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BIOMEDICAL A N D ENVIRONMENTAL MASS SPECTROMETRY, Vol. 15, 157-161 (1988)
A Gas Chromatographic/Mass Spectrometric Assay
for Catechol Estrogens in Microsomal Incubations:
Comparison with a Radiometric Assay
D. J. Porubek and S. D. Nelson?
Department of Medicinal Chemistry BG-20, School of Pharmacy, University of Washington, Seattle, Washington98195, USA
A gas chromatographichass spectrometric assay for quantifying two catechol estrogens, 2-hydroxyestradiol and
4-hydroxyestradiol, in microsomal preparations is described. The assay employs deuterium-labeled analogs of the
catechol estrogens as internal standards and permits quantification of catechol estrogens, in microsomal incubations,
at low (1-2) p~ concentrations. The compounds are analyzed as their trimethylsilyl derivatives following separation
by capillary gas chromatography.
INTRODUCTION
EXPERIMENTAL
Aromatic hydroxylation of estradiol by microsomal oxygenases generates the catechol estrogens 2-hydroxyestradiol and 4-hydroxyestradiol (Fig. 1). Catechol
estrogen formation is a major metabolic route for
endogenous and exogeneous estrogens in animals and
man.'-3 While the liver is primarily responsible for metabolism of estrogens into catechol estrogens, a number
p l a ~ e n t a and
~'~
of other organs including the
kidney""
possess metabolic activity. While the
estrogens retain some of the physiological properties of
the classic estrogen, estradiol, they possess certain
activities that are unique (see reviews, Refs 12-14).
The catechol estrogens are further metabolized in vivo
to their corresponding 0-methyl ethers by the enzyme
catechol-0-methyl transferase (COMT)." Because
COMT is an abundant enzyme in many tissues, including
red blood cells, and because catechol estrogens are good
substrates for COMT,I6 only very low levels of catechol
estrogens circulate freely. The catechol estrogens are
also labile compounds that spontaneously auto-oxidize
under neutral and basic condition^.'^
As a result of these technical difficulties a number of
analytical methods have been developed to measure
catechol estrogens in ~ i v o "and
~ ~in~vitro.2'-2'Among
the methods that were developed the radiometric method
of Fishman ef al." appeared to circumvent many problems. Because the method measures the process of radiolabel release from appropriately tritiated substrates, further biotransformation or degradation of the catechol
estrogens should not influence the analytically sensitive
step. Recently however, the accuracy of the radiometric
method has been questioned. Non-specific and/or peroxidative radiolabel release have been suggested as possible complications.24~25
In order to compare the radiometric assay with direct product isolation assay, we
developed a gas chromatographic/mass spectrometric
assay which measures the catechol estrogens under conditions which allow their simultaneous quantification by
the radiometric assay.
Materials
Chemicals purchased from Aldrich Chemical Company
(Milwaukee, Wisconsin) included isopropenyl acetate,
bromine,
potassium
tert-butoxide,
acetone-&
(99.5 at% D), methanol-d, (99.5 at% D), choloroformd, (98 at% D), sodium borodeuteride (98 at% D),
potassium nitrosodisulfonate, L-ascorbic acid, and silica
gel (60-200 mesh). Chemicals from Sigma Corporation
(St Louis, Missouri) included estrone, estradiol, NADP,
glucose-6-phosphate (G-6-P), glucose-6-phosphate
dehydrogenase, bovine serum albumin and dimethyldichlorosilane. Phenol reagent (Fohn-Ciocalteau) used in
the Lowry protein assay was supplied by Fisher Scientific
(Fair Lawn, New Jersey) The derivatization reagents,
N, 0-bis(trimethylsily)trifluoroacetamide (BSTFA) and
silylation grade pyridine, were purchased from Pierce
Chemical Company (Rockford, Illinois). Concentration
tubes were 12 ml Pyrex (Corning Glass works, Corning,
New York). Aquasol I1 scintillation cocktail was purchased from New England Nuclear (Boston,
Massachusetts). All other solvents and chemicals were
of reagent grade from commercial suppliers.
?"
@
HO
OH
4- ti" d r o x v r s t r a d i 0 1
t Author to whom correspondence should be addressed.
0887-6 134/ 88/030 157-05 $05.00
@ 1988 by John Wiley & Sons Ltd
Figure 1. Formation of catechol estrogens from estradiol.
Received 12 January 1987
Revised 1 June 1987
158
D. J. PORUBEK A N D S. D. NELSON
The synthesis of unlabeled 2-hydroxyestradiol and
4-hydroxyestradiol was accomplished by the method of
Gelbke et al.26 The synthesis of (15,16,17-,H3)2hydroxyestradiol and ( 15,16,17-2H3)4-hydroxyestradiol
was accomplished by the method of Knuppen et al.27
up through the preparation of ( 15,16,17-2H3)estradiol.
