Original

Chromatography, Vol.23 No.2 (2002)

Original

Determination of Estrogens in Rat Brains Using
Gas Chromatography/Mass Spectrometry/Mass Spectrometry
Kuniko Mitamura, Nanae Mikami, Yukiko Kambara, Emi Ohno and Kazutake Shimada*
Faculty of Pharmaceutical Sciences, Kanazawa University, 13−1 Takara−machi, Kanazawa 920−0934, Japan
Received for Review November 30, 2001. Accepted February 4, 2002.

Abstract
A standard addition determination method of estrone, estradiol and guaiacol estrones in rat brains using gas chromatography/mass spectrometry/mass spectrometry (GC/MS/MS) was developed. Estrone and estradiol were extracted with liquid−liquid extraction, purified successively
with silica−gel mini−column chromatography, solid−phase extraction and preparative HPLC. The obtained estrogens were derivatized to dimethylethylsilyl ethers followed by GC/MS/MS analysis. The concentrations of estrone and estradiol were 0−44 and 0−159 pg/g tissue (n=4), respectively. On the other hand, an additional pretreatment procedure was necessary for determination of guaiacol estrones in rat brains.
After the liquid−liquid extraction, guaiacol estrones were acetylated with acetic anhydride−pyridine and purified successively with silica−gel
mini−column chromatography and solid−phase extraction. The obtained guaiacol estrone acetates were hydrolyzed, derivatized to dimethylethylsilyl ethers and then applied to GC/MS/MS. Although the absolute recovery rates of guaiacol estrones spiked in brain homogenate
were higher than 65%, these estrogens in rat brain were not determined.
Keywords: estrone, estradiol, guaiacol estrone, rat brain, standard addition method, gas chromatography/mass spectrometry/mass spectrometry

catechol estrogens (2− or 4−hydroxy metabolites) [6,7] in rat

1. Introduction
The term “neurosteroids” applies to the steroids that are bothbrains using gas chromatography/mass spectrometry/mass spec-

synthesized in the nervous system, either de novo from cholesterol

trometry (GC/MS/MS) and/or liquid chromatography/mass spec-or from steroid hormone precursors, and that accumulate in thetrometry (LC/MS). However, two or three whole brains were usednervous system to levels that are at least in part independent of thesteroidgenic gland secretion rates. It has been reported that thesesteroids are found in larger amounts in brain than in plasma [1]; recently, significant interest has been focused on the biological properties of neurosteroids, such as pregnenolone, dehydroepiandrosterone and their conjugates [2]. Also, the existence of estrogen receptor in brain has been clarified [3], and the usefulness of estrogen−replacement therapy for dementia of Alzheimer type has been reported [4]. Much interest is focused on the action of estrogens on
the central nervous system and on the existence of estrogens in thebrain. In previous papers, we reported the existence of the classicalestrogens {estrone (E1), estradiol (E2) and estriol}, the guaiacol estrones {2−hydroxyestrone 3−methyl ether (2OHE13Me) and 4−hydroxyestrone 3−methyl ether (4OHE13Me)} [5] (Figure 1) and the

Figure 1. Structures of E1, E2 and guaiacol estrones.

*Corresponding author: Tel/fax, +81−76−234−4459;
e-mail, shimada@dbs.p.kanazawa−u.ac.jp―６５―

Chromatography, Vol.23 No.2 (2002)

2.2. Determination of E1 and E2

for identification at a time; therefore, the quantitative analyticalmethod using part of an entire brain is necessary to clarify the

Wistar strain rat was decapitated and the entire brain (ca. 1.8

physiological role of these estrogens in the central nervous system.

g) was homogenized in 10% EtOH (10 volumes, w/v) using a Tef-

In this paper, we describe the development of the standard ad-lon homogenizer, and then divided into three parts so that the con-dition determination method of E1, E2 and guaiacol estrones in parttent of brain in each tube is approximately 0.6 g of tissue. Stan-of a rat brain using GC/MS/MS.

dard solutions of E1 and E2 (each 0, 50, 100 pg in EtOH) and ISs ([2H5]−E1 and −E2; each 1ng in EtOH) were added to the homogenate.

