml of 0.1 M acetate buffer, pH
5. Samples were sonicated and subsequently incubated
overnight in a shaking water bath at 37°C. The hydro-
lyzed aglycone isoflavones were extracted from the
aqueous hydrolysis solution by partitioning into ethyl
acetate. Three 2-ml washes of ethyl acetate were com-
bined, and 1 ml of this was aliquoted into a separate
vial and dried at 60°C under nitrogen.
Gas chromatography–mass spectrometry. Dried
samples were derivatized by adding 0.6 ml pyridine
followed by 0.4 ml N-(tert-butyldimethylsilyl)-N-methyl-
trifluoroacetamide (TBDMS) containing 1% TBDMS-
chloride catalyst. After 1 h at room temperature 1-ml
samples were injected on to the GC capillary column
(MD800, Fisons, UK) through an injector operated in
the split mode (25:1) and lined with silanized glass.
The gas chromatography capillary column (15 m of
DB1, J & W, Jones Chromatography, Mid Glamorgan,
UK) was used isothermally at a temperature of 320°C
and the mass spectrometer was operated in its selected
ion mode. The mass ions selected were identified from
the cracking pattern of pure synthetic standards (Apin
Chemicals Ltd., Abingdon, Oxon, UK) derivatized with
TBDMS, and the mass ions used to identify the isofla-
vones are set out in Table 1. The mass to charge ratio
of daidzein No. 2 (Table 1) is thus the singly charged
molecular ion of the derivatized compound (see Fig. 1)
and the other fragments. Positive identification of the
fragments is difficult, but likely candidates would be
formed by a single tertbutyl group (m/z 57) fragment-
3
ing off. Daidzein No. 1 and genistein No. 1 have lost a
single tertbutyl group and are singly charged, whereas
genistein No. 2 has lost two such groups and is doubly
charged.
The food isoflavones were identified and quantified
by comparison with authentic synthetic daidzein and
genistein (Apin Chemicals Ltd.) using a combination of
their retention time on the GC and a ratio of their m/z
No. 1 to m/z No. 2 mass ions. After identification a
response ratio of each isoflavone was formed by divid-
ing the m/z No. 1 integral by that of the external
standard (6,7-dihydroxyisoflavone); the response ratio
accounts for changes in instrument response between
injections. The concentration of isoflavones in the sam-
ples was determined by comparing their response ratio
with calibration charts formed from authentic syn-
thetic standards run simultaneously on the GC–MS.
Quality assurance and internal standards. Each
food sample was assayed for phytoestrogens in quadru-
plicate using a standard addition method. An initial
assay was used to estimate the concentration of isofla-
vones without correction for procedural losses. The es-
timated concentration was used to calculate the
amount of the internal standards (synthetic glucosides,
daidzin (7-O-glucosyl-49-hydroxyisoflavone) and genis-
tin (7-O-glucosyl-49,5-dihydroxyisoflavone, both pur-
chased from Plantech UK, Reading, UK)) used in the
subsequent assays. The calculation took into account
the carbohydrate component of these synthetic com-
pounds and was designed such that the amount of
daidzein and genistein added as spikes would be equal
to the estimated concentration in the food. An equal
amount of spike was used to avoid proportional errors
that occur with smaller or larger quantities.
A sample of each food was spiked in duplicate with
synthetic daidzin and genistin dissolved in 80% meth-
anol(aq), and the alcohol was allowed to evaporate over-
night. The spiked samples were subjected to the isofla-
vone extraction procedure alongside a third unspiked
sample.
The coefficient of variation between the two un-
spiked assays was used as an indicator of interassay
variation. Samples were assayed again if there were
large differences in the concentration of the two spiked
or two unspiked assays and/or low recoveries of the
spikes (,70%).
Procedural losses. Procedural losses of daidzein
and genistein from the food were accounted for using
the internal standards, spiked into two of the four
assays carried out on each food. The calculation is
summarized in Eqs. [1] and [2].
~@S# 2 @F#!
5r [1]
@I#
4 LIGGINS ET AL.

