Liquid chromatography-mass spectrometric assay of androstenediol in prostatic tissue: Influence of androgen deprivation therapy on its level

Article abstract of DOI:10.1016/j.steroids.2006.08.003

Androstenediol (Adiol, androst-5-ene-3β,17β-diol) is suspected of being an endogenous proliferation agent of prostate cancer (PCa) even after androgen deprivation therapy (ADT). A liquid chromatography-electron capture atmospheric pressure chemical ionization-mass spectrometric (LC-ECAPCI-MS) method for the determination of Adiol in prostatic tissue was developed and validated for evaluating the influence of ADT on the prostatic Adiol level. After derivatization of Adiol with 4-nitrobenzoyl chloride, the detection response of the derivative was increased 150 times more than that of intact Adiol. The LC-MS method was specific and reliable for the measurement of a trace amount of Adiol in 30 mg of tissue. The clinical study using the developed method showed that the prostatic Adiol level was not changed by ADT. That is, the prostatic Adiol levels of PCa patients with ADT (n = 12), benign prostate hypertrophy patients without ADT (n = 8) and bladder cancer patients (without prostatic disease) (n = 6) were 0.83 ± 0.28, 0.62 ± 0.31 and 0.71 ± 0.28 ng g^-1 tissue, respectively, and there was no significant difference between these groups.

Full text of DOI:10.1016/j.steroids.2006.08.003

steroids 7 1 ( 2 0 0 6 ) 1007–1013
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Liquid chromatography–mass spectrometric assay of
androstenediol in prostatic tissue: Influence of androgen
deprivation therapy on its level
Tatsuya Higashia,∗, Naoki Takayamaa, Mariko Kyutokua, Kazutake Shimadaa,
Eitetsu Kohb, Mikio Namikib
a
Division of Pharmaceutical Sciences, Graduate School of Natural Science and Technology, Kanazawa University,
Kakuma-machi, Kanazawa 920-1192, Japan
b
Department of Urology, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi,
Kanazawa 920-0934, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Androstenediol (Adiol, androst-5-ene-3␤,17␤-diol) is suspected of being an endogenous pro-
liferation agent of prostate cancer (PCa) even after androgen deprivation therapy (ADT). A
liquid chromatography–electron capture atmospheric pressure chemical ionization–mass
spectrometric (LC–ECAPCI–MS) method for the determination of Adiol in prostatic tissue
was developed and validated for evaluating the influence of ADT on the prostatic Adiol
level. After derivatization of Adiol with 4-nitrobenzoyl chloride, the detection response of
the derivative was increased 150 times more than that of intact Adiol. The LC–MS method
was specific and reliable for the measurement of a trace amount of Adiol in 30 mg of tissue.
The clinical study using the developed method showed that the prostatic Adiol level was not
changed by ADT. That is, the prostatic Adiol levels of PCa patients with ADT (n = 12), benign
prostate hypertrophy patients without ADT (n = 8) and bladder cancer patients (without pro-
static disease) (n = 6) were 0.83 0.28, 0.62 0.31 and 0.71 0.28 ng g−1 tissue, respectively,
and there was no significant difference between these groups.
Received 27 June 2006
Received in revised form
5 August 2006
Accepted 11 August 2006
Published on line 15 September 2006
Keywords:
Androstenediol
(androst-5-ene-3␤,17␤-diol)
Prostate
Liquid chromatography–mass
spectrometry
© 2006 Elsevier Inc. All rights reserved.
Androgen deprivation therapy
Little is known about the mechanisms behind the transi-
tion of the disease to an androgen-independent stage. One
of the probable causes is the mutation of the androgen
1.
Introduction
receptor (AR) [3], which leads to an anti-androgen with-
drawal phenomenon; in patients with PCa who manifest
disease progression during ADT, discontinuance of anti-
androgen treatment might result in prostate-specific antigen
decline, often associated with clinical improvement. Another
possibility exists that other steroids whose production
cannot be blocked by LH-RH agonists are involved in the
growth stimulation of DHT-independent prostate tumors
[4].
