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Metabolic studies of mesterolone in horses
Analytica Chimica Acta 596 (2007) 149–155
Metabolic studies of mesterolone in horses Emmie N.M. Ho a,∗ , David K.K. Leung a , Gary N.W. Leung a , Terence S.M. Wan a,∗∗ , Henry N.C. Wong b , Xiaohua Xu c , John H.K. Yeung c a
Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China b Department of Chemistry, The Chinese University of Hong Kong, Sha Tin, N.T., Hong Kong, China c Department of Pharmacology, The Chinese University of Hong Kong, Sha Tin, N.T., Hong Kong, China
Received 5 March 2007; received in revised form 28 May 2007; accepted 30 May 2007 Available online 3 June 2007
Abstract Mesterolone (1␣-methyl-5␣-androstan-17-ol-3-one) is a synthetic anabolic androgenic steroid (AAS) with reported abuses in human sports. As for other AAS, mesterolone is also a potential doping agent in equine sports. Metabolic studies on mesterolone have been reported for humans, whereas little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of mesterolone in racehorses with an objective to identify the most appropriate target metabolites for detecting mesterolone administration. In vitro biotransformation studies of mesterolone were performed by incubating the steroid with horse liver microsomes. Metabolites in the incubation mixture were isolated by liquid–liquid extraction and analysed by gas chromatography–mass spectrometry (GC–MS) after acylation or silylation. Five metabolites (M1–M5) were detected. They were 1␣-methyl-5␣-androstan-3␣-ol-17-one (M1), 1␣-methyl-5␣-androstan-3-ol-17one (M2), 1␣-methyl-5␣-androstane-3␣,17-diol (M3), 1␣-methyl-5␣-androstane-3,17-diol (M4), and 1␣-methyl-5␣-androstane-3,17-dione (M5). Of these in vitro metabolites, M1, M3, M4 and M5 were confirmed using authentic reference standards. M2 was tentatively identified by mass spectral comparison to M1. For the in vivo metabolic studies, Proviron® (20 tablets × 25 mg of mesterolone) was administered orally to two thoroughbred geldings. Preand post-administration urine samples were collected for analysis. Free and conjugated metabolites were isolated using solid-phase extraction and analysed by GC–MS as described for the in vitro studies. The results revealed that mesterolone was extensively metabolised and the parent drug was not detected in urine. Three metabolites detected in the in vitro studies, namely M1, M2 and M4, were also detected in post-administration urine samples. In addition, two stereoisomers each of 1␣-methyl-5␣-androstane-3,17␣-diol (M6 and M7) and 1␣-methyl-5␣-androstane-3,16-diol17-one (M8 and M9), and an 18-hydroxylated metabolite 1␣-methyl-5␣-androstane-3,18-diol-17-one (M10) were also detected. The metabolic pathway for mesterolone is postulated. These studies have shown that metabolites M8, M9 and M10 could be used as potential screening targets for controlling the misuse of mesterolone in horses. © 2007 Elsevier B.V. All rights reserved. Keywords: Metabolism; Horse; Mesterolone; Steroids; Gas chromatography–mass spectrometry (GC–MS)
1. Introduction Anabolic androgenic steroids include testosterone and substances with related structure and activity. They have been
∗
Corresponding author. Tel.: +852 2966 6521; fax: +852 2601 6564. Corresponding author. Tel.: +852 2966 6296; fax: +852 2601 6564. E-mail addresses: [email protected] (E.N.M. Ho), [email protected] (T.S.M. Wan). ∗∗
0003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2007.05.052
misused in sport to increase muscle strength and bulk, and to promote aggressiveness. Among them, oral anabolic steroids represent a class of highly potential doping agents in equine sports because of their ease of administration and normally shorter excretion times. Mesterolone (1␣-methyl-5␣-androstan17-ol-3-one) was first synthesised in 1965 by Wiechert [1]. It is used in treatment of hypogonadism and infertility [2]. Mesterolone abuses have been reported frequently in human sports [3]. The metabolic studies of mesterolone have been reported in humans [3–6], but little is known about their bio-
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transformation in horses. This paper describes the investigation of the in vitro and in vivo metabolisms of mesterolone in horses.
pH 7.4). All the preparation steps and reagents were kept at 4 ◦ C. 2.2. Microsomal incubation
2. Materials and methods 2.1. Materials Mesterolone, -nicotine adenine dinucleotide (-NAD), glucose-6-phosphate, glucose-6-phosphate dehydrogenase, magnesium chloride (MgCl2 ), dithioerythritol (DTE), ethylenediaminetetraacetic acid (EDTA), phosphate buffered saline (PBS), ammonium iodide (NH4 I) and -glucuronidase (from Patella vulgata, lyophilised powder), protease (from bovine pancreases, type I, 6.9 units mg−1 solid), Tris (TRIZMA® ) and sodium hydroxide (pellets, analytical grade) were obtained from Sigma (St. Louis, MO, USA). -Glucuronidase (E. Coli) was from Roche (Indianapolis, USA). d3 -Androstanediol was synthesised in-house. 1␣-Methyl-5␣-androstan-3␣-ol-17-one (M1) and 1␣-methyl-5␣-androstane-3␣,17-diol (M3) were acquired from National Analytical Reference Laboratory (Pumble, Australia). The preparation used for the administration experiments, Proviron® (25 mg of mesterolone per tablet) was obtained from P.T. Schering AG (Indonesia, Germany). 1␣-Methyl-5␣-androstane-3,17-dione (M5) was obtained from Steraloids (Rhode Island, USA). 1␣-Methyl-5␣androstane-3,17-diol (M4) was synthesized from reduction of mesterolone with lithium aluminium hydride by The Chinese University of Hong Kong. Anhydrous methanolic hydrogen chloride used for methanolysis was prepared according to the procedures reported previously [7]. Acetic acid (96%), potassium chloride, potassium phosphate and sodium chloride (GR grade) were obtained from Merck (Darmstadt, Germany). Sodium hydroxide was purchased from Riedel-de Haen (Seelze, Germany). Sodium sulfate was purchased from Farco Chemical Supplies (Beijing, China). LiChrosolv® grade acetonitrile, ethyl acetate, n-heptane and methanol and GR grade diisopropyl ether, chloroform, dichloromethane and n-hexane were provided by Merck (Darmstadt, Germany). HPLC grade water was from an in-house water purification system (Milli-Q, Molsheim, France). The HPLC mobile phases were filtered through a 0.45 m Nylon-66 filter (Agilent Technologies, USA) before use. C18 Sep-Pak cartridges (3 mL, 500 mg) and Supelclean LC-Si cartridges (3 mL) were supplied by Waters (Massachusetts, USA) and Supelco (Pennsylvania, USA), respectively. Bond Elut Certify® cartridges (130 mg, 3 mL) were purchased from Varian (Harbor City, CA, USA). Pentafluoropropionic acid anhydride (PFPA) and N-methyl-Ntrimethylsilylfluoroacetamide (MSTFA) were obtained from Pierce (Illinois, USA). Horse liver microsomes were isolated from fresh horse liver. Fresh horse liver was supplied by the Equine Hospital of The Hong Kong Jockey Club. Small pieces of horse liver were homogenised in Tris/KCl buffer (0.05 M, pH 7.4). The homogeneous mixture was centrifuged at 11300 rpm for 25 min. Microsomes were isolated from the supernatant by centrifugation at 36,600 rpm for 1 h. The pellet of microsomes obtained was then washed twice with Tris/KCl buffer (0.05 M,
