Journal of Analytical Toxicology, Vol. 14, March/April 1990

Urinary Excretion of Furazabol Metabolite Claire Y. G r a d e e n , Siu C. Chan ~ and P e t e r S. Przybylski

Calgary Olympic Doping Control Centre, Department of Laboratory Medicine, Foothills Hospital, 1403, 29th Street N.W., Calgary, Alberta, Canada T2N 279

Abstract J

Experimental

Furazabol, a newer anabollc steroid, is metabolized into 16-hydroxyfurazabol and excreted in urine. The presence of this compound in urine can be monitored with a GC/MS procedure. It can be Incorporated into the general dope testing protocol for anabolic steroids.

Introduction Furazabol (17-/3-hydroxy- 17-t~-methylandrostano[2,3 -c] furazan) is an anabolic steroid first synthesized in 1965 (l). In animal studies, it was shown to be more anabolic than androgenic (2). In subcutaneous administration, the myotrophic/androgenic ratio of furazabol was determined to be 5.8, with testosterone propionate as the reference which has a ratio of 1. This compared favorably to stanozolol which has a ratio of 4.0. Stanozolol (17-/3-hydroxy-17-a-methylandrostano[2,3-c]pyrazole)has a structure very similar to that of furazabol (Figure 1). Stanozolol has become a household word since the 1988 Olympic Summer Games in Seoul, Korea. A high profile Canadian athlete tested positive for this anabolic steroid. Consequently, the Canadian Government conducted an inquiry, under the direction of Commissioner the Honorable Charles L. Dubin, into the use of drugs and banned practices intended to increase athletic performance. It was alleged, in the inquiry, that some track and field athletes had been using furazabol since the fall of 1985. However, no positive furazabol case had been reported to the Medical Commission of the International Olympic Committee (3). This arose because furazabol was not on the list of anabolic steroids screened for by most, if not all the doping control laboratories. A metabolic study of furazabol has been performed on rats (4). Several metabolites were identified in bile and feces after oral and subcutaneous administration. However, urinary metabolites were not characterized. This paper reports an excretion study in which a urinary metabolite is identified. The presence of this metabolite can be used as proof of use of the anabolic steroid.

" Author to whom correspondence should be addressed.

120

Excretion study A healthy male received a Miotolon (Daiichi Seiyaku Co.) tablet, 1 mg of furazabol, orally. Urine samples were collected at 0, 2, 4, 10, 24, 48, and 75 h post-dose. The samples were stored at 4~ until analysis. Equipment A Hewlett-Packard 5890A GC linked to an HP 5970B mass selective detector (MSD) were used for analysis. The linear velocity of the helium carrier gas through the DB-I (J&W Scientific) column was 45.5 cm/s at 200~ The dimensions of the column were 12.5 m x 0.25 mm i.d., with a film thickness of 0.25 #m. Injections were performed in the splitless mode. The temperature program was as follows: Initial temperature was 170~ increased to 215~ at a rate of 3~ then to 228~ at a rate of 4~ and finally to 280~ at a rate of 15~ The temperature was held at 280~ for 4 rain. The injection port temperature was set at 280~ The MSD was autotuned to the specifications of the manufacturer, and the electron voltage of the secondary electron multiplier was set at 200 eV above the tuned value. Materials All chemicals and solvents used were of the best commercial grades available. B-Glucuronidase (Type HP-2 crude solution) from Helix pomatia, N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), and trimethylsilyliodide (TMSI) were obtained from Sigma. Methyltestosterone was from Ciba-Geigy of Canada Ltd. Reagents 0.2M acetate buffer. Solutions of 0.2M acetic acid and 0.2M sodium acetate were mixed in proportion 1:4 (v/v) and the pH was then adjusted to 5.2. TMSI solution. A mixture of TMSl-dichloromethane-triethylamine was prepared in a 1-mL autosampler vial in proportion 70:430:1 (v/v/v). The mixture was only exposed to an atmosphere of nitrogen and protected from light. Sample preparation Free steroids. 250 ng of methyltestosterone was added to 5 mL of urine as an internal standard. The sample was extracted