Prostate cancer (PCa) is the most common malignancy in
aged males in the United States and is also sharply increas-
ing in Japan. Because PCa depends initially on androgens,
primarily 5␣-dihydrotestosterone (DHT), androgen depriva-
tion therapies [ADT, usually a combination of luteinizing
hormone-releasing hormone (LH-RH) agonists and anti-
androgens] often are the first choice of several therapeutic
procedures for PCa [1,2]. However, PCa eventually recurs
during ADT despite castrate levels of serum androgens.
∗
Corresponding author. Tel.: +81 76 234 4460; fax: +81 76 234 4459.
E-mail address: higashi@p.kanazawa-u.ac.jp (T. Higashi).
0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2006.08.003

1008
steroids 7 1 ( 2 0 0 6 ) 1007–1013
(Section 2.8)] were prepared from [7,7-2H2]-DHEA [13] in
our laboratories according to known methods [14]. Both of
the deuterium-labeled Adiols showed single peaks in their
LC-positive APCI–MS chromatograms (chemical purities:
more than 99%) and their isotopic purities were as follows;
IS (99.5% 2H5, 0% 2H4, 0.3% 2H3, 0.2% 2H2, 0% 2H1 and 0%
2H0) and D3-Adiol (99.0% 2H3, 1.0% 2H2, 0% 2H1 and 0% 2H0).
Stock solutions of each steroid were prepared as 100 ␮g ml−1
solutions in ethanol. Subsequent dilutions were carried out
with ethanol to prepare 1, 2, 5 and 10 ng ml−1 solutions.
4-Nitrobenzoyl chloride (NBC) was obtained from Tokyo Kasei
Kogyo (Tokyo, Japan). Strata-X cartridges (60 mg adsorbent;
Phenomenex, Torrance, CA, USA) were successively washed
with ethyl acetate (2 ml), methanol (2 ml) and water (2 ml)
prior to use. All other reagents and solvents were of analytical
grade.
Androstenediol (Adiol, androst-5-ene-3␤,17␤-diol) derived
from dehydroepiandrostenone (DHEA) is convertible into
testosterone (T) and further metabolized into DHT. Mitamura
et al. reported that Adiol is the major metabolite of DHEA in
human prostate homogenate in vitro [5]. It has been demon-
strated that Adiol is a strong activator of the AR in several
cell lines [1,6], and some anti-androgens fail to block com-
pletely the androgenic activity of Adiol [6]. These findings
indicate that Adiol may cause the proliferation of PCa even
after ADT. However, the influence of ADT on the Adiol level in
the prostate is poorly understood.
Liquid chromatography–mass spectrometry (LC–MS) has
been recently used for prostatic androgen analysis due to its
specificity and versatility [1,2,7,8]. However, Adiol has a rather
low response using either electrospray ionization or atmo-
spheric pressure chemical ionization (APCI) due to their low
proton-affinitive properties, which leads to a lack of sensi-
tivity. Electron capture APCI (ECAPCI)–MS using a commercial
APCI interface operating in the negative-ion mode is a highly
sensitive technique for electron-affinitive compounds [9–12].
We have demonstrated that LC–ECAPCI–MS coupled with the
introduction of a nitrobenzene moiety enables the analysis of
trace levels of neuroactive steroids [11] and estrogens [12] in
various biological samples.
2.2.
LC–MS(–MS)
LC–MS(–MS) was performed using a ThermoQuest LCQ (San
Jose, CA) liquid chromatograph–ion trap–mass spectrometer
connected to a Jasco PU-980 (Tokyo) chromatograph. A YMC-
Pack Pro C18 RS (5 ␮m, 150 mm × 4.6 mm i.d.; YMC, Kyoto,
Japan) column was used at 40 ◦C. Methanol–water (100:1, v/v)
was used as the mobile phase at a flow rate of 1.0 ml min−1
.
With this background information, in the present paper, a
new LC–ECAPCI–MS method combined with derivatization for
the determination of prostatic Adiol is described. Using the
method, the influence of ADT on the Adiol level in the prostate
is also examined.
Adiol and IS were analyzed as their 3,17-bis-nitrobenzoates
(NB-derivatives, Fig. 1) by APCI–MS–MS [selected reaction
monitoring (SRM)] in the negative-ion mode. Helium was used
as the collision gas, and the relative collision energy was set
at 20%. The sheath gas flow rate was set at 60 units, and the
capillary voltage and tube lens offset were −10 and −15 V,
respectively. The heated capillary temperature and vaporizer
temperature were 200 and 425 ◦C, respectively. The precursor
and monitoring ions of the NB-derivatives were as follows:
Adiol-NB, m/z 588.2 and 588.2; IS-NB, m/z 593.2 and 593.2.