@F# TABLE 2
5 @A# [2] Percentage Recovery of Isoflavones Upon Extraction Using
r
a Variety of Methods to Remove the Conjugated Carbohy-
drate Component
Equation [1] calculates the recovery (r) of each of the
internal standards; [S] is the average concentration of White flour spiked
the isoflavone determined for the spiked samples, [F] is with synthetic
the average unspiked samples isoflavone concentration Soy flour glucosides
Method of hydrolysis
and [I] is the initial concentration of the aglycone in the
(enzyme source or acid) Daidzein Genistein Daidzein Genistein
spike. Equation [2] then uses r to adjust the average
concentration of the isoflavone determined for the un- Almonds 77 (611) 77 (612) 37 (624) 40 (614)
spiked samples to account for procedural losses. Aspergillus niger 83 (64) 91 (67) 85 (63) 95 (66)
Helix pomatia 82 (69) 80 (68) 70 (616) 60 (620)
3 M HCl 84 (67) 74 (614) 37 (68) 30 (612)
Method Development
Investigation of lipid removal by hexane partitioning. Note. Parentheses contain the interassay coefficient of variation.
The loss of synthetic daidzein and genistein (Apin
Chemicals) dissolved in 1 ml of 80% aqueous methanol 100 Fishman units of cellulase from A. niger in 5 ml of 0.1
upon partitioning into hexane (three 3-ml washes) was M acetate buffer, pH 5.0; or 5 ml of 0.1 M phosphate
investigated. After evaporation of the alcohol, the re- buffer, pH 6.8, containing 100 Fishman units of b-gluco-
maining isoflavones were quantified after derivatiza- sidase from almonds. The enzyme hydrolysis solutions
tion using GC–MS. were heated to 37°C overnight in a shaking water bath.
Hydrolysis of glycosidic bond. The efficiencies of dif- Also investigated was acid hydrolysis using 5 ml of hy-
ferent procedures for hydrolysis of the glycosidic bond, of drochloric acid at a variety of concentrations; samples
the isoflavone glycosides, were compared using two sam- were heated to 80°C for 2 h after which the samples were
ple types: an in house stock soy flour with a known allowed to cool to room temperature (30 min) and the acid
isoflavone content (independently determined to contain neutralized with sodium hydroxide. Results for the con-
a total of 1.05 mg g21 daidzein and 1.15 mg g21 genistein, centration of hydrochloric acid that gave the highest yield
after the carbohydrate component is taken into account) of isoflavones (3 M HCl) are presented along with the
and white wheat flour spiked with known amounts of the data for enzymatic hydrolysis. Unconjugated isoflavones
synthetic glucosides, daidzin (7-O-glucosyl-49-hydroxy- were isolated from the final aqueous solvents by parti-
isoflavone) and genistin (7-O-glucosyl-49,5-dihydroxy- tioning into ethyl acetate (three 2-ml washes), drying a
isoflavone, both purchased from Plantech). The white portion of this under nitrogen, derivatization with TB-
wheat flour was spiked with 1.67 mg g21 of each of DMS, and analysis by GC-MS.
daidzin and genistin (the flour therefore contained Validation of the isoflavone extraction protocol. To
;1 mg g21 of the aglycones). Two and a half grams of food test the validity of the assay over a wide concentration
sample was prepared for hydrolysis, as described above. range, the in-house stock soy flour (of known isoflavone
To the aqueous solution remaining after evaporation of composition) was diluted into white wheat flour con-
the alcohol was added either; 5000 Fishman units of taining little or no isoflavones to form a series of test
b-glucuronidase, from H. pomatia, with some aryl sulfa- foods. Each of the test foods was assayed in quadrupli-
tase activity, in 5 ml of 0.1 M acetate buffer, pH 5.0; or cate for their isoflavone content.

RESULTS
Investigation of Lipid Removal by Hexane
Partitioning
The remaining concentration of daidzein and
genistein in aqueous methanol after partitioning with
hexane is illustrated in Fig. 2. Both compounds illus-
trated a concentration-dependent loss upon washing
with the hexane.

Hydrolysis of Glycosidic Bond

Enzyme hydrolysis of two food types, soy flour of
known isoflavone composition and white flour contain-
FIG. 2. Loss of daidzein and genistein from 80% methanol(aq) upon ing synthetic daidzin and genistin, is compared to acid
partitioning with hexane. hydrolysis in Table 2. For reasons of clarity, only the
 EXTRACTION AND QUANTIFICATION OF DAIDZEIN AND GENISTEIN 5

highest yield of isoflavones from acid hydrolysis is il-

lustrated. Of all the methods of hydrolysis examined,
only enzyme hydrolysis using a preparation of A. niger
produced recoveries that were both good and consistent
across both food types investigated.