A mixture of horse liver microsomes (20 mg), -NAD (4 mM), glucose-6-phosphate (10 mM), magnesium chloride (5 mM), EDTA (1 mM) and glucose-6-phosphate dehydrogenase (2 U mL−1 ) in phosphate buffer (5 mL; pH 7.4) was incubated with mesterolone (250 g) at 37 ◦ C for 2 h. The reaction was terminated by adding ice-cold acetonitrile (5 mL). The mixture was centrifuged at 3000 rpm for 10 min, extracted twice with ethyl acetate (5 mL), and the combined organic extract was evaporated to dryness. The residue was analysed by gas chromatography–mass spectrometry (GC–MS) as pentafluoropropionyl (PFP) or trimethylsilyl (TMS) derivatives. Control experiments in the absence of either (a) mesterolone or (b) microsomes were performed in parallel. 2.3. Drug administration studies Proviron® (20 tablets × 25 mg of mesterolone) was administered orally to two thoroughbred geldings by stomach tubing. Urine samples were collected before administration and then at least twice daily for up to 11 days post-administration. Urine samples were extracted with and without prior hydrolysis in order to identify, respectively, conjugated and free mesterolone and its metabolites. The extracts were analysed by GC–MS after acylation or trimethylsilylation. 2.4. Extraction procedures for administration urine samples of mesterolone 2.4.1. Methanolysis Urine (5 mL) was spiked with d3 -androstandiol (250 ng) as the internal standard and loaded onto a C18 Sep-Pak cartridge which had been pre-conditioned with methanol (5 mL) followed by deionised water (5 mL × 2). The cartridge was rinsed with deionised water (5 mL × 2) and n-hexane (5 mL), then eluted with methanol (3 mL). The eluate was evaporated to dryness at 60 ◦ C under nitrogen. Anhydrous methanolic hydrogen chloride (1 M, 0.5 mL) was added and the solution was heated at 60 ◦ C for 10 min. Diisopropyl ether (3.0 mL) was added and the mixture was transferred to a 15-mL graduated centrifuge tube containing NaOH/NaCl (1 M/0.15 M, 2 mL). The mixture was then rotated for 2 min and centrifuged at 3000 rpm for 0.5 min. The organic layer was passed through an anhydrous sodium sulfate drying tube, and then evaporated to dryness at 60 ◦ C under nitrogen. The residue was reconstituted in ethyl acetate (50 L) and loaded onto a Supelclean LC-Si normal-phase extraction cartridge, which had been pre-conditioned with chloroform/ethyl acetate (1:1, v/v, 3 mL). The cartridge was eluted with chloroform/ethyl acetate (1:1, v/v, 3 mL). The first 0.5 mL chloroform/ethyl acetate was discarded. The eluate (2.5 mL) was collected and evaporated to dryness at 60 ◦ C under nitrogen. The residue was analysed by GC–MS after acylation or trimethylsilylation.
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2.4.2. Enzyme hydrolysis Urine (3 mL) was spiked with d3 -androstanediol (150 ng) as the internal standard and diluted with potassium phosphate buffer (pH 6.0, 0.1 M, 1 mL). A solution of protease (5 mg mL−1 , 60 L) and -glucuronidase (Patella vulgata, 18,000 U mL−1 , 360 L) was added and the urine sample was incubated at 65 ◦ C for 3.5 h. The enzyme treated urine was diluted with potassium phosphate buffer (pH 6.0, 0.1 M, 1.6 mL) before loading onto a Bond Elut Certify® cartridge, which had been pre-conditioned with methanol (2 mL), deionised water (2 mL), and potassium phosphate buffer (pH 6.0, 0.1 M, 2 mL). The cartridge was then washed with phosphate buffer (pH 6.0, 0.1 M, 2 mL) followed by acetic acid (1.0 M, 2 mL), and then eluted with dichloromethane/ethyl acetate (4:1, v/v, 3 mL). The eluate was washed with NaOH/NaCl (1 M/0.15 M, 2 mL). The organic extract was passed through an anhydrous sodium sulfate drying tube and evaporated to dryness under nitrogen at 60 ◦ C. The dried residue was derivatised by acylation or trimethylsilylation for GC–MS analysis. 2.5. Extraction procedures for phase II metabolism studies 2.5.1. Unconjugated steroids Urine (5 mL) was adjusted to pH 6.8 and extracted with diisopropyl ether (5 mL). The organic layer was base-washed (1 M NaOH/0.15 M NaCl, 2.0 mL) and centrifuged (3000 rpm for 1 min). The organic layer was passed through an anhydrous sodium sulfate drying tube, and then evaporated to dryness under nitrogen at 60 ◦ C. 2.5.2. Glucuronide-conjugated steroids The remaining urine was then adjusted to pH 6.4 and incubated overnight at 37 ◦ C with -glucuronidase from E. Coli (100 L). The enzyme-hydrolysed steroids were extracted according to the procedures for unconjugated steroids. 2.5.3. Sulfate and sulfate–glucuronide conjugated steroids The remaining sulfate conjugates in the aqueous layer after enzyme hydrolysis was extracted using SPE (C18 Sep-Pak cartridge), hydrolysed using anhydrous methanolic hydrogen chloride and extracted as described previously in the section on methanolysis. 2.6. Derivatization for GC–MS analysis Pentafluoropropionyl derivatives were prepared by adding acetonitrile (100 L) and pentafluoropropionic acid anhydride (30 L) to the dry residue. The mixture was incubated at 60 ◦ C for 15 min, and then evaporated to dryness at 60 ◦ C under nitrogen. The residue was reconstituted in n-heptane (35 L). Trimethylsilyl (TMS) derivatives were prepared by adding MSTFA/NH4 I/DTE (1000:2:4, v/w/w, 30 L) to the dry residue. The mixture was incubated at 60 ◦ C for 15 min. The resulting solution was then injected directly into the GC–MS.