2. Experimental

After standing for 10min at room temperature, AcOEt (6ml) was

2.1. Apparatus, materials and animals

added to the samples and the mixture was then sonicated for 20min

GC/MS/MS was performed on a GCQ gas chromatograph/ion

in an ultrasonic bath, stirred at room temperature for 30min andtrap mass spectrometer (ThermoQuest, San Jose, CA, USA)centrifuged at 1500g for 20min. The organic layer was collected

equipped with a 5% diphenyl−95% dimethylpolysiloxane capillary

and the residue was re−extracted with AcOEt (6ml), and the entire

column (Rtx−5MS, 30m x 0.25mm i.d., 0.25µm df; Restek, Belle-organic layer was evaporated under a N2 gas stream. The residue

fonte, PA, USA), which was used with the following conditions:

was dissolved in hexane−AcOEt (20:1, 0.5ml) and subjected to sil-electron ionization was set at 70 eV and detected with positive ion

ica−gel mini−column chromatography. After washing with hexane

mode. Carrier gas was helium at a flow rate of 40cm/s. The col-(5ml) and hexane−AcOEt (10:1, 5ml), the fraction eluted with hex-

umn oven temperature was set at 60℃ for 4 min, ramped to 280℃

ane−AcOEt (2:1, 8ml) was evaporated under a N2 gas stream. The

at 40℃/min and held at 280℃ for 10min. The split−less injector

residue was dissolved in 10% MeOH (1ml) and applied to an ISO-

temperature was set at 60℃ for 1min, ramped to 280℃ at 180℃/

LUTE C18 (EC) cartridge. After washing with H2O (5ml) andmin and held at 280℃ for 18 min. The transfer line temperature

20% MeOH (2 ml), the eluate with MeOH (3ml) was evaporated

and the ion source temperature were set at 275℃ and 200℃, re-under a N2 gas stream. The obtained residue was purified by prepa-

spectively.

rative HPLC and the fraction containing E1, E2 and ISs (5.5−7.5

Preparative HPLC was performed on a LC−6A (Shimadzu,min) was evaporated in vacuo and the residue was derivatized with

Kyoto, Japan) equipped with a J’sphere ODS−H80 (4µm, 150 x

DMESI (50µl) for 30min at 60℃. The reaction mixture was di-

4.6mm i.d.) (YMC, Kyoto) column and an SPD−10A UV detector

luted with CHCl 3 (0.5ml), applied to a silica−gel mini−column(280nm) (Shimadzu). The flow rate and the column temperaturechromatography, and the eluate with CHCl3 (5ml) was evaporated

were set at 1ml/min and 40℃, respectively. E1 and E2 were notseparated when MeOH−H2O (13:7) was used as the mobile phase,so that both compounds were obtained in the same fraction.
E1 and E2 were supplied by Teikoku Hormone Mfg. (Tokyo,
Japan). Guaiacol estrones (2OHE12Me, 2OHE13Me, 4OHE13Me,
4OHE14Me) and 16−oxoestradiol (16oxoE2) {internal standard(IS) for guaiacol estrones} were purchased from Steraloids (Newport, RI, USA). [2,4,6,6,9−2H5]−E1 and −E2 (ISs for E1 and E2) [8]were prepared in our laboratories from [6,6,9−2H3]−E1 and −E2, respectively, which were kindly supplied by Nippon Kayaku (Tokyo). Silica−gel mini−column chromatography (0.5g, 3 x 0.6cm i.
d.) was performed with Silica Gel 60 (70−230mesh; E. Merck,
Darmstadt, Germany). ISOLUTE C18 (EC) cartridges (500mg;
International Sorbent Technology, Hengoed, Mid Glamorgan, UK)
were purchased from Uniflex (Tokyo). Dimethylethylsilylimidazole (DMESI) was obtained from Tokyo Kasei Kogyo (Tokyo),and all the other reagents were analytical grade and commerciallyavailable.
Male Wistar strain rats (7weeks old) were obtained from Japan S.L.C. (Hamamatsu, Japan).
Figure 2. Procedure for determination of E1, E2 and guaiacol estrones.

