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Home , Epiandrosterone, Epitestosterone, Epitiostanol, Gestrinone, Mepitiostane, Mestanolone

In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strauß - Köln 2007

Masato Okano, Ayako Ikekita, Mitsuhiko Sato, Shinji Kageyama

Detection of in Doping Analysis

Anti-doping center, Mitsubishi Chemical Medience Corporation, Tokyo Japan

Introduction Hematopoetics, anabolic androgenic used in doping purposes are prohibited by WADA. Table-1 shows most of the clinically used hematopoietics in Japan except for iron preparations 1, our laboratory already has readied analytical methods for EPO and almost anabolic steroids, however, analytical methods have not been conducted for mepitiostane and . Table-1 Hematopoietics used clinically in Japan 1 Commercial name Active compound Epogin, Exprex Erythropoetin alfa Espo, Recomon Erythropoetin beta Aranesp Darbepoetin alfa Hemataide Synthetic peptide Mircera Erythropoietin receptor activator Duran Duramin Nandrolone cyclohexylpropionate Deca Durabolin, Deca Duramin Durabolin Nandrolone phenylpropionate Mesanolon Primobolan Thiodol Epitiostanol Thioderon Mepitiostane

(except for sideropenia anemia medicine)

Mepitiostane (Thioderon®, 2α, 3α-epithio-17β-(1-methoxycyclopentyloxy)-5α-) -17β-ol), the prodrug of epitiostanol (2α, 3α-epithio-5α-androstane-17β-ol) is an epithiosteroid having anti-estrogenic activity, a concomitant weak androgenic and myotropic activity, and have been used in the treatment of mammary cancer. Mepitiostane and epitiostanol increase hemoglobin in the circulation by stimulating the maturation of CFU-E in

123 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strauß - Köln 2007

the bone marrow. Thioderon® was developed and produced by Shionogi Pharmaceutical Co. Ltd. (Osaka, Japan) 2. Production of Thiodol® was discontinued in 2001, however, Thiodol® has been therapeutically used for the same purposes as Thioderon®. After oral administration of mepitiostane, it is metabolized to the active hematopoetic compound epitiostanol via detachment of methoxycyclopentyloxy from the 17th position and epitiostanol is metabolized to 2,(5α)-androsten-17β-ol (M-1) and 2, (5α)-androsten-17-one (M-2) mainly by oxygenation (Fig.1) 3, 4.

CH3O

O OH H H H H S HH S HH H H Epitiostanol Mepitiostane

oxygenation oxygenation/dehydrogenation

OH O H H H

SO HH SO HH

H H Epitiostanol S-oxide

dethionylation dethionylation

OH O H H H

HH HH

H M-1 H M-2

Epitiostanol Olefin

Fig.1 The metabolic pathway of mepitiostane3, 4

Mepitiostane and epitiostanol are not mentioned in the WADA prohibited List in 20075. There were no reports concerning the abuse of this substance by athletes, however, it is possible that some athletes use them for doping purposes. Therefore the aim of this study was to establish adequate GC/MS and LC/MS parameters for the screening of mepitiostane and also to start the screening in routine doping tests.

124 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strauß - Köln 2007

Chemicals All reagents were analytical grade, acetic acid, hydrochloride, diethyl ether, ethanol, potassium carbonate anhydrous, formic acid, sulphuric acid, ethyl acetate, sodium acetate trihydrate, sodium sulfate, sodium sulfate anhydrous and HPLC grade methanol were purchased from Kokusan Chemical Works (Tokyo, Japan). Dithioerytritol was purchased from Nakalai Tesuque (Osaka, Japan), iodotrimethylsilane (TMSI) was purchased from Aldrich (Steinheum, Germany) and N-methyl-N-trimethylsilylfluoroacetamide (MSTFA) was

purchased from Marchery Nagel (Düren, Germany). XAD-2 and Bond Elut C18 3 cc/500 mg were products of Sigma-Aldrich Japan (Tokyo, Japan) and Varian (Walnut Creek, CA, USA). Glufatase type A-2(β-glucuronidase; arylsulfatase = 42,000 Fishman U: 21,000 Roy U per ml each) from Ampullaria was purchased from Nippon Bio-Test (Tokyo, Japan), β-glucuronidase from E.coli (80 unit/ml) was purchased from Boehringer Manheim (Düsseldorf, Germany). Thioderon® and mepitiostane were purchased from Shionogi Pharmaceutical Co. Ltd. (Osaka, Japan). Epitiostanol was from Society of Japanese Pharmacopoeia (Tokyo, Japan). 2, (5α)-androsten-17β-ol and 2, (5α)-androsten-17-one were purchased from Steraloids Inc. (Newport, RI, USA).