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Journal of Analytical Toxicology, Vol. 14, March/April 1990

with 5 mL of distilled diethyl ether after the addition of 3 g of sodium sulfate. The organic layer was then dried under a stream of N2 at 40~ 200 #L of methanol was added to pick up the residue and transferred to a conical autosampler vial. Again the methanol was dried under a stream of N2. Totalsteroids. A C,8 Sep-Pak cartridge (Millipore) was used to extract the drugs from the urine matrix. The cartridge was activated by rinsing with 5 mL of HPLC grade methanol, followed by 5 mL of distilled deionized water. To l0 mL of urine as an internal standard, 250 ng of methyltestosterone was added and the sample was passed through the cartridge. The cartridge was washed with 5 mL of distilled water, and the drugs were then eluted with l0 mL of methanol. The methanol was dried, and the residue was reconstituted with l mL of 0.2M acetate buffer; 100/zL of/~-glucuronidase, 10,000 units of activity, was added. The solution was incubated at 55~ for two hours to hydrolyze the conjugates. To the cooled hydrolysate, 100 mg of potassium carbonate and 5 mL of distilled diethyl ether were added to extract the drugs. The organic layer was first dried over about 100 mg of anhydrous sodium sulfate and then under a stream of nitrogen at 40~ 200 #L of methanol was added and then transferred to a conical autosampler vial. Again the methanol was dried under a stream of nitrogen. Derivatizatlon

For better chromatographic properties and volatility, the steroids were derivatized into trimethylsilyl (TMS) derivatives. To the residue in the conical vial, 100/zL of MSTFA and 0.2 mg of dithioerythritol were added and the vial was then capped with a Teflon-lined rubber seal. Then, 2/~L of the TMSI solution was introduced into the vial with a Hamilton syringe. The mixture was heated at 70~ for 20 min.

zabol. The presence of this compound in the feces of rat after administration of furazabol was reported (4). The structural identification was supported by evidence from IR, NMR, and chemical manipulation. Authentic sample of this metabolite was synthesized (5), and its structure was confirmed. In a study (6) of the urinary excretion in men of 4-chloro-l-dehydromethyltestosterone (Oral Turinabol, VEB Jenapharm) (6) which is also a 17-/3-hydroxy-17-o~-methyl steroid, the major metabolites found were the 6-/3-hydroxy and the 16-/3-hydroxy derivatives. The interpretation of the mass spectrum (Figure 2) is as follows: m / z 490 is the molecular ion; m / z 218 and 231 originate from cleavage of two carbon-carbon bonds in the cyclopentane ring of the steroid molecule; and m / z 400 is [M+-TMSOH]. However, the structure of the TMS derivative of this metabolite cannot be conclusively established because the analysis of the authentic substance is required. In the rat metabolic study (4), one of the other metabolites was the 17-c~-hydroxymethyl derivative of furazabol. The mass spectrum can also be interpreted in terms of the TMS derivative of this metabolite. However, in our work with other 17-/3-hydroxy-17-ta-methyl steroids, the presence of 17-ta-hydroxymethyl derivative in human urine has never been established. This metabolite is excreted in men in the conjugated form, as it is not detected in our free steroids procedure. Figure 3A shows typical SIM chromatograms, with m/z 490, 231, and 218 for a positive furazabol urine sample. The ion with m/z 446 (Figure 3B) is the molecular ion of the di-TMS

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Analysis

2/zL of this extract was injected into the GC-MSD. The MSD was in the full scan mode for metabolite identification and in the selected ion monitoring (SIM) mode in the screening process. The ions chosen were m / z 490, 231, and 218. The dwell time for all these ions was 50 ms.

