... Center, Korea Advanced Institute of Science and Technology, 144-29, Samsung-dong, Kangnam-ku,. Seoul, Korea. Abstract. The amount of stanozolol and 3'-hydroxystanozolol in urine ... and the maximum of 3'-hydroxystanozolol in 19 h.
Journal of Analytical Toxicology, Vol. 14, March/April 1990
Quantitative Determination of Stanozolol and Its Metabolite in Urine by Gas Chromatography/ Mass Spectrometry H e a - Y o u n g P. C h o o * ,
Oh-Seung
Kwon, and Jongsei Park**
Doping Control Center, Korea Advanced Institute of Science and Technology, 144-29, Samsung-dong, Kangnam-ku, Seoul, Korea
Abstract The amount of stanozolol and 3'-hydroxystanozolol in urine was determined after oral administration of atanozolol. A gas chromatograph/mass selective detector equipped with a capillary column was used for these determinations. The GC/MS was operated in the SlM mode, and m/z 581,669, and 315 were monitored for stanozolol, 3'-hydroxystanozolol, and calusterone (internal standard), respectively. The detection limit was approximately 1 ng/mL for both steroids9 The maximum excretion rate of stanozolol was reached in 8 h and the maximum of 3'-hydroxystanozolol in 19 h. However, only 3% of the administered amount was recovered in urine9
Introduction
The abuse of anabolic steroid drugs by athletes has increased over the last decade, even though the International Olympic Committee (IOC) has banned their use since 1974. Stanozolol, 17~-methyl-17/3-hydroxy-5ct-androstano-(3,2-C)-pyrazole,has been one of the most abused anabolic steroids. Since stanozolol is structurally different from most anabolic steroids, its gas chromatographic behavior is poor and its detection in urine is relatively difficult. However, there are some reports on the detection of stanozolol in urine using gas chromatography/mass spectrometry after derivatization to the trimethylsilylether derivative (1,2) and isotope dilution-mass fragmentography (3). Recently Donike and co-workers identified several stanozolol metabolites in urine: 3 '-hydroxystanozolol, 3 '-hydroxyepistanozolol, 4~-hydroxystanozolol, 16/3-hydroxystanozolol, 16-c~hydroxystanozolol, 3',16-dihydroxystanozolol, and 4fl,16dihydroxystanozolol. Another reason for the difficulty in detecting stanozolol is its low excretion in urine. When excretion of stanozolol was investigated with radio-labeled drug, only 16~ of the radioactivity was excreted in the urine during the first day, while 40-600/o was excreted in the feces (5). In this paper the amounts of stanozolol and 3'-hydroxystanozolol in urine were determined after oral administration 9Current address: Ewha Women's University, School of Pharmacy ** Author to whom requests for reprints should be addressed,
of stanozolol. In order to obtain high sensitivity and characteristic ions in the mass spectrum, the two compounds were derivatized to their trimethylsilylether (OTMS) and heptafluorobutyramide (NHFB) as reported (6). Calusterone, another anabolic steriod, was used as the internal standard (Figure 1).
Materials and Methods
Chemicals and reagents. Stanozolol was supplied by Hanil Co9 3'-Hydroxystanozolol was a kind gift from Prof. M. Donike (Cologne, FRG). Amberlite XAD-2 resin (particle size 0.150.2 mm) was supplied by Serva Co. fl-Glucuronidase (activity 200 U / m L ) was purchased from Boehringer Co. The derivatizing agents MSHFB (N-methyl-N-trimethylsilylheptafluorobutyramide) and MBHFB(N-methylbisheptafluorobutyramide) were obtained from Macherey Nagel. All other chemicals and solvents were reagent grade and used without purification. Standards and calibration curve. For the preparation of calibration samples, methanolic solutions of stanozolol, 3'hydroxystanozolol, and internal standard were fortified into blank urine. Samples were at concentrations of 10, 20, 50, 100, 150, and 200 n g / m L of stanozolol and 3 '-hydroxystanozolol. The internal standard was fortified at a concentration of 150 n g / m L for all samples. Drug administration. Stanozolol 20 mg (2 mg • l0 tablets) was administered to a healthy male (60 Kg) orally. Urine samples were collected at up to 72 h post-dose and stored at 4~ Extraction. The procedure published in the literature was adopted (7-9). Into a pasteur pipet (i.d. 0.5 cm), prewashed