Sample preparations 2 mL of human sample was applied to a solid-phase an Amberlite XAD-2. And the solid-phase was washed with 2 mL of distilled water. Elution was performed with 2.5 mL of methanol. The elute was dried under nitrogen stream at 40 °C. The dried residue was dissolved in 1 mL of 0.2 M sodium acetate buffer (pH 5.2) followed by addition of 50 μL of internal standard solution (20 μg/ml 17α-). Hydrolysis was performed with

30 μL of Glufatase type A-2 at 60 °C for 1 hr. After addition of 0.25 mL of 7 % K2CO3, the steroids were extracted with 5 mL of diethyl ether. The organic layer was evaporated under nitrogen stream at 40 °C. The dried residue was dissolved in 50 μL of MSTFA/TMSI/DTE, 1000/2/2(v/v/w) and heated at 60 °C for 15 min for GC-MS analyses and was dissolved in

1 % CH3COOH/CH3CN: 65/35(V/V) for LC/MS analyses.

Instrumentation and GC-MS and LC-MS parameters The GC-MS system was an Agilent 6890/5973 inert mass equipped with an Ultra-1 capillary column, length 17 m, 0.25 mm internal diameter, 0.11 μm film thickness (Agilent Technologies, Palo Alto, CA, USA). The LC-MS experiments were performed using an

125 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strauß - Köln 2007

Agilent HPLC 1100 Series (Agilent Technologies, Palo Alto, CA, USA), which was interfaced to a QSTAR XL MS/MS (Applied Biosystems, Foster City, CA, USA). The analytical column

was a Supelco Discovery C18 column (id = 4.0 mm; length = 50 mm). Table-2 shows the detailed analytical parameters of GC-MS and LC-MS system.

Excretion study Two capsules of Thioderon® containing mepitiostane 10mg were administered to a 37-year-old healthy male volunteer. Blank sample was collected before an oral administration. Urine samples obtained were collected for the first 49 hours after the administration to detect target metabolites of mepitiostane.

Table-2 GC-MS and LC-MS parameters

GC/MS system parameters: Volume: 2 μL, Temp.: 280 °C Split mode (11:1) Carrier gas: Helium at constant flow rate of 1mL/min. Oven temp. program: Initial 180 °C hold 1 min, 3 °C/min to 229 °C and 40 °C/min to 300 °C, hold 2 min Ionisation: 70 eV, electron impact (EI) Data acquisition: Scan range 70 to 500, SIM mode Monitored and dwell time: 129, 256 and 346 m/z; 10, 20, 25 mesec.respectively LC/MS system

Mobile phase: A: 1 % CH3COOH, B: CH3CN, Flow rate: 250 μL/min Temp: 25 °C Gradient: 0 min - 10 min: A 65 % to 10 %, 10.1 min: A 65 % Run time: 11 min Ionization condition(Polarity): ESI (Positive mode) APPI (Positive mode) spray Temp.: 450 °C 400 °C Ion spray Voltage: 5,500 V 1,300 V

Neblizer Gas: 2.85 L/min(Zero Air) 3.94 L/min(N2) Dopant: Toluene 20 μL/mim

Aux. Gas: 4.80 L/min(Zero Air) 2.00 L/min(N2) TOF MS range: m/z 140 to 600 m/z 140 to 600

Results and Discussion The total ion chromatograms and mass spectrum of TMS-derivatives of epitiostanol, M-1 and M-2 are shown in Fig.2. The relative retention time of eptitiostanol, M-1 and M-2 were 0.93, 0.43 and 0.41 respectively. The typical fragment ions of epitiostanol-O-TMS were at m/z 378(M+), 346(M+ -S) and 129 (D-ring fragment) , those of M-1 as TMS derivative were at m/z 346(M+), 256(M+ -TMSOH) and 129 (D-ring fragment) and those of M-2 as TMS derivative

126 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strauß - Köln 2007

+ + were at m/z 344(M ), 329(M -CH3) and 169 (D-ring fragment).