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Figure 2. Mass spectrum of the di-TMS derivative of furazabol metabolite, 16-fl-hydroxyfurazabol.

Results

and

Discussion

By comparing the total ion chromatogram of the pre-dose urine against those of the samples collected post-dose, one metabolite with a retention time of 22.8 min was detected. The mass spectrum of the TMS derivative of this metabolite is shown in Figure 2. The metabolite is assumed to be 16-hydroxyfura-

A.

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Figure 1. Molecular structures of (A) stanozolol and (B) furazabol.

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Figure 3. SIM chromatograms of the di-TMS derivative of (A) 16-fl-hydroxyfurazabol and (B) methyltestosterone in the 10-h post-dose urine sample.

121

Journal of Analytical Toxicology, Vol. 14, March/April 1990

derivative of the internal standard, methyltestosterone (retention time, 19.2 min). The ratio of the peak area of m / z 490 to that of m / z 446 in their respective ion chromatograms reflects the concentration of the metabolite. Table I shows the variation of these ratios with time after dosing. The specific gravity and creatinine concentration of these samples are also included. It can be seen that the concentration of the metabolite was the highest in the first post-dose sample (2 hours) and decreased steadily with time. It remained detectable at 48 hours after the dose was administered, but not at 75 hours.

Acknowledgment We would like to thank Ciba-Geigy of Canada Ltd. for the gift of methyltestosterone pure substance and Mr. Makoto Ueki

Table I. Variation of Furazabol Metabolite Excretion with Time* Time post-dose (h) 0 2 4 10 24 48 9Dose: 1 rng, p.o.

122

Specific gravity 1.017 1.018 1.026 1.024 1.010 1.015

Creatlnlne (mmollL) 8.8 9.5 18.0 15.7 3.2 10.0

Area (m/z 490) Area (m/z 446) 0 4.20 0.52 0.31 0.13 0.05

of the Mitsubishi Yuka Bio-Clinical Laboratories Inc. for the Miotolon tablets.

References 1. G. Ohta, T. Takegoshi, K. Ueno, and M. Shimizu. Investigations on steroids. IV. Syntheses of androstano[2,3-c]furazans and related compounds. Chem. Pharm. Bull. 13:1445-49 (1965). 2. A. Kasahara, T. Onodera, M. Mogi, Y. Oshima, and M. Shimizu. Investigations of steroids. V. Pharmacological studies. (1). Anabolic and androgenic activities of 17-/~-hydroxy-17-(~-methyl-5-~-androstano[22-c]furazan (androfurazanol), a new active anabolic steroid. Chem. Pharm. Bull. 13:1460-69 (1965). 3. Communication from the Sub-commission of Doping and Biochemistry of Sport of the Medical Commission of the international Olympic Committee, 1989. 4. T. Takegoshi, H. Tachizawa, and G. Ohta. Investigations on steroids. XII. Metabolites of furazabol (17-/~-hydroxy-17-a-methyl-5-a-androstano[2,3-c]furazan) administered to rats. Chem. Pharm. Buff. 20: 1243-59 (1972). 5. T. Takegoshi. Investigations on steroids. XIII. Synthesis of the compounds related to the metabolites of furazabol (17-/J-hydroxy-7-amethyl-5-a-androstano[2,3-c]furazan). Chem. Pharm. Bull. 20: 1260-71 (1972). 6. V.K. Schubert and G. Schumann. Stoffwechsel von Steroid-pharmaka II1. Ausscheidung von Tritiummarkiertem 4-chlor-17-=-methyl17-/~-hydroxy-1,4-androstadien-3-on und dessen Metaboliten beim Menschen. Endokrinologie 56:1-10 (1970). Manuscript received March 31, 1989; revision received December 4, 1989.

Urinary excretion of furazabol metabolite.

Furazabol, a newer anabolic steroid, is metabolized into 16-hydroxyfurazabol and excreted in urine. The presence of this compound in urine can be moni...
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