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2.7. Instrumentation An Agilent 6890N Network GC system coupled to an Agilent 5973 Network Mass Selective Detector (Agilent Technologies, California, USA) was used. Separation was performed on an HP1-MS (∼ 25 m × 0.25 mm, 0.25 m film thickness) column with a constant helium flow of 1.2 mL min−1 . The oven temperature was set initially at 120 ◦ C for 0.5 min, increased to 180 ◦ C at 30 ◦ C min−1 , to 230 ◦ C at 4 ◦ C min−1 and to 300 ◦ C at 30 ◦ C min−1 , and then held at 300 ◦ C for 5 min. Samples (1 L) were injected at 250 ◦ C in the splitless mode. All GC–MS analyses were performed in the electron ionisation (EI) mode with full-scan acquisition. 3. Results and discussion 3.1. In vitro biotransformation studies Five metabolites (M1–M5) were detected in the in vitro biotransformation study of mesterolone. Fig. 1 shows the total-ion chromatogram of the PFP derivatives of mesterolone and the five metabolites obtained from a microsomal incubation mixture. Their structures are shown in Fig. 2. Of these metabolites, 1␣-methyl-5␣-androstan-3␣ol-17-one (M1), 1␣-methyl-5␣-androstane-3␣,17-diol (M3), 1␣-methyl-5␣-androstane-3,17-diol (M4) and 1␣-methyl5␣-androstane-3,17-dione (M5) were confirmed with authentic reference standards. Fig. 3 shows the extracted-ion chromatograms (m/z 450, 406, 286) and the EI mass spectra of the mono-PFP derivatives of M1 and M2 obtained from the microsomal incubation mixture. Based on the similarity between the two mass spectra, M2 was assigned to be the 3-epimer of M1. Both M1 and M2 had been reported as metabolites of mesterolone in human [3,4]. 3.2. Oral administration studies 3.2.1. Phase I metabolism Eight phase I urinary metabolites were detected in the in vivo study (Fig. 4). Of these metabolites, M1, M2 and M4 were also detected in the in vitro studies. The mass spectra of the PFP derivatives of both M6 and M7 were almost identical to the corresponding data obtained for M4 and the in vitro metabolite M3 (Fig. 5), suggesting that M6 and M7 are the 17-epimers of M3 and M4. Indeed, 17-epimerization of 17-hydroxy steroids is a well reported metabolic pathway for horses [8,9]. It has been determined in the authors’ laboratory that the PFP derivatives of 3␣-hydroxy steroids invariably eluted earlier than their 3counterparts on methylsilicone phase. Based on these findings, it can be further postulated that M6 and M7 are respectively the 3␣,17␣-diol and 3,17␣-diol of 1-methyl-5␣-androstane3,17␣-diol. Metabolites M6 and M7 have never been reported previously as metabolites of mesterolone in other species. The TMS derivatives of metabolites M8, M9, and M10 all gave a molecular ion at m/z 536, which is consistent with the addition of a hydroxyl group to mesterolone. M8 and M9 are proposed to be isomers of 1␣-methyl-5␣-androstane-3,16-
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Fig. 1. Total-ion chromatogram of the pentafluoropropionyl derivatives of mesterolone and the five metabolites identified after incubation of mesterolone with horse liver microsomes.
Fig. 2. Metabolites identified after incubation of mesterolone with horse liver microsomes.
Fig. 3. Extracted-ion chromatograms and EI mass spectra of the mono-pentafluoropropionyl derivatives of 1␣-methyl-5␣-androstan-3␣-ol-17-one (M1) and 1␣methyl-5␣-androstan-3-ol-17-one (M2) obtained from the in vitro incubation mixture of mesterolone with horse liver microsomes.
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Fig. 4. Metabolites identified in equine urine samples collected after an oral administration of Proviron® (20 tablets × 25 mg of mesterolone) to a thoroughbred gelding.
Fig. 5. Extracted-ion chromatograms and EI mass spectra of the bis-pentafluoropropionyl derivatives of 1␣-methyl-5␣-androstane-3,17-diol of an authentic standard of M3 and of M4, M6 and M7 obtained from a urine sample collected 6 h after an administration of 500 mg of Proviron® (20 tablets × 25 mg of mesterolone) to a thoroughbred gelding.