Oxidation of ( 15,16,17-2H3)estradiol according to the
method of Gelbke et a1.26 afforded the deuterated
catechol estrogens. Mass spectrometric analysis of
(15,16,17-'H3)2-hydroxyestradiol revealed a labeling
pattern of 2.0% 'Ho, 14.9% 2H,, 10.3% 'H2, 72.2% 2H,
and 0.5% 'H,. Analysis of (15,16,17-'H,)4-hydroxyestradiol revealed 0.8% 'Ho, 10.2% ,HI, 11.2% ,H2,
77.6% ,H, and 0.2% 'H4.
The synthesis of the radiolabeled estrogrens (2,H)estradiol and (4-3H)estradiol was reported previouslyZ8with the verification of position of label and
determination of radiochemical purity (98% ) being
described therein. The specific activity of the substrates
employed in the metabolic incubations was 32 mCi/mol
for (2-3H)estradiol and 34 mCi/mmol for (43H)estradiol.
Methods
Microsomes. Microsomes and microsomal stock sol-
utions were prepared according to Wheeler et ~ 1 . ' Male
~
Sprague-Dawley rats (180-200 g) were used. It was
possible to store the microsomes at -80°C in 0.1 M
phosphate buffer (0.2 mM EDTA, pH 7.4) containing
20% glycerol for at least a month. Therefore, frozen
microsomes were used in these studies.
Preparation of standards. Standard solutions of catechol
estrogens were prepared by dissolving 500,250, 125 and
25 pg of 2-hydroxyestradiol and 62.5, 31.2, 15.6 and
7.8 pg of 4-hydroxyestradiol in 1-ml volumes of
methanol-acetic
acid-ascorbic
acid
(MAA)
(10 ml: 1 ml :60 mg).I7 Standard solutions of the deuterated internal standards were similarly prepared by dissolving 125 pg ( 15,16,17-2H3)2-hydroxyestradioland
15.6 pg (15,16,17-2H3)4-hydroxyestradiol in 1-ml
volumes of the MAA mixture. All stock solutions were
stored in silanized glass vials at -80 "C in the dark and
were stable over a perod of at least 3 m o n t h ~ . ' ~
Incubation conditions. Incubation mixtures were made up
to 1 ml combining 610 pl buffer (0.1 M phosphate,
0.8 mM ascorbate, pH 7.4, prepared freshly), 100 FI
microsomes (10 mg/ml), 250 p1 cofactor mix (10 pmol
G-6-P, 3 pmol NADP, 3 units G-6-P dehydrogenase and
5 pmol MgCl,), 20 p1 substrate (5 mM estradiol in
ethanol) and 20 p1 internal standard (deuterated 2- or
4-hydroxyestradiol). For the construction of standard
curves, 2 0 4 of standard stock solutions of 2- or 4hydroxyestradiol were added in place of substrates. All
incubations were conducted for 10 minutes at 37 "C with
shaking in a Dubnoff Incu-Shaker (Lab-Line Instruments Inc., Melrose Park, Illinois). Incubations and
subsequent work-ups were conducted with glassware
that had been silanized.
then centrifuged at 1000 x g for 10 min and the aqueous
layer separated from the organic. The aqueous layer was
successively extracted with chloroform-acetone (4 x
6 ml, see Radiometric assay) while 4-ml aliquots of the
initial organic extracts were transferred to concentration
tubes containing anhydrous sodium sulfate (- 1 g).
After drying for 1 h the organic extracts were transferred
to clean concentration tubes and the solvent removed
under a stream of nitrogen. To the dry extracts were
added 0.1 ml BSTFA and 0.01 ml pyridine. The tubes
were capped, gently vortexed and heated at 60°C for
30 min.
Gas chromatography mass spectrometry assay. Capillary gas
chromatography was performed with a Hewlett-Packard
(Avondale, Pennsylvania) 5700 gas chromatograph
fitted with a wide-bore DB-5 bonded-phase (normal film
thickness) fused silica column (30m) from J. & W.
Scientific Inc. (Rancho Cordova, California). Quantitative gas chromatographic/mass spectrometric measurements were conducted on a VG Analytical Micromass
7070H mass spectrometer (VG Analytical Inc.,
Manchester, UK) with a VG 2000 data system interfaced
with a Hewlett-Packard 5700 gas chromatograph and
capillary injector.