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Chromatography, Vol.23 No.2 (2002)

Kuniko Mitamura, Nanae Mikami, Yukiko Kambara,
Emi Ohno and Kazutake Shimada

under a N2 gas stream. The residue was dissolved with hexane (50µl) and an aliquot of which was subjected to GC/MS/MS {precursor ions, m/z 356 and 361 (10−14min, for E1 and [2H5]−E1), 444
and 449 (14−18min, for E2 and [2H5]−E2); excitation voltage, 1.0V
for E1 and [2H5]−E1 and 1.2V for E2 and [2H5]−E2; monitoring ions,m/z 271 (E1), 276 ([2H5]−E1), 340 (E2) and 344 ([2H5]−E2)} (Figure2).
2.3. Determination of guaiacol estrones
Standard solutions of guaiacol estrones (each 25−400 pg in
EtOH) and IS (16oxoE2;10 ng in EtOH) were added to the Wistar
rat brain homogenate corresponding to 0.5 g of tissue, and the estrogens were extracted with AcOEt as described above. After the
Figure 3. Product ion mass chromatogram of DMES derivatives of
guaiacol estrones (100pg/tube).
Peaks: 1, 4OHE14Me; 2, 2OHE13 Me; 3, 2OHE12Me; 4,
4OHE13Me. GC/MS/MS conditions were described in
the experimental section.

solvent was evaporated under a N2 gas stream, the extract was
treated with acetic anhydride−pyridine (1:2, 0.3ml) at room temperature for 30min. After addition of H2O (1ml), the entire solution was extracted with AcOEt, and the organic layer was successively washed with 5% HCl, 5% NaHCO3 and H2O. After evaporation of the solvent, the residue was dissolved in hexane−AcOEt

heptafluorobutyric anhydride and pentafluorobenzyl bromide (for(10:1, 1ml), subjected to silica−gel mini−column chromatographyderivatization of the hydroxy group). Among these derivatization

and washed with hexane−AcOEt (10:1, 5ml). The fraction eluted

reagents, DMESI appeared to be most promising with respect towith hexane−AcOEt (1:1, 6ml) was evaporated under a N2 gas

resolution, sensitivity and feasibility, and the product ion mass

stream, the residue was dissolved in 10% MeOH (2ml) and applied

chromatogram was shown in Figure 3.

to an ISOLUTE C18 (EC) cartridge. After washing with H2O (5The mass spectra of DMES derivatives of E1, E2, 2OHE12Meml) and 30% MeOH (2ml), the eluate with MeOH (3ml) was

and 16oxoE2 showed an ion at m/z 356 ([M]+), m/z 444 ([M]+), m/z

evaporated under a N2 gas stream. The residue was hydrolyzed

342 ([M−CH3−C2H5]+) and m/z 429 ([M−C2H5]+), respectively, aswith 1% KOH/MeOH (0.5ml) for 30min at room temperature.

the base peak ion, so that these ions were chosen as the precursor

After the neutralization with 5% HCl, the entire solution was ex-ions in the GC/MS/MS (Figure 4a). The product ion mass spectra

tracted with AcOEt and the organic layer was washed with H2O.

of E1− and 2OHE12Me−DMES showed the prominent ion ([precur-

The solvent was evaporated under a N2 gas stream, the obtained

sor ion−85]+) at m/z 271 and m/z 257, respectively, which were as-

residue was derivatized with DMESI, treated as above, and thensigned as the ions formed by dissociation of the steroid C− andsubjected to GC/MS/MS analysis {precursor ions, m/z 342 (14−16

D−ring and rearrangement of hydrogen (Figure 4b) [9]. The massmin, for guaiacol estrones), 429 (16−18min, for 16oxoE2); excita-

spectra and product ion mass spectra of the other guaiacol estrone

tion voltage, 1.0 V; monitoring ions, m/z 257 (guaiacol estrones),isomers did not show such a clear difference from those of

337 (16oxoE2)} (Figure 2).