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Fig. 6. EI mass spectrum of the tris-trimethylsilyl derivatives of 1␣-methyl-5␣-androstane-3,18-diol-17-one (M10) obtained from a urine sample collected 25 h after an administration of 500 mg of Proviron® (20 tablets × 25 mg of mesterolone) to a thoroughbred gelding.
diol-17-one based on the similarity of their TMS derivative mass spectra to that reported by Goudreault and Ayotte [3]. Fig. 6 shows the mass spectrum of the TMS derivative of M10, which compares very well to the TMS derivative of 1␣-methyl5␣-androstane-3,18-diol-17-one reported in the literature [5]. Diagnostic ion at m/z 433 (M-(CH3 )3 SiOCH2 ) supports that hydroxylation is at C-18. C-16 hydroxylation has been reported as a common metabolic pathway for many anabolic steroids
(such as nandrolone, testosterone and stanozolol) in horses [10], however, to the best of our knowledge, the hydroxylation of steroids at the C-18 has not been reported previously in horses, although its had been observed in humans [5,11]. The major phase I metabolic processes of mesterolone are summarised in Fig. 7. They include (i) reduction of the 3-keto function in the A-ring to yield M3/M4; (ii) oxidation at C17 to give M1 and M2, possibly via intermediates M3/M4 or M5;
Fig. 7. A proposed pathway for the metabolism of mesterolone in horse.
E.N.M. Ho et al. / Analytica Chimica Acta 596 (2007) 149–155 Table 1 Detection periods of the urinary metabolites of mesterolone Metabolites of mesterolone
Horse A (h)
Horse B (h)
1␣-Methyl-5␣-androstan-3␣-ol-17-one (M1) 1␣-Methyl-5␣-androstan-3-ol-17-one (M2) 1␣-Methyl-5␣-androstane-3,17-diol (M4) 1␣-Methyl-5␣-androstane-3␣,17␣-diol (M6) 1␣-Methyl-5␣-androstane-3,17␣-diol (M7) 1␣-Methyl-5␣-androstane-3,16-diol-17-one (M8) 1␣-Methyl-5␣-androstane-3,16-diol-17-one (M9) 1␣-Methyl-5␣-androstane-3,18-diol-17-one (M10)
Not detected 29 25 Not detected 31 71 71 54
31 31 29 31 47 47 55 47
(iii) subsequent reduction of the 17-oxo function of M1/M2 to yield the corresponding 17 epimers M6/M7; and (iv) C16 and C18 hydroxylation of M1/M2 to yield respectively M8/M9 and M10. No parent mesterolone was detected in any of the postadministration urine. 3.2.2. Phase II metabolism The phase II metabolism study showed that M1, M6, M7, M9 and M10 were mainly excreted in the glucuronide fraction while the rest (M2, M4 and M8) were mostly present in the sulfate fraction. The findings were consistent with the observations reported by Houghton [10] that the prefer mode of conjugation for17hydroxyl metabolites is by sulfation, whereas 17␣-hydroxyl metabolites tend to form glucuronide conjugates. 3.2.3. Analytes of choice for routine screening The ultimate goal of this project is to identify potential screening targets for controlling the misuse of mesterolone. The duration for the detection of their metabolites is therefore of great interest. The periods for which these metabolites could be detected in post-administration urine by GC–MS after enzyme hydrolysis or methanolysis are summarised in Table 1. Metabolites M8, M9 and M10 could be detected for the longest in both horses and are therefore suitable targets for detecting mesterolone administration. 4. Conclusion In vitro biotransformation using horse liver microsomes and in vivo metabolism of mesterolone were studied. For
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the in vitro biotransformation of mesterolone, five metabolites (M1–M5) were detected. They were 1␣-methyl-5␣androstan-3␣-ol-17-one (M1), 1␣-methyl-5␣-androstan-3-ol17-one (M2), 1␣-methyl-5␣-androstane-3␣,17-diol (M3), 1␣-methyl-5␣-androstane-3,17-diol (M4), and 1␣-methyl5␣-androstane-3,17-dione (M5). For the oral administration of mesterolone, the parent drug was not detected. However, eight metabolites, M1, M2, M4, two stereoisomers each of 1␣-methyl-5␣-androstane-3,17␣-diol (M6 and M7) and 1␣methyl-5␣-androstane-3,16-diol-17-one (M8 and M9), and an 18-hydroxylated metabolite 1␣-methyl-5␣-androstane-3,18diol-17-one (M10), could be detected. The metabolic pathway for mesterolone is postulated. Based on the detection times, metabolites M8, M9, and M10 could be used as potential screening targets for detecting mesterolone administration in horses. Acknowledgements The authors wish to thank the veterinary surgeons of The Hong Kong Jockey Club for their assistance in performing drug administration experiments and for arranging sample collection, and to Anne Fan, Chris Szeto, L.H. Yau, Coco Ng and Celia Wong for their technical assistance. References [1] R. Wiechert, Proceedings of 1st International Congress, Vol. 2, Academic Press, New York, 1965, p. 77. [2] W.H. Martindale, in: J.E.F. Reynolds (Ed.), The Extra Pharmacopoeia, 31st ed., Royal Pharmaceutical Society, London, 1996, p. 1497. [3] D. Goudreault, C. Ayotte, in: M. Donike (Ed.), Proceedings of the 13th Cologne workshop, 1995, Cologne: Sport und Buch Straub, 1996, p. 55. [4] D. De Boer, E.G. De Jong, R.A.A. Maes, J.M. Van Rossum, J. Steroid Biochem. Mol. Biol. 42 (1992) 411. [5] R. Mass´e, D. Goudreault, J. Steroid Biochem. Mol. Biol. 42 (3/4) (1992) 399. [6] G.P. Cartoni, M. Ciardi, A. Giarrusso, F. Rosati, J. Chromatogr. 279 (1983) 515. [7] P.W. Tang, D.L. Crone, Anal. Biochem. 182 (1989) 289. [8] O. Edlund, L. Bowers, J. Henion, T.R. Covey, J. Chromatogr. 497 (1989) 49. [9] H.W. Hagedorn, R. Schulz, A. Friedrich, J Chromatogr. 577 (2) (1992) 195. [10] E. Houghton, in: C.R. Short (Ed.), Proceedings of the 9th International Conference on Racing Analysts Veterinarians, Vol. 1, Department of Veterinary Physiology, Louisiana State University, 1992, p. 3. [11] D.K. Fukushima, H.L. Bradlow, L. Hellman, J. Biol. Chem. 237 (1962) 3359.