Validation of the Isoflavone Extraction Protocol

The log charts, Figs. 3A and 3B, plot the assayed
concentration of daidzein and genistein against that
calculated from the dilution. Figure 3A illustrates the
results of the unspiked assays, i.e., without adjustment
for procedural losses. Figure 3B illustrates the same
results, after adjustment, for procedural losses, using
the recovery of the internal standard spikes of syn-
thetic daidzin and genistin. Figure 3C shows the dif-
ference between the assayed and calculated concentra-
tions used in Fig. 3B plotted against their average. The
lack of a trend in the regression of the isoflavones
(daidzein illustrates only a weak trend, r2 5 0.007)
combined with the clustering of points around the zero
of the y-axis indicates that the quantification method is
accurate (36).

DISCUSSION
The isoflavone extraction method presented in this ar-
ticle has been developed for use in any laboratory where
an extensive study of phytoestrogen content in foods
might be carried out. As a result, procedures were de-
signed to be simple and avoided, where possible, the use
of special techniques. Derivatization of compounds with
TMS followed by drying under nitrogen and reconstitu-
tion in a small amount of solvent prior to injection into a
GC–MS is a common technique. The procedure effec-
tively concentrates the sample but also increases the risk
of loss of the target compounds. To simplify this extrac-
tion procedure, the drying down of silanized samples was
omitted and they were injected directly into the GC–MS.
While this increases the maintenance necessary on the
mass spectrometer, it also beneficially increases the life
span of the gas chromatography column and decreases
the time necessary for sample preparation. Silanization
in this manner with TMS did not prove satisfactory; the
isoflavones were not stable and significant decay oc-
curred in vials of an autosampler during the course of an
FIG. 3. Recovery of isoflavones in test foods. Daidzein is abbreviated
overnight run. Switching to derivatization of the isofla- to Da and genistein to Ge; the regression lines are illustrated and their
vones with TBDMS improved the stability, volatility, and value (r2) quoted alongside the gradient (slope) and its standard error
chromatographic resolution of the ethers. In addition, the (SE). (A) Plot of the raw data, produced without adjustment for proce-
mass spectra exhibit high-intensity ions at mass num- dural losses of the compounds. (B) The same results adjusted for pro-
cedural losses, using the recovery of the internal standards. (C) Bland
bers likely to be specific to these compounds in a complex and Altman plot of the data from B; along with the regression lines are
matrix (Table 1). The cracking pattern stability of the plotted the 95% limits of agreement (36).
TBDMS ethers is good, offering more than one candidate
for quantification should the need arise.
The removal of lipid from an initial alcoholic extrac- primarily removed to prevent micelle formation in
tion of a food sample, by partitioning into hexane is later steps of an extraction procedure. Interaction be-
common in the literature (5, 18 –21, 30, 37). The lipid is tween isoflavones, both conjugated to carbohydrate
6 LIGGINS ET AL.
and unconjugated, and the micelles could obstruct the
extraction of those compounds from the solution. How-
ever, the solubility of the isoflavones in hexane is
poorly addressed in the literature. The results illus-
trated in Fig. 2 clearly show a concentration-dependent
loss of daidzein and genistein from aqueous methanol
upon hexane partitioning. Back extraction of the hex-
ane with fresh aqueous methanol would probably re-
cover the isoflavones, but such a step is not specified in
the literature (5, 18 –21, 30, 37). Furthermore, agly-
cone isoflavones have been used to check for losses in
extraction procedures, whereas the compounds occur
naturally as glycosides which are likely to be much less
soluble in hexane. The lipid removing step was there-
fore omitted from the isoflavone extraction procedure.
No adverse effects have been observed during isofla-
vone quantification in other foods, and the sample
preparation time is reduced substantially.
The removal of water from a biological sample
containing compounds to be silanized and analyzed
by GC–MS is desirable to ensure complete reaction of
the silanizing reagent with the compounds of inter-
est. Excess water sequesters the silanizing reagent,
making complete derivatization of all of the isofla-
vone present very difficult. Solid-phase extraction
(SPE) both concentrates and purifies samples, and
using synthetic compounds, protocols efficient at iso-
lating daidzein and genistein from acetate buffer