2OHE12Me. The product ion mass spectrum of E2−DMES showedions at m/z 299 and m/z 340. The former product ion was formed

3. Resultsby dissociation of the D−ring and the latter ion corresponded to

3.1. GC/MS analysis of estrogens[M−DMESOH]+. Although the intensity of the latter ion was lower

In the previous paper, we chose the methyloxime (MO) and/or

than that of the former, the ion at m/z 340 was chosen as the moni-trimethylsilyl ether (TMS) as the derivatization form for identifica-

toring ion because of responsible peak purity. 16oxoE2−DMES

tion of classical estrogens and guaiacol estrones in rat brain using

produced an ion at m/z 337 in GC/MS/MS and it was used as a

GC/MS/MS [5]. Although derivatization to MO−TMS was simple

monitoring ion, but the origin was not assigned. Using these prod-

and convenient, the complete separation of the four guaiacol es-

uct ions as the monitoring ions, the signal to noise ratio (S/N) of

trone isomers was not achieved chromatographically. To find suit-every authentic estrogen at 5 pg/injection was higher than 5.

able derivatization for E1, E2 and guaiacol estrones, we examined
3.2. Determination of E 1 and E 2 in rat brains

O −methylhydroxylamine, O −pentafluobenzylhydroxylamine (forderivatization of the carbonyl group), N, O−bis (trimethylsilyl)

A pilot study was performed to optimize the extraction andtrifluoroacetamide, DMESI, dimethyl−iso−propylsilylimidazole,

purification methods. The recovery rates of authentic E1, E2 (50−―６７―Chromatography, Vol.23 No.2 (2002)

Figure 4. Mass spectra (a) and product ion mass spectra (b) of DMES derivatives of E1, E2, 2OHE12Me and 16oxoE2.

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Chromatography, Vol.23 No.2 (2002)

Kuniko Mitamura, Nanae Mikami, Yukiko Kambara,
Emi Ohno and Kazutake Shimada

400pg) and their ISs ([2H5]−E1 and −E2; each 1 ng) from silica−gel

say was lower than 13%, the recovery rate varied in the individualmini−column chromatography and solid−phase extraction wereassay (48−68%, n=3). For the assessment of linearity, increasingaround 60−80%. To examine the effect of endogenous substances,amounts of E1 and E2 (0, 50, 100pg) and ISs (1ng) were added to

ISs were added to the brain homogenate, and then extracted andthe brain homogenate (corresponding to 0.6g of tissue) and as-

purified successively by silica−gel mini−column chromatography,

sayed, and the typical product ion mass chromatograms were

solid−phase extraction, and then subjected to GC/MS/MS. The

shown in Figure 5. The peak area ratios of the DMES derivatives

peaks corresponding to the DMES ethers of ISs were detectedof E1 and E2 toward those of ISs (y) were plotted versus the added

clearly without interference by other peaks. However, the areas ofamount of E1 and E2 (x) to obtain the regression lines (Figures 6a,these peaks tended to decrease as the analyses were repeated owingb). These showed almost satisfactory linearity with correlation co-to endogenous interfering substances; therefore, further purification

efficient values of more than 0.981 and 0.977, respectively. How-

using preparative HPLC is necessary prior to derivatization to aever, the slope of the lines varied in the individual assay {E1: 7.15 x

DMES ether. E1 and E2 were separated using the ODS column and

10−4, 20.5%; E2: 1.05 x 10−3, 49.7% (mean, RSD, n=4)}, and differ-

MeCN−H2O system as a mobile phase. On the other hand, theseence from that of standard sample (E1: 9.00 x 10−4, 4.9%; E2: 8.82 x

were not separated using a MeOH−H2O system, so that both estro-

10−4, 6.5%, n=3) was observed.

gens and ISs were obtained in the same fraction.

Therefore, the standard addition method was applied for theThen the reproducibility and accuracy were examined in order

determination of E1 and E2 in rat brains (n=4), and the procedure

to validate the method. The overall absolute recovery rates were

was shown in Figure 2. The concentration of E1 and E2 in rat brains

examined using ISs (1ng) which were added to the brain homogen-was determined from the distance between the intersection of theate. Although the relative standard deviation (RSD) in the same as-line with the x axis and the origin of the coordinates. When thepeak corresponding to E1 or E2 was not detectable on the chromatogram, without addition of estrogens, the concentration of E1 or E2
was expressed as 0 pg/g tissue. The measured brain concentrationof E1 and E2 were 0, 0, 0, 44.2pg/g tissue and 0, 15.4, 159.0, 37.2
pg/g tissue, respectively.
3.3. Determination of guaiacol estrones in rat brains
First of all, we attempted to apply the above pretreatment
method for determination of guaiacol estrones in order to analyze
E1 and E2 simultaneously. Authentic samples (0.1−100ng) were
subjected to silica−gel mini column chromatography, solid phaseextraction, and the elution conditions were examined. Although
the silica−gel mini−column chromatography was effective for purification of biological samples, the recovery rates of low amounts ofguaiacol estrones (<10ng) were not satisfactory (<10%). Theselow recovery rates might be caused by irreversible adsorption of
the guaiacol estrones on silica−gel, which may depend on theguaiacol structure of the target compound. In the previous paper,
we reported that the derivatization to acetate was useful for thetreatment of catechol estrogens in the complex matrix and prevention of adsorption [6, 7]. These data prompted us to apply acetylation for determination of guaiacol estrones. Authentic samples(100pg) were acetylated with acetic anhydride−pyridine, subjected
to silica−gel mini−column chromatography, solid−phase extraction, and then hydrolyzed with 1% KOH in MeOH. After derivati-