2.
Experimental
2.1.
Materials and reagents
Adiol was synthesized by reducing DHEA with NaBH4.
[7,7,16,16,17␣-2H5]-Adiol (IS) and [7,7,17␣-2H3]-Adiol [D3-
Adiol, which was used for the determination of the recovery
rate during the pretreatment (Section 2.7) and the influence
of the prostate components on derivatization efficiency
2.3.
Calibration curve for Adiol
Standard solutions of Adiol (10, 20, 50, or 100 pg) and IS (200 pg)
placed in a tube were derivatized as described below and
Fig. 1 – Structures of Adiol, IS and their derivatized forms with NBC. D = deuterium (2H).

steroids 7 1 ( 2 0 0 6 ) 1007–1013
1009
subjected to LC–MS–MS, and the calibration curves were con-
structed by plotting the peak area ratios (Adiol/IS) against the
amounts of Adiol (pg).
and the resulting residue was dissolved in ethanol (30 ␮l), 10 ␮l
of which was subjected to LC–MS–MS.
2.7.
Recovery rate of Adiol during pretreatment
2.4.
Prostatic tissue
procedure
Prostatic tissues were obtained from 12 PCa patients with
neoadjuvant hormone therapy (combination of LH-RH agonist
and anti-androgen drug) for 3–6 months before radical prosta-
tectomy, 8 benign prostate hypertrophy (BPH) patients without
any hormone therapy and 6 bladder cancer (BCa) patients (rad-
ical cystoprostatectomy for BCa) and stored at −20 ◦C prior to
use. Some of the BCa patients had received chemotherapy with
platinum preparations but no patient had received hormone
therapy. All the patients gave informed consent at Kanazawa
University Hospital (Kanazawa, Japan).
Because prostate tissue in which Adiol was not detected could
not be obtained, the recovery rate of Adiol during the pre-
treatment procedure was determined using the tissue spiked
with D3-Adiol. The ethanolic solution of D3-Adiol (50 pg in
10 ␮l) or ethanol (10 ␮l, control sample) was added to the
prostatic tissue (30 mg), and the resulting samples were pre-
treated. D3-Adiol (50 pg) was then added to only the control
sample, and IS (200 pg) was added to both samples. After
derivatization, the samples were subjected to LC–MS–MS. The
contaminant of D3-Adiol in IS and the isotopic ion of D3-
Adiol might have some influence on the peak areas of D3-
Adiol (the analyte for the recovery determination) and IS,
respectively. However, the influence of the contaminant on
the measurement of D3-Adiol was not observed due to the
extremely high isotope purity of IS. The influence of the iso-
topic ion at m/z 593 of D3-Adiol on the peak area of IS could
be also neglected (less than 1%). Based on these results, the
recovery rate was calculated from the peak area ratios (D3-
Adiol/IS) of the spiked and control samples without any cor-
rection.
2.5.
Pretreatment procedure for analysis of Adiol in
human prostate
Prostatic tissue was minced by scissors and crushed by a glass
homogenizer on ice. IS (200 pg) was added to the crushed
tissue (30 mg) and further homogenized in methanol–water
(3:7, v/v, 0.2 ml) using a glass homogenizer. The homogenate
was heated at 60 ◦C for 30 min and centrifuged at 1500 × g
(4 ◦C, 5 min). The supernatant was saved and the precipitate
was suspended with methanol–water (3:7, v/v, 0.2 ml) and
heated at 60 ◦C for 30 min. After centrifugation at 1500 × g
(4 ◦C, 5 min), the supernatants were combined (this solution
is called the prostate extract). The prostate extract was added
to acetonitrile (1 ml), vortex-mixed for 30 s and centrifuged
at 1500 × g (4 ◦C, 5 min). The supernatant was diluted with
water (3 ml) and purified using a Strata-X cartridge. After suc-
cessive washing with water (2 ml), methanol–water (7:3, v/v,
2 ml) and hexane (1 ml), the steroids were eluted with ethyl
acetate (1 ml). After evaporation, the residue was dissolved
in hexane–isopropanol (19:1, v/v, 40 ␮l) containing proges-
terone (20 ng) and subjected to high-performance liquid chro-
matography (HPLC) [Hitachi L-7110 chromatograph (Tokyo)
with Shimadzu SPD-10A UV detector; column, Develosil (3 ␮m,
100 mm × 4.6 mm i.d., Nomura Chemical, Seto, Japan); mobile
2.8.