Metabolic studies of mesterolone in horses Emmie N.M. Ho a,∗ , David K.K. Leung a , Gary N.W. Leung a , Terence S.M. Wan a,∗∗ , Henry N.C. Wong b , Xiaohua Xu c , John H.K. Yeung c a
Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China b Department of Chemistry, The Chinese University of Hong Kong, Sha Tin, N.T., Hong Kong, China c Department of Pharmacology, The Chinese University of Hong Kong, Sha Tin, N.T., Hong Kong, China
Received 5 March 2007; received in revised form 28 May 2007; accepted 30 May 2007 Available online 3 June 2007
Abstract Mesterolone (1␣-methyl-5␣-androstan-17-ol-3-one) is a synthetic anabolic androgenic steroid (AAS) with reported abuses in human sports. As for other AAS, mesterolone is also a potential doping agent in equine sports. Metabolic studies on mesterolone have been reported for humans, whereas little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of mesterolone in racehorses with an objective to identify the most appropriate target metabolites for detecting mesterolone administration. In vitro biotransformation studies of mesterolone were performed by incubating the steroid with horse liver microsomes. Metabolites in the incubation mixture were isolated by liquid–liquid extraction and analysed by gas chromatography–mass spectrometry (GC–MS) after acylation or silylation. Five metabolites (M1–M5) were detected. They were 1␣-methyl-5␣-androstan-3␣-ol-17-one (M1), 1␣-methyl-5␣-androstan-3-ol-17one (M2), 1␣-methyl-5␣-androstane-3␣,17-diol (M3), 1␣-methyl-5␣-androstane-3,17-diol (M4), and 1␣-methyl-5␣-androstane-3,17-dione (M5). Of these in vitro metabolites, M1, M3, M4 and M5 were confirmed using authentic reference standards. M2 was tentatively identified by mass spectral comparison to M1. For the in vivo metabolic studies, Proviron® (20 tablets × 25 mg of mesterolone) was administered orally to two thoroughbred geldings. Preand post-administration urine samples were collected for analysis. Free and conjugated metabolites were isolated using solid-phase extraction and analysed by GC–MS as described for the in vitro studies. The results revealed that mesterolone was extensively metabolised and the parent drug was not detected in urine. Three metabolites detected in the in vitro studies, namely M1, M2 and M4, were also detected in post-administration urine samples. In addition, two stereoisomers each of 1␣-methyl-5␣-androstane-3,17␣-diol (M6 and M7) and 1␣-methyl-5␣-androstane-3,16-diol17-one (M8 and M9), and an 18-hydroxylated metabolite 1␣-methyl-5␣-androstane-3,18-diol-17-one (M10) were also detected. The metabolic pathway for mesterolone is postulated. These studies have shown that metabolites M8, M9 and M10 could be used as potential screening targets for controlling the misuse of mesterolone in horses. © 2007 Elsevier B.V. All rights reserved. Keywords: Metabolism; Horse; Mesterolone; Steroids; Gas chromatography–mass spectrometry (GC–MS)
1. Introduction Anabolic androgenic steroids include testosterone and substances with related structure and activity. They have been
∗
Corresponding author. Tel.: +852 2966 6521; fax: +852 2601 6564. Corresponding author. Tel.: +852 2966 6296; fax: +852 2601 6564. E-mail addresses: [email protected] (E.N.M. Ho), [email protected] (T.S.M. Wan). ∗∗
0003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2007.05.052
misused in sport to increase muscle strength and bulk, and to promote aggressiveness. Among them, oral anabolic steroids represent a class of highly potential doping agents in equine sports because of their ease of administration and normally shorter excretion times. Mesterolone (1␣-methyl-5␣-androstan17-ol-3-one) was first synthesised in 1965 by Wiechert [1]. It is used in treatment of hypogonadism and infertility [2]. Mesterolone abuses have been reported frequently in human sports [3]. The metabolic studies of mesterolone have been reported in humans [3–6], but little is known about their bio-
150
E.N.M. Ho et al. / Analytica Chimica Acta 596 (2007) 149–155
transformation in horses. This paper describes the investigation of the in vitro and in vivo metabolisms of mesterolone in horses.
pH 7.4). All the preparation steps and reagents were kept at 4 ◦ C. 2.2. Microsomal incubation
2. Materials and methods 2.1. Materials Mesterolone, -nicotine adenine dinucleotide (-NAD), glucose-6-phosphate, glucose-6-phosphate dehydrogenase, magnesium chloride (MgCl2 ), dithioerythritol (DTE), ethylenediaminetetraacetic acid (EDTA), phosphate buffered saline (PBS), ammonium iodide (NH4 I) and -glucuronidase (from Patella vulgata, lyophilised powder), protease (from bovine pancreases, type I, 6.9 units mg−1 solid), Tris (TRIZMA® ) and sodium hydroxide (pellets, analytical grade) were obtained from Sigma (St. Louis, MO, USA). -Glucuronidase (E. Coli) was from Roche (Indianapolis, USA). d3 -Androstanediol was synthesised in-house. 1␣-Methyl-5␣-androstan-3␣-ol-17-one (M1) and 1␣-methyl-5␣-androstane-3␣,17-diol (M3) were acquired from National Analytical Reference Laboratory (Pumble, Australia). The preparation used for the administration experiments, Proviron® (25 mg of mesterolone per tablet) was obtained from P.T. Schering AG (Indonesia, Germany). 1␣-Methyl-5␣-androstane-3,17-dione (M5) was obtained from Steraloids (Rhode Island, USA). 1␣-Methyl-5␣androstane-3,17-diol (M4) was synthesized from reduction of mesterolone with lithium aluminium hydride by The Chinese University of Hong Kong. Anhydrous methanolic hydrogen chloride used for methanolysis was prepared according to the procedures reported previously [7]. Acetic acid (96%), potassium chloride, potassium phosphate and sodium chloride (GR grade) were obtained from Merck (Darmstadt, Germany). Sodium hydroxide was purchased from Riedel-de Haen (Seelze, Germany). Sodium sulfate was purchased from Farco Chemical Supplies (Beijing, China). LiChrosolv® grade acetonitrile, ethyl acetate, n-heptane and methanol and GR grade diisopropyl ether, chloroform, dichloromethane and n-hexane were provided by Merck (Darmstadt, Germany). HPLC grade water was from an in-house water purification system (Milli-Q, Molsheim, France). The HPLC mobile phases were filtered through a 0.45 m Nylon-66 filter (Agilent Technologies, USA) before use. C18 Sep-Pak cartridges (3 mL, 500 mg) and Supelclean LC-Si cartridges (3 mL) were supplied by Waters (Massachusetts, USA) and Supelco (Pennsylvania, USA), respectively. Bond Elut Certify® cartridges (130 mg, 3 mL) were purchased from Varian (Harbor City, CA, USA). Pentafluoropropionic acid anhydride (PFPA) and N-methyl-Ntrimethylsilylfluoroacetamide (MSTFA) were obtained from Pierce (Illinois, USA). Horse liver microsomes were isolated from fresh horse liver. Fresh horse liver was supplied by the Equine Hospital of The Hong Kong Jockey Club. Small pieces of horse liver were homogenised in Tris/KCl buffer (0.05 M, pH 7.4). The homogeneous mixture was centrifuged at 11300 rpm for 25 min. Microsomes were isolated from the supernatant by centrifugation at 36,600 rpm for 1 h. The pellet of microsomes obtained was then washed twice with Tris/KCl buffer (0.05 M,