Figure 5. Product ion mass chromatograms of DMES derivativesof E1, E2 (50pg/tube) and ISs (1ng/tube).
a) Authentic samples, b) extracted E1, E2 and ISs added
to rat brain homogenate. Peaks: 1, E1; 2, [2H5]−E1; 3, E2;
4, [2H5]−E2.

zation with DMESI, the sample was analyzed with GC/MS/MS asshown in Figure 2. The recovery rate was improved and raised
higher than 90%.
It was obvious that a stable isotope labeled compound, such as―６９―Chromatography, Vol.23 No.2 (2002)

Figure 6. Determination of E1 (a), E2 (b) and guaiacol estrones (c−f) in rat brains using standard addition method.

Table 1. Absolute recovery of guaiacol estrones and IS
Add(pg/tube)
0100
400(10ng)

Recovery (%)a)
2OHE12Me

2OHE13Me

4OHE13Me

4OHE14Me

16oxoE2

N.D.b)
65.9
74.3

N.D.
75.5
71.0

N.D.
74.2
70.7

N.D.
65.8
75.7

N.D.

63.0

a) Mean (n=3).
b) N.D. = below the detection limit (5pg/ injection; S/N>5).

―７０―Chromatography, Vol.23 No.2 (2002)

Kuniko Mitamura, Nanae Mikami, Yukiko Kambara,
Emi Ohno and Kazutake Shimada

the deuteride, was superior for IS in GC/MS analysis; however, thefemale rats which have an estrous cycle. Additional experiments

hydrolysis step could cause deuterium−hydrogen exchange. We

are required in order to clarify this ambiguity, and the developed

chose 16oxoE2 as an IS for analysis of guaiacol estrones, which ismethod would be helpful to elucidate the relationship between es-

not an endogenous compound and is clearly separated fromtrogen levels and the neural state.
Although we reported the existence of guaiacol estrones using

guaiacol estrones in the GC/MS.
To estimate the overall recovery rates, guaiacol estrones (0,two or three whole brains [5], the developed method using a small

100, and 400pg) and IS (10ng) were added to rat brain homogenate

sample of the brain could not give the reliable determination data(corresponding to 0.5g of tissue), extracted and treated as above,of guaiacol estrones. However, we have already demonstrated thefollowed by derivatization and GC/MS/MS analysis. The recovery

existence of hydroxylase and catechol−O −methyltransferase in therates were almost satisfactory taking into consideration the above

brain to give catechol estrogens and guaiacol estrogens, respec-

pretreatment method (Table 1); however, the peaks corresponding

tively [12]. The development of a more sensitive determination

to guaiacol estrones were not detectable on the chromatogram of

method, such as immunoassay, is necessary to overcome this dis-the samples where guaiacol estrones were not added. So the intra−

crepancy.

assay reproducibility was examined using brain homogenate towhich was added guaiacol estrones (100pg) and IS (10ng). RSD

Acknowledgement(n=4) of the peak area ratios of 2OHE12Me, 2OHE13Me, 4OHE13

We thank Teikoku Hormone Mfg. and Nippon Kayaku for

Me and 4OHE14Me toward that of IS were 9.4%, 12%, 5.8% andsupplying estrogens. This work was supported in part by a grant

8.4%, respectively.

from the Ministry of Education, Science, Sports and Culture of Japan.

In order to assess the linearity, increasing amount of guaiacol
estrones (25−400pg) and IS (10ng) were added to the brain homo-

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trogen levels in serum of male rats do not fluctuate unlike those of―７１―