Influence of prostate components on
derivatization efficiency
D3-Adiol (50 pg) was derivatized for preparation of a con-
trol sample (this sample was derivatized without prostate
components). D3-Adiol (50 pg) was added to the pretreated
prostate sample and then derivatized (test sample, deriva-
tization was carried out with prostate components). After
addition of the derivatized IS (200 pg of IS was derivatized
beforehand) to both the control and test samples, they
were subjected to LC–MS–MS. The influence of prostate
components on derivatization efficiency was evaluated from
the peak area ratios (D3-Adiol/IS) of the test and standard
samples.
phase, hexane–isopropanol (19:1, v/v); flow rate, 1.0 ml min−1
;
column temperature, 40 ◦C and detection, 240 nm]. Because
Adiol and IS were eluted just behind progesterone, which
shows an intense absorption at UV 240 nm (retention time,
4.6 min), the reproducible collection of the fraction containing
Adiol and IS (retention time, 5.1–6.4 min) could be done by use
of the peak of progesterone as a mark. After evaporation, the
residue was subjected to derivatization with NBC as described
below.
2.9.
recovery)
Assay accuracy (matrix effect and analytical
The matrix effect was examined by comparing the slope of
the calibration curve constructed as described above and that
of a curve prepared by adding Adiol (10, 20, 50 and 100 pg) to
prostatic tissue (30 mg) (matrix sample). The matrix samples
were prepared using five different tissues.
The analytical recovery was determined as follows. Ethanol
(10 ␮l, unspiked sample) or an ethanolic solution of Adiol
(30 pg in 10 ␮l, spiked sample) was added to the prostatic
tissue (30 mg) (the spiked concentrations of Adiol was 0 or
1.0 ng g−1 tissue, respectively). After the addition of IS (200 pg),
the resulting samples were pretreated, derivatized and ana-
lyzed by LC–MS–MS. The analytical recovery of Adiol was
defined as F/(F0 + 1.0) × 100%, where F and F0 are the Adiol
concentrations in the spiked and unspiked samples (0.64 or
1.12 ng g−1 tissue, see Table 1), respectively.
2.6.
Derivatization reaction
The calibration curve samples or prostate samples were dried
and added with reagent, NBC (20 ␮g) in benzene (40 ␮l) and cat-
alyst, quinuclidine (20 ␮g) in benzene (10 ␮l). The mixture was
kept at 80 ◦C for 10 min, the additional reagent and catalyst
(20 ␮g each) were then added, and the entire mixture was fur-
ther kept at 80 ◦C for 30 min. After addition of ethanol (30 ␮l) to
decompose the excess reagent, the solvents were evaporated

1010
steroids 7 1 ( 2 0 0 6 ) 1007–1013
Table 1 – Assay precision and accuracy
BPH tissue
PCa tissue
Measureda
Expectedb
Precision/
recovery (%)
Measured
Expected
Precision/
recovery (%)
(ng g−1 tissue)
(ng g−1 tissue)
(ng g−1 tissue)
(ng g−1 tissue)
Intact (intra-assay)
Intact (inter-assay)
Spiked sampled
0.63 0.044
0.64 0.048
1.56
–
–
1.64
R.S.D.c: 7.0
R.S.D.: 7.5
Recoverye: 95.1
1.03 0.083
1.12 0.057
2.26
–
–
2.12
R.S.D.: 8.1
R.S.D.: 5.1
Recovery: 106.5
a
Mean S.D. (n = 5) for the intra- and inter-assay variation tests and mean of duplicate assays for the analytical recovery test.
Expected values were calculated based on the values obtained in the inter-assay variation test.
S.D./mean × 100.
b
c
d
e
Adiol (30 pg) was spiked into the prostatic tissue (30 mg) and then analyzed.