A mixture of horse liver microsomes (20 mg), -NAD (4 mM), glucose-6-phosphate (10 mM), magnesium chloride (5 mM), EDTA (1 mM) and glucose-6-phosphate dehydrogenase (2 U mL−1 ) in phosphate buffer (5 mL; pH 7.4) was incubated with mesterolone (250 g) at 37 ◦ C for 2 h. The reaction was terminated by adding ice-cold acetonitrile (5 mL). The mixture was centrifuged at 3000 rpm for 10 min, extracted twice with ethyl acetate (5 mL), and the combined organic extract was evaporated to dryness. The residue was analysed by gas chromatography–mass spectrometry (GC–MS) as pentafluoropropionyl (PFP) or trimethylsilyl (TMS) derivatives. Control experiments in the absence of either (a) mesterolone or (b) microsomes were performed in parallel. 2.3. Drug administration studies Proviron® (20 tablets × 25 mg of mesterolone) was administered orally to two thoroughbred geldings by stomach tubing. Urine samples were collected before administration and then at least twice daily for up to 11 days post-administration. Urine samples were extracted with and without prior hydrolysis in order to identify, respectively, conjugated and free mesterolone and its metabolites. The extracts were analysed by GC–MS after acylation or trimethylsilylation. 2.4. Extraction procedures for administration urine samples of mesterolone 2.4.1. Methanolysis Urine (5 mL) was spiked with d3 -androstandiol (250 ng) as the internal standard and loaded onto a C18 Sep-Pak cartridge which had been pre-conditioned with methanol (5 mL) followed by deionised water (5 mL × 2). The cartridge was rinsed with deionised water (5 mL × 2) and n-hexane (5 mL), then eluted with methanol (3 mL). The eluate was evaporated to dryness at 60 ◦ C under nitrogen. Anhydrous methanolic hydrogen chloride (1 M, 0.5 mL) was added and the solution was heated at 60 ◦ C for 10 min. Diisopropyl ether (3.0 mL) was added and the mixture was transferred to a 15-mL graduated centrifuge tube containing NaOH/NaCl (1 M/0.15 M, 2 mL). The mixture was then rotated for 2 min and centrifuged at 3000 rpm for 0.5 min. The organic layer was passed through an anhydrous sodium sulfate drying tube, and then evaporated to dryness at 60 ◦ C under nitrogen. The residue was reconstituted in ethyl acetate (50 L) and loaded onto a Supelclean LC-Si normal-phase extraction cartridge, which had been pre-conditioned with chloroform/ethyl acetate (1:1, v/v, 3 mL). The cartridge was eluted with chloroform/ethyl acetate (1:1, v/v, 3 mL). The first 0.5 mL chloroform/ethyl acetate was discarded. The eluate (2.5 mL) was collected and evaporated to dryness at 60 ◦ C under nitrogen. The residue was analysed by GC–MS after acylation or trimethylsilylation.
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2.4.2. Enzyme hydrolysis Urine (3 mL) was spiked with d3 -androstanediol (150 ng) as the internal standard and diluted with potassium phosphate buffer (pH 6.0, 0.1 M, 1 mL). A solution of protease (5 mg mL−1 , 60 L) and -glucuronidase (Patella vulgata, 18,000 U mL−1 , 360 L) was added and the urine sample was incubated at 65 ◦ C for 3.5 h. The enzyme treated urine was diluted with potassium phosphate buffer (pH 6.0, 0.1 M, 1.6 mL) before loading onto a Bond Elut Certify® cartridge, which had been pre-conditioned with methanol (2 mL), deionised water (2 mL), and potassium phosphate buffer (pH 6.0, 0.1 M, 2 mL). The cartridge was then washed with phosphate buffer (pH 6.0, 0.1 M, 2 mL) followed by acetic acid (1.0 M, 2 mL), and then eluted with dichloromethane/ethyl acetate (4:1, v/v, 3 mL). The eluate was washed with NaOH/NaCl (1 M/0.15 M, 2 mL). The organic extract was passed through an anhydrous sodium sulfate drying tube and evaporated to dryness under nitrogen at 60 ◦ C. The dried residue was derivatised by acylation or trimethylsilylation for GC–MS analysis. 2.5. Extraction procedures for phase II metabolism studies 2.5.1. Unconjugated steroids Urine (5 mL) was adjusted to pH 6.8 and extracted with diisopropyl ether (5 mL). The organic layer was base-washed (1 M NaOH/0.15 M NaCl, 2.0 mL) and centrifuged (3000 rpm for 1 min). The organic layer was passed through an anhydrous sodium sulfate drying tube, and then evaporated to dryness under nitrogen at 60 ◦ C. 2.5.2. Glucuronide-conjugated steroids The remaining urine was then adjusted to pH 6.4 and incubated overnight at 37 ◦ C with -glucuronidase from E. Coli (100 L). The enzyme-hydrolysed steroids were extracted according to the procedures for unconjugated steroids. 2.5.3. Sulfate and sulfate–glucuronide conjugated steroids The remaining sulfate conjugates in the aqueous layer after enzyme hydrolysis was extracted using SPE (C18 Sep-Pak cartridge), hydrolysed using anhydrous methanolic hydrogen chloride and extracted as described previously in the section on methanolysis. 2.6. Derivatization for GC–MS analysis Pentafluoropropionyl derivatives were prepared by adding acetonitrile (100 L) and pentafluoropropionic acid anhydride (30 L) to the dry residue. The mixture was incubated at 60 ◦ C for 15 min, and then evaporated to dryness at 60 ◦ C under nitrogen. The residue was reconstituted in n-heptane (35 L). Trimethylsilyl (TMS) derivatives were prepared by adding MSTFA/NH4 I/DTE (1000:2:4, v/w/w, 30 L) to the dry residue. The mixture was incubated at 60 ◦ C for 15 min. The resulting solution was then injected directly into the GC–MS.