XAD-2 resin was poured up to 3 cm of height. Urine (5 mL) and internal standard solution (calusterone l0 ppm, 75 #L) were applied to the column. The XAD-2 column was washed with 5 mL of water. Then the adsorbed drugs were eluted with 3 mL of methanol. The methanol solution was evaporated to dryness in vacuo. Phosphate buffer (0.2M, pH 7.0, 1 mL) and 25 #L of the/3-glucuronidase (100 U/mL) were added and heated to 55~ for I h. After the solution was cooled to room temperature, 100 mg of potassium carbonate and 5 mL of diethylether were added. The mixture was shaken mechanically for 5 min and two layers were separated after centrifugation (2500 rpm, 5 rain). The ether layer was transferred to another tube and the solvent was evaporated in vacuo. The residue was dried in a vacuum
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1 09
Journal of Analytical Toxicology, Vol. 14, March/April 1990
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Determination of steroids Figure 2B shows a chromatogram obtained from the analysis of stanozolol and 3'-hydroxystanozolol. Stanozoiol-OTMSNHFB eluted at 7.4 min, and 3 '-hydroxystanozolol-bis-OTMSNHFB eluted at 8.2 min. Ion 684 was additionally used to confirm the 3 '-hydroxystanozolol because ion 669 was present at low abundances. There were no interfering peaks at the retention times of those peaks in blank urine (Figure 2A). Internal standard, calusterone-OTMS-enol-TMS, showed a sharp peak at 5.8 min (Figure 2D). Figure 2C shows a typical chromatogram obtained from the urine after drug administration. The small peak at 7.0 min in Figure 2C was identified as 3 '-hydroxyepistanozolol (4). Figures 3 and 4 show the full spectra of these compounds. There was a linear relationship when the ratios of the peak area of stanozolol (ion 581) or 3 '-hydroxystanozolol (ion 669) to the peak area of calusterone (ion 315) were plotted against the various concentrations of stanozolol and 3'-hydroxystanozolol (r = 0.989 and 0.997 respectively). When six samples were tested, the overall recovery was 48~ (RSD = 6%) for stanozolol and 7307o (RSD = 7%) for 3'hydroxystanozolol. When a series of samples at low concen110
Figure 2. Selected ion chromatograms of stanozolol (581), 3'-hydroxystanozolol (669 and 684), and calusterone (315): (A) Blank urine, (B) after addition of 2 ng/mL stanozolol and 3'-hydroxystanozolol to blank urine, (C) urine at 17 h after oral administration of 20 mg stanozolol to human, and (D) internal standard added to blank urine.
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Journal of AnalyticalToxicology,Vol. 14, March/April 1990
Table I. C o n c e n t r a t i o n of Stanozolol and 3 ' - H y d r o x y a t a n o z o l o l
in H u m a n Urine after Oral A d m i n i s t r a t i o n
3'-OH.$1anozolol
Stanoz0101 Time (h)
Udne volume (mL)
Conc* (nglmL)
0- 5 5-10 10-17 17-20 20-24 24-29 29-34 34-42 42-48 48-53 53-58 58-64 64-70 70-72
418 245 250 490 530 245 200 185 465 480 435 250 500 285
70.76 240.8 163.03 35.57 26.47 34.96 68.24 60.71 21.34 19.02 20.57 20.50 17.66 16.75
RSD
Amount ~ug)
Excretion rate ~uglh)
Conc" (nglmL)
1.7 0.7 2.3 1.0 2.5 3.0 1.0 0.8 3.0 0.5 0.5 7.3 2.1 0.3
29.58 58.99 40.76 18.41 14.03 8.57 13.65 11.23 9.92 9.13 8.95 5.13 8.83 4.77
5.91 11.80 5.82 6.14 3.15 1.71 2.73 1.40 1.65 1.83 1.79 0.86 1.47 2.39
42.85 218.67 328.90 79.64 31.63 81.49 28.47 276.2 40.01 21.54 51.79 67.52 18.01 16.91
RSD
Amount (~g)
Excretion rate ~glh)
2.6 0.5 1.7 1.0 3.2 6.9 1.6 2.6 3.6 8.4 3.9 3.0 13.5 1.8
17.90 53.57 82.23 39.12 16.76 19.96 56.94 51.46 18.60 10.34 22.53 16.88 9.00 4.82
3.58 10.71 11.75 13.04 4.19 3.99 11.36 6.45 3.10 2.07 4.51 2.81 1.29 2.41
9 Eachconcentrationrepresentsthe meanof threemeasurements.
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trations was analyzed, the samples with higher than l - n g / m L concentration produced stanozolol and 3 '-hydroxystanozolol peaks clearly by GC/MS. However, only the samples within the calibration range were quantitated in this study. Day-to-day precision for the stanozolol and 3'-hydroxystanozolol indicated the standard deviation values were 12.25 and 10.58 respectively. Each was determined for three consecutive days at 2-ng/mL concentration and three samples per day.