Analytical recovery: measured value/expected value × 100.
2.10. Assay precision
The intra-assay precision was assessed by determining two
prostate samples at different concentration levels (n = 5 for
each sample) on a day. The inter-assay precision was assessed
by determining these samples over 5 days. The precision was
determined as the relative standard deviation (R.S.D., %).
2.11. Determination of prostatic DHT
The DHT level in PCa or BPH tissue was determined by the
previously developed method [7].
3.
Results and discussion
3.1.
LC–ECAPCI–MS(–MS) of NB derivatives
For the APCI–MS operating in the negative-ion mode, the
NB-derivatives of Adiol and IS provided only their molecular
anions [M]− at m/z 588.2 and 593.2, respectively (Fig. 2a and b).
This demonstrated that the derivatives underwent electron
capture in the APCI source. The limit of detection [signal to
noise ratio (S/N) = 5] of the derivatized Adiol (6.8 fmol per injec-
tion) was 150 times greater than that of intact Adiol [1.0 pmol
(300 pg) per injection] analyzed by positive APCI–MS.
The derivatization rate of nanogram amounts of Adiol with
NBC was almost quantitative, because after derivatization of
Adiol (10 ng), intact steroid was not detected in LC–positive
APCI–MS [the minimum detectable amount of intact Adiol was
300 pg (3% of the initial amount)]. Next, 10 ng and 50 pg of Adiol
were individually derivatized and then dissolved in 6 ml and
30 ␮l of ethanol, respectively. Ten microliters of the respec-
tive solutions were subjected to LC–MS. As a result, the peak
areas obtained from both the solutions were practically equal.
This result demonstrates that derivatization rate is quantita-
tive even when picogram amounts of Adiol are derivatized.
The use of the SRM mode may allow for the discrimination
and quantification of Adiol from prostate components without
the need for a long chromatographic separation, due to its high
specificity. Based on this, MS–MS analysis of the derivative
was also examined, where [M]− was used as the precursor ion.
However [M]− of the derivative was very stable, and therefore,
no characteristic product ion was formed in MS–MS (Fig. 2c).
Based on this result, the following SRM mode was employed
Fig. 2 – Mass spectra of derivatized (a) Adiol, (b) IS and (c)
product ion mass spectrum of derivatized Adiol.

steroids 7 1 ( 2 0 0 6 ) 1007–1013
1011
for the determination of Adiol in the prostatic tissue; after col-
lision of [M]− with 20% relative collision energy, the residual
same ion was monitored, by which the noise ions derived from
the prostate components were reduced while comparatively
maintaining the intensity of the monitoring ion.
3.2.
Pretreatment of prostate tissue
The extraction of Adiol from the prostatic tissues was per-
formed by the previously developed method [7] with
a
slight modification. The prostate extract was purified using
a Strata-X cartridge and normal-phase HPLC. The Adiol
fraction was then treated with excess NBC. The recovery
rate [mean standard deviation (S.D.), five different tissues]
of Adiol during the extraction and purification steps was
61.7 2.6%. The above pretreatment procedure was useful
to remove endogenous components that interfere with the
derivatization reaction, so that the derivatization efficiency of
the prostate sample was almost equal to that of the standard
sample (96.1 2.4%, mean S.D., n = 3).
Typical chromatograms of the prostate samples obtained
from patients with PCa, BPH and BCa are shown in Fig. 3.
The peaks corresponding to the derivatized Adiol and IS were
clearly observed at 5.0 and 4.9 min, respectively. This slight
difference in their retention times is due to isotope effect.
3.3.
Calibration curve and limit of quantitation
The calibration curve was constructed by plotting the peak
area ratio (Adiol/IS, y) versus the amount (pg) of Adiol per
tube (x) using the standard solutions. The regression line
obtained from the combination of five standard curves was
y = 0.00516x + 0.00209 with a correlation coefficient (r2) of 0.999
within the range of 10–100 pg per tube. The R.S.D. value of the
slope was 1.0%.