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2.7. Instrumentation An Agilent 6890N Network GC system coupled to an Agilent 5973 Network Mass Selective Detector (Agilent Technologies, California, USA) was used. Separation was performed on an HP1-MS (∼ 25 m × 0.25 mm, 0.25 m film thickness) column with a constant helium flow of 1.2 mL min−1 . The oven temperature was set initially at 120 ◦ C for 0.5 min, increased to 180 ◦ C at 30 ◦ C min−1 , to 230 ◦ C at 4 ◦ C min−1 and to 300 ◦ C at 30 ◦ C min−1 , and then held at 300 ◦ C for 5 min. Samples (1 L) were injected at 250 ◦ C in the splitless mode. All GC–MS analyses were performed in the electron ionisation (EI) mode with full-scan acquisition. 3. Results and discussion 3.1. In vitro biotransformation studies Five metabolites (M1–M5) were detected in the in vitro biotransformation study of mesterolone. Fig. 1 shows the total-ion chromatogram of the PFP derivatives of mesterolone and the five metabolites obtained from a microsomal incubation mixture. Their structures are shown in Fig. 2. Of these metabolites, 1␣-methyl-5␣-androstan-3␣ol-17-one (M1), 1␣-methyl-5␣-androstane-3␣,17-diol (M3), 1␣-methyl-5␣-androstane-3,17-diol (M4) and 1␣-methyl5␣-androstane-3,17-dione (M5) were confirmed with authentic reference standards. Fig. 3 shows the extracted-ion chromatograms (m/z 450, 406, 286) and the EI mass spectra of the mono-PFP derivatives of M1 and M2 obtained from the microsomal incubation mixture. Based on the similarity between the two mass spectra, M2 was assigned to be the 3-epimer of M1. Both M1 and M2 had been reported as metabolites of mesterolone in human [3,4]. 3.2. Oral administration studies 3.2.1. Phase I metabolism Eight phase I urinary metabolites were detected in the in vivo study (Fig. 4). Of these metabolites, M1, M2 and M4 were also detected in the in vitro studies. The mass spectra of the PFP derivatives of both M6 and M7 were almost identical to the corresponding data obtained for M4 and the in vitro metabolite M3 (Fig. 5), suggesting that M6 and M7 are the 17-epimers of M3 and M4. Indeed, 17-epimerization of 17-hydroxy steroids is a well reported metabolic pathway for horses [8,9]. It has been determined in the authors’ laboratory that the PFP derivatives of 3␣-hydroxy steroids invariably eluted earlier than their 3counterparts on methylsilicone phase. Based on these findings, it can be further postulated that M6 and M7 are respectively the 3␣,17␣-diol and 3,17␣-diol of 1-methyl-5␣-androstane3,17␣-diol. Metabolites M6 and M7 have never been reported previously as metabolites of mesterolone in other species. The TMS derivatives of metabolites M8, M9, and M10 all gave a molecular ion at m/z 536, which is consistent with the addition of a hydroxyl group to mesterolone. M8 and M9 are proposed to be isomers of 1␣-methyl-5␣-androstane-3,16-
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Fig. 1. Total-ion chromatogram of the pentafluoropropionyl derivatives of mesterolone and the five metabolites identified after incubation of mesterolone with horse liver microsomes.
Fig. 2. Metabolites identified after incubation of mesterolone with horse liver microsomes.
Fig. 3. Extracted-ion chromatograms and EI mass spectra of the mono-pentafluoropropionyl derivatives of 1␣-methyl-5␣-androstan-3␣-ol-17-one (M1) and 1␣methyl-5␣-androstan-3-ol-17-one (M2) obtained from the in vitro incubation mixture of mesterolone with horse liver microsomes.
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Fig. 4. Metabolites identified in equine urine samples collected after an oral administration of Proviron® (20 tablets × 25 mg of mesterolone) to a thoroughbred gelding.
Fig. 5. Extracted-ion chromatograms and EI mass spectra of the bis-pentafluoropropionyl derivatives of 1␣-methyl-5␣-androstane-3,17-diol of an authentic standard of M3 and of M4, M6 and M7 obtained from a urine sample collected 6 h after an administration of 500 mg of Proviron® (20 tablets × 25 mg of mesterolone) to a thoroughbred gelding.