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oxandrolone, which has half lives of 0.5 h and 9.4 h in serum (10). The excretion half life of 3 '-hydroxystanozolol was 15.1 h. The cumulative urinary excretion of stanozolol and 3 '-hydroxystanozolol over 72 h (Figure 5) showed an excretion amount of 3'-hydroxystanozolol about twice that of stanozolol. In conclusion, the excreted amount of stanozolol and 3'hydroxystanozolol was determined and the described procedure was simple and specific for the quantitation of stanozolol and its metabolite in urine. This procedure can also be adopted for the analysis of similar steroids in biological samples.
Excretion in urine
The amounts of stanozolol and 3 '-hydroxystanozolol excreted in urine after oral adminstration of 20 mg stanozolol are shown in Table I. The total amounts recovered in 72 h were 242 #g for stanozolol and 420 #g for 3'-hydroxystanozolol. This amount is only about 3~ of the administered stanozolol. Ward et al. also reported the low level of urinary excretion of stanozolol in man and suggested that stanozolol may be eliminated in the hepatic portal system (5). The majority of the stanozolol was detected only after enzymatic hydrolysis. The excretion of stanozolol showed two phases: the first rapid phase with half life of 7.7 h and the subsequent slow phase with half life of 38.5 h. Similar elimination pattern was also observed with another anabolic steriod,
References 1. R.J. Ward, C.H.L. Shackleton, and A.M, Lawson. Gas chromatographic-mass spectrometric methods for the detection and identification of anabolic steroid drugs. Br. J. Sports ivied. 9(2): 93-97 (1975). 2. M. Bertrand, R. Masse, and R. Dugal. GC-MS approach for the detection and characterization of anabolic steroids and their metabolites in biological fluids at major international sporting events, Farm. Tijdschr. Belg. 55(3): 85-101 (1978). 3. O. Lantto, I. Bjorkhem, H. Ek, and D. Johnston. Detection and 111
Journal of AnalyticalToxicology,Vol. 14, March/April 1990
quantitation of stanozolol (STROMBA) in urine by isotope dilutionmass fragmantography. J. Steroid Biochem. 14:721-27 (1981). 4.W. Schanzer, G. Opfermann, and M. Donike. Metabolism of stanozolol. Identification of urinary metabolites. In press. 5. R.J. Ward, A.M. Lawson, and C.H.L. Shackleton. Screening by gas chromatography-mass spectrometry for metabolites of five commonly used anabolic steroid drugs. Mass Spectrum Drug Meteb. [Proc. InL Symp.]. 465-74, 1977. 6. W. Schanzer. Untersuchungen Zum Nachweis und Metabolismus von Hormonen und Dopingmitteln, insbesondere mit Hilfe der Hochdruckflussigkeitschromatographie (1984), Dissertation, Institut fur Biochemie, Deutsche Sporthochschule Koln, FRG. 7. M. Donike, J. Zimmermann, K.-R. Barwalf, W. Schanzer, V. Christ, K. Klowtermann, and G. Opfermann. Routinebestimmung von
112
Anabolika in Ham. Deutsche Zeitschrift fur Sportmedizin 35: 14-23 (1984). 8. M. Donike, N-MethyI-N-trimethylsilyl-trifluoracetamid, ein nsues Silylierungsmittel aus der Reihe der silylisrtan Amide. J. Chromatogr. 42:103-104 (1969). 9. M. Donike. N-Trifluoroacetyl-O-trimethylsilyl-phenolalkyllamine. Darstellung und massenspezifischer gaschromatographischer Nachwsis. J. Chromatogr. 103:91-112 (1975). 10. A. Kalim, R.E. Ranney, J. Zagerella, and H.I. Maibach. Oxandrolone disposition and metabolism in man. C/in. Pharm. Therapeutics 14(6): 862-69 (1973). Manuscript received April 20, 1989; revision received January 3, 1990.