To determine the extent to which the prostate matrix
affects the quantification, the slope of the above calibra-
tion curve was compared to that of the curve prepared with
the matrix sample. As a result, the slope of the latter was
0.00524 0.00015 (mean S.D., n = 5, R.S.D. 2.9%), which was
practically identical to the slope of the curve constructed
with standard solutions. This result clearly revealed that the
prostate matrix did not affect the calibration curve. Based on
this result and the fact that it is impossible to obtain Adiol-free
prostatic tissue, the calibration curve was constructed using
the standard solutions in the following studies. The applicabil-
ity of this curve to the prostatic Adiol assay was also examined
in the analytical recovery test that will be discussed later.
Because Adiol-free prostatic tissue was not available, the
limit of quantitation of this assay could not be determined.
However, at least 0.35 0.05 ng g−1 tissue (mean S.D., n = 5)
of prostatic Adiol could be determined with acceptable repro-
ducibility (R.S.D. 15.0%) and S/N value (more than 6).
Fig. 3 – Representative chromatograms obtained from
prostatic tissue of (a) PCa, (b) BPH and (c) BCa patients.
Adiol concentrations were (a) 1.00, (b) 0.64 and (c)
1.08 ng g−1 tissue, respectively.
The prostatic tissues with the addition of a known amount
of Adiol were pretreated and analyzed in order to exam-
ine the analytical recovery, and a satisfactory recovery was
obtained (Table 1). Although the calibration curve was con-
structed using the standard solutions of Adiol in the present
study as mentioned above, this result demonstrates that the
prostatic Adiol can be accurately determined using the curve.
3.4.
Assay precision and accuracy
The intra-assay (n = 5) R.S.D. values were less than 8.1% and
good inter-assay (n = 5) R.S.D. values (less than 7.5%) were also
obtained, as shown in Table 1.

1012
steroids 7 1 ( 2 0 0 6 ) 1007–1013
Table 2 – Prostatic Adiol and DHT levels
Adiol
DHT
Mean S.D. (ng g−1 tissue)
Mean S.D. (ng g−1 tissue)
Range (ng g−1 tissue)
Range (ng g−1 tissue)
PCa (n = 12)
BPH (n = 8)
BCa (n = 6)
0.83 0.28
0.62 0.31
0.71 0.28
0.48–1.45
0.26a–1.22
0.35–1.08
Not detected
4.95 1.39
Not examined
–
3.10–7.41
–
a
When the Adiol level was below 0.35 ng g−1 tissue, 50 mg of tissue was used for its determination.
androstenediol is present in prostate cancer tissue after
androgen deprivation therapy and activates mutated
androgen receptor. Cancer Res 2004;64:765–71.
[2] Titus MA, Schell MJ, Lih FB, Tomer KB, Mohler JL.
Testosterone and dihydrotestosterone tissue levels
in recurrent prostate cancer. Clin Cancer Res 2005;11:
4653–7.
[3] Feldman BJ, Feldman D. The development of
androgen-independent prostate cancer. Nat Rev Cancer
2001;1:34–45.
[4] Ha¨rko¨nen P, To¨rn S, Kurkela R, Porvari K, Pulkka A, Lindfors
A, Isomaa V, Vihko P. Sex hormone metabolism in prostate
cancer cells during transition to an androgen-independent
state. J Clin Endocrinol Metab 2003;88:705–12.
[5] Mitamura K, Nakagawa T, Shimada K, Namiki M, Koh E,
Mizokami A, Honma S. Identification of
These data indicate that the present method is highly repro-
ducible and accurate.
3.5.
Influence of ADT on prostatic Adiol level
The influence of ADT on the Adiol level in the prostate was
examined using the developed method. The prostatic Adiol
levels in the patients with PCa, BPH and BCa are summarized
in Table 2. There was no significant difference in the level
between the three groups. This result demonstrates that Adiol
is present in PCa tissue after ADT at the same levels of tissues
without hormone treatment and contrasts with the prostatic
DHT level, which is significantly reduced by ADT (Table 2). As
mentioned in Section 1, several reports proved that Adiol is a
strong activator of the AR in PCa cells without being metab-
olized to T or DHT [1,6] and that some anti-androgens fail to
block completely the androgenic activity of Adiol [6]. From our
results and the above findings, Adiol is strongly suspected of
being a proliferation agent of PCa even after ADT. Therefore,
the development of new therapeutic approaches that block
Adiol’s androgenic action is worth investigating. Regarding
this, Miyamoto et al. [15,16] recently reported new steroidal
anti-androgens, which can block the Adiol-induced AR trans-
activation.
dehydroepiandrosterone metabolites formed from prostate
homogenate using liquid chromatography–mass
spectrometry and gas chromatography–mass spectrometry. J
Chromatogr A 2002;961:97–105.