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Fig. 6. EI mass spectrum of the tris-trimethylsilyl derivatives of 1␣-methyl-5␣-androstane-3,18-diol-17-one (M10) obtained from a urine sample collected 25 h after an administration of 500 mg of Proviron® (20 tablets × 25 mg of mesterolone) to a thoroughbred gelding.
diol-17-one based on the similarity of their TMS derivative mass spectra to that reported by Goudreault and Ayotte [3]. Fig. 6 shows the mass spectrum of the TMS derivative of M10, which compares very well to the TMS derivative of 1␣-methyl5␣-androstane-3,18-diol-17-one reported in the literature [5]. Diagnostic ion at m/z 433 (M-(CH3 )3 SiOCH2 ) supports that hydroxylation is at C-18. C-16 hydroxylation has been reported as a common metabolic pathway for many anabolic steroids
(such as nandrolone, testosterone and stanozolol) in horses [10], however, to the best of our knowledge, the hydroxylation of steroids at the C-18 has not been reported previously in horses, although its had been observed in humans [5,11]. The major phase I metabolic processes of mesterolone are summarised in Fig. 7. They include (i) reduction of the 3-keto function in the A-ring to yield M3/M4; (ii) oxidation at C17 to give M1 and M2, possibly via intermediates M3/M4 or M5;
Fig. 7. A proposed pathway for the metabolism of mesterolone in horse.
E.N.M. Ho et al. / Analytica Chimica Acta 596 (2007) 149–155 Table 1 Detection periods of the urinary metabolites of mesterolone Metabolites of mesterolone
Horse A (h)
Horse B (h)
1␣-Methyl-5␣-androstan-3␣-ol-17-one (M1) 1␣-Methyl-5␣-androstan-3-ol-17-one (M2) 1␣-Methyl-5␣-androstane-3,17-diol (M4) 1␣-Methyl-5␣-androstane-3␣,17␣-diol (M6) 1␣-Methyl-5␣-androstane-3,17␣-diol (M7) 1␣-Methyl-5␣-androstane-3,16-diol-17-one (M8) 1␣-Methyl-5␣-androstane-3,16-diol-17-one (M9) 1␣-Methyl-5␣-androstane-3,18-diol-17-one (M10)
Not detected 29 25 Not detected 31 71 71 54
31 31 29 31 47 47 55 47
(iii) subsequent reduction of the 17-oxo function of M1/M2 to yield the corresponding 17 epimers M6/M7; and (iv) C16 and C18 hydroxylation of M1/M2 to yield respectively M8/M9 and M10. No parent mesterolone was detected in any of the postadministration urine. 3.2.2. Phase II metabolism The phase II metabolism study showed that M1, M6, M7, M9 and M10 were mainly excreted in the glucuronide fraction while the rest (M2, M4 and M8) were mostly present in the sulfate fraction. The findings were consistent with the observations reported by Houghton [10] that the prefer mode of conjugation for17hydroxyl metabolites is by sulfation, whereas 17␣-hydroxyl metabolites tend to form glucuronide conjugates. 3.2.3. Analytes of choice for routine screening The ultimate goal of this project is to identify potential screening targets for controlling the misuse of mesterolone. The duration for the detection of their metabolites is therefore of great interest. The periods for which these metabolites could be detected in post-administration urine by GC–MS after enzyme hydrolysis or methanolysis are summarised in Table 1. Metabolites M8, M9 and M10 could be detected for the longest in both horses and are therefore suitable targets for detecting mesterolone administration. 4. Conclusion In vitro biotransformation using horse liver microsomes and in vivo metabolism of mesterolone were studied. For
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the in vitro biotransformation of mesterolone, five metabolites (M1–M5) were detected. They were 1␣-methyl-5␣androstan-3␣-ol-17-one (M1), 1␣-methyl-5␣-androstan-3-ol17-one (M2), 1␣-methyl-5␣-androstane-3␣,17-diol (M3), 1␣-methyl-5␣-androstane-3,17-diol (M4), and 1␣-methyl5␣-androstane-3,17-dione (M5). For the oral administration of mesterolone, the parent drug was not detected. However, eight metabolites, M1, M2, M4, two stereoisomers each of 1␣-methyl-5␣-androstane-3,17␣-diol (M6 and M7) and 1␣methyl-5␣-androstane-3,16-diol-17-one (M8 and M9), and an 18-hydroxylated metabolite 1␣-methyl-5␣-androstane-3,18diol-17-one (M10), could be detected. The metabolic pathway for mesterolone is postulated. Based on the detection times, metabolites M8, M9, and M10 could be used as potential screening targets for detecting mesterolone administration in horses. Acknowledgements The authors wish to thank the veterinary surgeons of The Hong Kong Jockey Club for their assistance in performing drug administration experiments and for arranging sample collection, and to Anne Fan, Chris Szeto, L.H. Yau, Coco Ng and Celia Wong for their technical assistance. References [1] R. Wiechert, Proceedings of 1st International Congress, Vol. 2, Academic Press, New York, 1965, p. 77. [2] W.H. Martindale, in: J.E.F. Reynolds (Ed.), The Extra Pharmacopoeia, 31st ed., Royal Pharmaceutical Society, London, 1996, p. 1497. [3] D. Goudreault, C. Ayotte, in: M. Donike (Ed.), Proceedings of the 13th Cologne workshop, 1995, Cologne: Sport und Buch Straub, 1996, p. 55. [4] D. De Boer, E.G. De Jong, R.A.A. Maes, J.M. Van Rossum, J. Steroid Biochem. Mol. Biol. 42 (1992) 411. [5] R. Mass´e, D. Goudreault, J. Steroid Biochem. Mol. Biol. 42 (3/4) (1992) 399. [6] G.P. Cartoni, M. Ciardi, A. Giarrusso, F. Rosati, J. Chromatogr. 279 (1983) 515. [7] P.W. Tang, D.L. Crone, Anal. Biochem. 182 (1989) 289. [8] O. Edlund, L. Bowers, J. Henion, T.R. Covey, J. Chromatogr. 497 (1989) 49. [9] H.W. Hagedorn, R. Schulz, A. Friedrich, J Chromatogr. 577 (2) (1992) 195. [10] E. Houghton, in: C.R. Short (Ed.), Proceedings of the 9th International Conference on Racing Analysts Veterinarians, Vol. 1, Department of Veterinary Physiology, Louisiana State University, 1992, p. 3. [11] D.K. Fukushima, H.L. Bradlow, L. Hellman, J. Biol. Chem. 237 (1962) 3359.