Abundance

TIC: 0400004.D 15.30 1400000 I.S 1300000 1200000 TIC 1100000

1000000

900000

800000

6.57 700000 M-1

600000 500000 M-2 Epitiostanol 400000 6.31 10.31 300000

200000 14.21 6.42 100000 9.43

6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 Time-->

Abundance

Scan 203 (6.563 min): 0400004.D 46000 129

44000

42000

40000

38000 202 36000 73 34000

32000

30000

28000 M-1

26000 241

24000

22000 91 256

20000 346 18000

16000

14000 105 12000

10000 145 331 55 8000 161

6000 187

4000 292

217 2000 277 313 397 0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 m/z--> Abundance

Scan 168 (6.307 min): 0400004.D 60000 329

55000

50000

45000

40000

35000 73 344 M-2 30000

25000

20000

15000

10000 91 169

5000 55 107 131 155 183 207 221 239 275 253 290 313 364381 0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380

Am/z-->bundance

Scan 1249 (14.215 min): 0400004.D 129 10000

9500

9000 73 202 8500

8000

7500

7000

6500

6000 241 5500 Epitiostanol 5000

4500 346 91105 4000 256 3500

3000

2500 331 55 147 2000 161 1500 187 292 1000 217 500 273 378 307 363 0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 m/z--> Fig.2 The total ion chromatograms and mass spectrum of standard mixture (TMS-derivatives of epitiostanol, M-1 and M-2)

127 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strauß - Köln 2007

Abundance

TIC: 0100001.D 15.32 1600000 1500000 I.S 1400000

1300000

1200000

1100000

1000000 900000 Degradation product 800000

700000

6.58 600000 Epitiostanol 500000

400000 10.33

300000 14.23 200000

100000 9.44

0 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 Time-->

Abundance Abundance

Scan 206 (6.585 min): 0100001.D 129 202 Scan 1250 (14.222 min): 0100001.D 36000 129 34000 15000 73 32000 14000

30000 13000 73 28000 12000 26000 11000 202 24000 241 10000 22000 9000 20000 241 18000 8000 91 256 16000 7000 91 346 14000 6000 105 12000 5000 346 10000 4000 331 107 8000 55 147 3000 55 145 6000 187 161 2000 4000 257 162 217 292 1000 185 292 2000 221 311 329 378 272 316 369 0 0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 m/z--> m/z-->

Fig.3 TIC and the mass spectrums obtained after delivatization of epitiostanol

Epitiostanol M-1 M-2

m/z 129 m/z 129 m/z 169

Std. m/z 346 m/z 256 m/z 329

m/z 378 m/z 346 m/z 344

Pre

7.7hr

Fig. 4 Detection of mepitiostane metabolites by GC/MS

128 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strauß - Köln 2007

The peak at the relative retention time of 0.43 was also observed on the total ion chromatogram of epitiostanol, and its mass spectrum agreed with that of M-1. Therefore, our interpretation is that epitiostanol was pyrolized to M-1 during the derivatization or GC/MS analysis (Fig.3). Metabolite M-1 could be detected in the urine sample collected 7.7 hours after a 10 mg oral administration of mepitiostane (Fig.4), however, epitiostanol and M-2 could not be detected.

Table-3 Metabolites of mepitiostane and their proposed TMS derivatives Chemical name Derivatives MW Epitiostanol 2α,3α-epitio-5α-androstan-17β-ol mono-TMS 378 M-1 2,(5α)-androsten-17β-ol mono-TMS 346 M-2 2,(5α)-androsten-17-one mono-TMS 344 M-3 2α-hydroxy-epi- tris-TMS 522 M-4 2β,16β-dihydroxy-androsterone tris-TMS 538 M-5 2-keto-epi-androsterone tris-TMS 520 M-6 5α-androstan-2β,3α,17β-triol tris-TMS 524 M-7 2β-OH- tris-TMS 522 M-8 2β-OH-androsterone tris-TMS 522 M-9 5α-androstan-2β,3α,17β-triol-16-one tetra-TMS 610

The other metabolites of mepitiostane are shown in Table-33, 4, but the reference standards are not available in our laboratory. The ions m/z 522 and 520 are extracted from full scans, and four peaks could be observed in the administrated urine sample (Fig.5). These peaks were not observed in the urine before the administration. It may be suggested that these were metabolites of mepitiostane listed in Table-3.