[6] Miyamoto H, Yeh S, Lardy H, Messing E, Chang C.
ꢀ5-Androstenediol is a natural hormone with androgenic
activity in human prostate cancer cells. Proc Natl Acad Sci
USA 1998;95:11083–8.
[7] Higashi T, Yamauchi A, Shimada K, Koh E, Mizokami A,
Namiki M. Determination of prostatic androgens in 10 mg of
tissue using liquid chromatography–tandem mass
spectrometry with charged derivatization. Anal Bioanal
Chem 2005;382:1035–43.
[8] Nishiyama T, Hashimoto Y, Takahashi K. The influence of
androgen deprivation therapy on dihydrotestosterone levels
in the prostatic tissue of patients with prostate cancer. Clin
Cancer Res 2004;10:7121–6.
[9] Singh G, Gutierrez A, Xu K, Blair IA. Liquid
chromatography/electron capture atmospheric
pressure chemical ionization/mass spectrometry:
analysis of pentafluorobenzyl derivatives of biomolecules
and drugs in the attomole range. Anal Chem
4.
Conclusion
In this study, we developed the LC–MS method for the deter-
mination of Adiol in prostatic tissue after converting Adiol to
a highly detectable derivative in ECAPCI–MS. The method was
specific and reproducible, and enabled the analysis of a trace
amount of Adiol using a small amount of sample.
The clinical study using the developed method found that
reduction of the prostatic Adiol level by ADT was not observed.
This result and the in vitro experimental data previously
reported [1,6] strongly suggest that Adiol may be one of the
agents that advance PCa even when DHT is exhausted by ADT.
2000;72:3007–13.
[10] Higashi T, Takido N, Yamauchi A, Shimada K.
Electron-capturing derivatization of neutral steroids for
increasing sensitivity in liquid chromatography/negative
atmospheric pressure chemical ionisation–mass
spectrometry. Anal Sci 2002;18:1301–7.
[11] Higashi T, Takido N, Shimada K. Studies on neurosteroids
XVII. Analysis of stress-induced changes in neurosteroid
levels in rat brains using liquid chromatography–electron
capture atmospheric pressure chemical ionisation–mass
spectrometry. Steroids 2005;70:1–11.
[12] Higashi T, Takayama N, Nishio T, Taniguchi E, Shimada K.
Procedure for increasing the detection responses of
estrogens in LC–MS based on introduction of a nitrobenzene
moiety followed by electron capture atmospheric pressure
chemical ionization. Anal Bioanal Chem, in press.
[13] Blair IA, Phillipou G, Seaborn C. Synthesis of C7-2H2-steroids
for human metabolism studies. J Label Compd Radiopharm
1978;15:645–55.
Acknowledgment
Part of this work was supported by grants from the Japan Soci-
ety for the Promotion of Science (JSPS).
r e f e r e n c e s
[1] Mizokami A, Koh E, Fujita H, Maeda Y, Egawa M, Koshida K,
Honma S, Keller ET, Namiki M. The adrenal androgen

steroids 7 1 ( 2 0 0 6 ) 1007–1013
1013
[14] Dehennin L, Reiffsteck A, Scholler R. Simple methods for the
a potent antiandrogen with marginal agonist activity. Proc
synthesis of twenty different, highly enriched
deuterium-labeled steroids, suitable as internal standards
for isotope dilution mass spectrometry. Biomed Mass
Spectrom 1980;7:493–9.
Natl Acad Sci USA 2003;100:4440–4.
[16] Miyamoto H, Marwah P, Marwah A, Yang Z, Chung CY,
Altuwaijri S, Chang C, Lardy H. Identification of steroid
derivatives that function as potent antiandrogens. Int J
Cancer 2005;117:866–72.
[15] Miyamoto H, Marwah P, Marwah A, Lardy H, Chang C.
3␤-Acetoxyandrost-1,5-diene-17-ethylene ketal functions as