Typically 1-pl aliquots of the derivatized extracts were
directly injected into the gas chromatograph/mass spectrometer for analysis. The gas chromatographic conditions were: helium carrier gas head pressure of 15 psi,
splitless injection, injector temperature of 280 "C, initial
oven temperature of 1OO"C, initial heating rate of
30deg.C/min for 4min, a second heating rate of
5 deg. C/min until 280 "C was attained and a 5-min hold
at 280°C. Under these conditions the trimethylsilyl ethers
of 2-hydroxyestradiol and 4-hydroxyestradiol eluted at
14.4 and 15.0 min, respectively; whereas that of estradiol
eluted at 12.6 min The capillary column was run directly
into the mass spectrometer source with the direct inlet
transfer line held at 280 "C. The mass spectrometer conditions included a source temperature of 200 "C, 70 eV
electron energy (electron impact), 4 kV accelerating voltage, 200 pA emission current, 2400-3800 V multiplier
setting. The instrument was run in the selected ion
monitoring (SIM) mode and calibrated for the measurement of ions at 504.2898 and 507.3084 a.m.u. Deuterated
analogs of the catechol estrogens were employed as
internal standards and by using trideuterated analogs
interfering ion signals from the multiple isotopes of
silicon derivatives were minimized.
Radiometric assay. Following extraction of the aqueous
incubation media with chloroform-acetone, the samples
were analyzed by transfer of 0.2-ml aliquots to scintillation vials containing 10 ml scintillation cocktail. Samples
were counted for 10min with a Beckmann LS-7500
instrument (Beckmann Instruments, Inc., Fullerton,
California) utilizing a counting program with an external
standard to correct for quench in all samples.
RESULTS AND DISCUSSION
Sample preparation. Upon completion of incubation the
reactions were terminated by addition of 6 ml
chloroform -acetone (4: 1). The mixtures were vortexed
It was possible to separate the catechol estrogens of
estradiol from each other and estradiol itself by capillary
AN ASSAY FOR CATECHOL ESTROGENS
159
b c
FID
RESPONSE
I
*
I
.
.
2
I
,
.
6
4
.
.
8
.
.
a
10
,
'
.
12
a
s
14
.
.
16
18
TINE(min)
Figure 2. Separation of estradiol(a), 2-hydroxyestradiol(b), and 4-hydroxyestradiol(c) as their tri-trimethylsilyl derivatives by capillary gas
chromatography.
gas chromatography (Fig. 2). Derivatization was
required to protect the labile catechol estrogens and
render them volatile Conversion of the catechol
tri-trimethylsilyl derivatives by treatment with BSTFA
proved to be very convenient. The method is rapid and
requires a minimum amount of sample manipulation.
Shown in Fig.3(a) is the mass spectrum of the tritrimethylsilyl derivative of 2-hydroxyestradiol above
300 a.m.u. The spectrum of the tri-trimethylsilyl derivative of 4-hydroxyestradiol is nearly identical in this1
region (Fig. 3(b)). The relative intensities of the
molecular ions substantiate their use for quantification.
The standard curves of peak area ratio versus weight
ratio of the unlabeled and deuterated metabolites are
presented in Fig. 4. For construction of the standard
curve for 2-hydroxyestradiol the amount of internal standard (( 15,16,17-2H3)2-hydroxyestradiol)
was held constant at 2500 ng ml-' of microsomal incubation and the
amount of unlabeled metabolite varied between
250 ng ml-' and 25 000 ng m1-I. At both extremes of the
standard curve significant deviation from linearity
occurs, indicating the limits of reliable quantification
for the assay (Fig. 4(a)). The corresponding standard
curve for 4-hydroxyestradiol (Fig. 4(b)) was constructed
by holding constant the amount of internal standard
(( 15,16,17-2H3)4-hydroxyestradiol) at 625 ng m1-l of
microsomal incubation and the amount of unlabeled
metabolite varied between 62.5 ng and 6250 ng ml-'. Significant deviation from linearity again occurred at both
extremes of the standard curve but restriction of quan-
(a)
RELATIVE
ION
INTENSITY
"1432
0
r
446
489
, .
.
I,
7_
620
608
480
460
448
640
MOLECULAR WEIGHT
Figure 3a. Mass spectrum of the tri-trimethylsilyl derivative of 2-hydroxyestradiol above 300 a.m.u.
1
1'"l
RELATIVE
ION
INTENSITY
5:1, .
483
,
440
,
,
,
,
468
,
I
,
I
,
460
MOLECULAR
,
, Illl,
688
,
~
628
,
I
. ,
640
.
I
WEIGHT
Figure 3b. Mass spectrum of the tri-trimethylsilyl derivative of 4-hydroxyestradiol above 300 a.rn.u.