Pre After administration

m/z 522 m/z 522

m/z 520 m/z 520

Fig. 5 Other proposed metabolites of mepitiostane

129 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strauß - Köln 2007

1600 1400 1200 1000 800 600 400 Conc. (ng/mL) 200 0 0 1020304050 Time after administration (hr)

Fig.6 Urinary concentration of 2, (5α)-androsten-17β -ol (M-1) after administration of mepitiostane

M-1 was detected up to 49 hours after a 10 mg administration of mepitiostane (Fig.6). Epitiostanol and M-2 were not detected over a time period of 49 hours. Furthermore, the metabolic profile of mepitiostane was investigated according to Axelson’s ion-exchange method 6. The result showed that M-1 was excreted as its glucuronide.

Table-4 Concentrations of endogenous steroids after administration

Time course 0'00'' 1'20'' 4'40'' 7'40'' 11'40'' 16'50'' 23'10'' 25'40'' 32'15'' 40'50'' 49'35'' Androsterone 508 1149 1531 2158 1247 997 1151 1240 1743 1556 1200 756 1527 1884 2595 1400 1410 1610 1802 2131 1965 1655 6.0 5.6 3.2 3.8 3.8 2.9 3.7 4.0 4.2 5.3 4.8 8.4 6.9 10.9 13.7 8.6 6.2 11.3 7.8 12.3 11.0 12.9 5α- 0.0 0.4 0.7 1.5 1.0 1.3 0.7 1.3 1.2 1.8 1.6 5β-androstan-3α,17βdiol 12.4 29.1 37.9 45.5 38.4 32.6 29.0 30.4 36.3 56.4 33.8 5α-androstan-3α,17βdiol 10.0 20.0 33.2 32.0 28.0 38.3 22.8 22.7 35.3 49.4 39.0 70.8 136.0 107.4 192.4 154.5 126.1 137.3 190.5 193.8 222.5 203.7 6-OH- 3.2 3.6 1.6 1.7 1.8 1.7 2.0 2.8 2.0 2.8 2.5 Unit: ng/mL

Steroid profiles after administration are shown in Table-4 in order to investigate the effects of anti- substances on parameters. It seems that concentrations of androsterone and etiocholanolone were increased by the administration, however, we are not able to judge

130 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strauß - Köln 2007

whether it was significantly influenced by the administration. Further studies (more volunteers and multiple dosages) must be performed. In addition, it was necessary to perform LC/MS identification in order to verify the existence of epitiostanol in the urine after administration. Epitiostanol, M-1 and M-2 could not be detected by our screening procedure for heat labile compounds (THG and ) by LC/MS analysis in positive ESI mode, because the proton affinity of the metabolites is too low for ionization. Atmospheric photo spray ionization (APPI) is known to be more effective for non-polar compounds than ESI. Hence, LC/MS analysis in APPI mode was performed + (Fig.7). The monitoring ions of epitiostanol, M-1 and M-2 were m/z 289 [M+H-H2O] , 257 + + [M+H-H2O] , and 255[M+H-H2O] respectively. Mepitiostan was detected successfully as epitiostanol over a time period of 4.7 hours after the administration. M-1 could be detected 7.7 hours after the administration.

Epitiostanol M-1 M-2

m/z m/z m/z 289 257 255 100ng/ml Spiked

Pre.

4.7hr

7.7hr

Fig.7 Detection of metabolites of mepitiostane by LC/APPI (+)-TOFMS

131 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (15). Sport und Buch Strauß - Köln 2007

Conclusion The common steroid screening procedure by GC/MS is suitable for the detection of mepitiostan abuse, and then the target substance should be 2, (5α)-androsten-17β-ol as TMS derivative. The detection of specific metabolite epitiostanol by LC/MS in APPI-mode could be proposed for the confirmation analysis. The mentioned analytical methods for the hydroxy-metabolites are still in investigation.

References 1. Drugs in Japan Forum (2007) Drugs in Japan Ethical drugs. 2. Shionogi Pharmaceutical Co. Ltd. Osaka, Japan (2005) Drug information leaflet 3. Pharmaceutical Co. Ltd. Osaka, Japan (1982) A study about of Mepitiostane. 4. T. Ichihashi, H. Kinoshita, K. Shimamura, H. Yamada (1991) Absorption and disposition of in rats (1): Route of administration and plasma levels of epitiostanol. Xenobiotica 21, 865-872. 5. World Anti-Doping Agency. The 2007 prohibited list. International Standard, Montreal (2007) http://www.wada-ama.org/rtecontent/document/2007_LIST.pdf (access date 10.01.2007) 6. M. Axelson, B.-L. Sahlberg, J. Sjovall (1981) Analysis of profiles of conjugated steroids in urine by ion-exchange separation and gas chromatography-mass spectrometry. J Chromatogr 224, 355-370.

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