were developed. However, the application of these
SPE protocols to daidzein and genistein from hydro-
lyzed food failed; the results are not shown but the
trihydroxyflavonoids were particularly problematic.
A simple method to extract isoflavones from an aque-
ous solution is to partition it with an organic solvent
immiscible in water, but it requires that the com-
pounds be soluble in the solvent. Diethyl ether is used
for this purpose in the literature, but in our hands
ethyl acetate, which is safer to handle, was more effi-
cient (results not shown) (18).
The hydrolytic removal of the carbohydrate com-
ponent of isoflavone glycosides simplifies the quan-
titative analysis of the isoflavone. Different hydro-
lytic procedures were investigated on two food types,
and the results of four of these methods are reported
in Table 2. The crude preparation of H. pomatia
produced reasonable recoveries from the soy flour
but not from synthetic standards spiked into white
flour. Similar results were obtained when b-glucosi-
dase from almonds or acid hydrolysis was used. The
low recovery of the synthetic standards from white
flour could be due to a variety of reasons from incom-
plete hydrolysis of the glycosides to degradation of
the unconjugated isoflavones. The low recovery indi-
cates that these methods of hydrolysis are not appli-
cable to differing sources of the isoflavone glycosides;
thus, quantitative results for differing foods using
these methods would have to be treated with caution.
A. niger was chosen as a potential source of enzyme
useful for the hydrolysis of isoflavone glycosides from
foods, because of its use in producing fermented soy
products. Such products have been shown to be rich
in unconjugated isoflavones and contain little of the
isoflavone glycosides (21, 34). Enzyme hydrolysis us-
ing the preparation of A. niger produced recoveries
that were reasonably consistent between different
sources of the isoflavone glycosides and it was there-
fore adopted into the extraction procedure reported
in this article.
The method described in this article for quantifica-
tion of daidzein and genistein in food was developed
using relatively rich sources of isoflavones, such as soy
flour and synthetic isoflavones. The foods common to
the traditional western-style diet are, however, likely
to contain much lower concentrations of the isoflavones
than those sources; thus, the method had to be vali-
dated over a wide concentration range much lower
than that in soy-based foods. The range of test foods
devised to examine the method over such a concentra-
tion range produced the results illustrated in Figs.
3A–3C. In the corrected results, Fig. 3B, the gradient of
the regression indicates that assayed results are very
similar to the calculated values. In Fig. 3C, the lack of
trend in the regression indicates that the difference
between the calculated and analytical data is not bi-
ased in any particular direction and that the assayed
concentration closely resembles that of the calculated.
The recoveries of the internal standards, for these test
foods, were in the range of 70 to 99%, the lower end of
this scale occurred in the samples containing little
isoflavone. Within these limitations, the method was
accurate to at least 100 ng of daidzein or genistein per
gram of freeze-dried food.
The figure of 100 ng g21 is not the limit of detection
of the technique, as defined by the amount of isoflavone
per gram of dried food, rather it was the lowest con-
centration tested using the diluted soy flour. This still
produced good recoveries of both food isoflavones and
synthetic daidzin and genistin internal standards. Any
limit of detection depends upon the detector itself. In
our hands, using a bench-top quadrupole GC–MS, the
practical on-column limit of detection would approxi-
mately be 250 pg. Thus, if the method described in this
article were adjusted further, by derivatizing more of
the residue of ethyl acetate followed by concentration
of the derivatized isoflavones and injection of more
than one microlitre on to the GC column, the limit of
detection could potentially fall to around 20 pg per
gram of food.
ACKNOWLEDGMENTS
Dr. P. Murphy, Iowa State University, is thanked for the indepen-
dent isoflavone analysis of the stock soy flour. Mrs. M. Harding, Miss
E. Neeley, and Miss C. Jeffray are also thanked for their advice and
technical assistance. This work was supported by the United King-
 EXTRACTION AND QUANTIFICATION OF DAIDZEIN AND GENISTEIN
dom Medical Research Council, Ministry for Agriculture Fisheries
and Food (Contract FS2034) and the United States of America Army
(Contract DAMD17-97-1-7028).
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