160
D. J. PORUBEK AND S. D. NELSON
i\i iciii it11 i o
Figure 4a. Standard curve of weight ratio versus peak area ratio for 2 hydroxyestradiol with [2H,]2-hydroxyestradiol held constant at
2500 ng m1-l
(b)
/
10.0
PEAK
AREA
1.0
RATIO
0.1
1.0
0.1
10.0
I E I G t i T RATIO
Figure 4b. Standard curve of weight ratio versus peak area ratio for 4-hydroxyestradiol with [2H,]4-hydroxyestradiol held constant at
625 ng ml-'.
tification of metabolites within these extremes proved
reliable. The corresponding linear regression parameters
for each metabolite are given in Table 1. The reproducibility and accuracy of the assay were tested by replicate
analysis of microsomes spiked with known amounts of
each metabolite (Table 2).
As mentioned in the introduction the development of
a gas chromatographic/mass spectrometric assay
intrinsically compatible with the radiometric assay was
desired. Such methodology would enable direct assessment of the accuracy of the radiometric assay. Additionally, the methodology could provide a n accurate
means for the quantification of specific catechol
estrogens in tissues that produce these products at moderate rates (0.1-10 nmol min-' mg-' tissue). Thus, the
gas chromatographic/mass spectrometric assay was
used in conjunction with the radiometric assay to
Table 1. Liner regression parameters calculated from standard
curves of peak area ratios versus weight ratios
2-Hydroxyestradiol
Slope
Y-int.
r2
*
0.97 0.02
0.08 0.04
0.99
*
4-Hydroxyestradioi
*
*
0.94 0.02
0.16 0.04
0.98
A N ASSAY FOR C A T E C H O L ESTROGENS
Table 2. Reproducibility and accuracy of quantification of 2hydroxyestradiol and 4-hydroxyestradiol in microsomes
Amount added
Amount recovered
(ndmll
(ng/mlla
2-OH-Estradiol (n=4)
1250
5000
4-OH-Estradiol (n=4)
156
625
CVb
Accuracy‘
1520 53
5198 f 319
3.5
6.1
21.6 4.2
5.6 4.5
176+11
672 43
6.3
6.4
12.7k7.1
9.0 5.6
*
*
Table 3. Rates of formation (w f SD, ng product mg protein-’
10 min-’) of 2-hydroxyestradiol and 4-hydroxyestradiol by liver microsomes obtained from male rats.
Multiple incubations from a single preparation of
microsomes obtained from 3 rats
Assay
*
*
*
MeaniSD.
CV=coefficient of variation.
c Mean percentage deviation of all concentrations from the theoretical value.
a
measure catechol estrogen formation by hepatic microsomes from male rats. The gas chromatographic/mass
spectrometric assay and radiometric assay were run in
tandem in the same incubations by replacing unlabeled
substrate estradiol with radiolabeled estradiol ((2- or
4-3H)estradiol). The catechol estrogens were isolated,
derivatized and analyzed as described herein and the
aqueous incubation media were processed and analyzed
for tritium content as previously described for the radiometric assay.’
A comparison of the rates of formation of 2- and
4-hydroxyestradiol appears in Table 3. As expected, both
assays revealed a greater rate of 2-hydroxylation than
4-hydroxylation. Additionally, the values obtained for
2-hydroxylation agree quite favorably between assays.
However, a major discrepancy between assays was
observed for rates of 4-hydroxylation, in that the radiometric assay gave a value at least three times that
afforded by the gas chromatographic/mass spectrometric assay. The origin of this major discrepancy is not
yet known; however, non-specific and/or peroxidative
161
GC/MS
Radiometric
2 Hydroxyestradiol
*
6715 188
5539 f40
4 Hydroxyestradiol
617*60
1691 *17
release of tritium from estrogens has been previously
suspected. 24,25330 Another possibility is binding of a reactive metabolite of estrogens to tissue macromolecules in
such a way that tritium is lost from the 4-p0sition.~’-~’
Alternatively, the discrepancy may in part lie in the fact
that during incubation of (4-’H)estradiol with microsomes 2-hydroxylation as well as 4-hydroxylation is
occurring. The formation of radiolabeled 2-hydroxyestradiol under such circumstances could lead to spurious
release of tritium if a small portion of the metabolite
decomposed or was further biotransformed. Such a process would not be expected to play a major role during
the incubation of (2-’H)estradiol since the amount of
radiolabeled 4-hydroxyestradiol thus formed would be
negligible.
In summary, a gas chromatographic/mass spectrometric assay suitable for measurement of catechol
estrogen formation in tissues such as liver has been
developed. The assay permits the direct quantification
of metabolites and, while it was primarily developed for
the purpose of assessing the accuracy of the radiometric
method , it should also be suitable for routine measurements of catechol estrogen production where more
specific measurements of 2- and 4-hydroxylation might
be warranted.
Acknowledgement
D. J. Porubek was supported by NIH Training Grant no. GM 07750.
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