Journal of Analytical Toxicology,Vol. 32, June 2008
Short Communication I
Detection of Urinary Metabolites Common to Structurally Related 17a-Alkyl Anabolic Steroids in Horses and Application to Doping Testsin Racehorses: Methandienone, Methandriol, and Oxymetholone MasayukiYamada1,', Sugako Aramaki 1, Masahiko Kurosawa1, Koichi Saito2, and Hiroyuki Nakazawa 2 1Laboratoryof Racing Chemistry, 1731-2 Tsuruta-machi, Utsunomiya City, Tochigi, 320-085l, Japan and 2Departmentof Analytical Chemistry,Hoshi University, 2-4-41 Ebara, Tokyo 142-8501,Japan
Abstract ] Methandienone, methandriol, and oxymetholone, which are anabolic steroids possessing 1 7a-methyl and 171S-hydroxygroups, were developed as oral formulations for therapeutic purposes. However, they have been used in racehorses to enhance racing performance. In humans, it has been reported that structurally related anabolic steroids having the 17a-methyl and 171~hydroxy groups, including 17a-methyltestosterone, mestanolone, methandienone, methandriol, and oxymetholone, have metabolites in common. In this study, we found that metabolites common to those of 17a-methyltestosterone and mestanolone were detected in horse urine after the administration of oxymetholone, methandienone, and methandriol. Based on analytical data, we confirmed these to be the common metabolites of five structurally related steroids, 17o.methyltestosterone, mestanolone, oxymetholone, methandienone, and methandriol. Furthermore, we detected hitherto unknown urinary metabolites of methandriol and oxymetholone in horses. The parent steroid itself was detected in horse urine after the administration of methandriol, other than metabolites common to 17a-methyltestosterone and mestanolone. On the other hand, the major metabolite of oxymetholone was mestanolone, aside from metabolites presumed to be the stereoisomers of 2-hydroxymethyl-17o.-methyl-5o.-androstan3,17l]-diol and 2,1 7a-di(hydroxymethyl)-5a-androstan-3,1 71~-diol. The simultaneous detection of common metabolites and other main metabolites would help us narrow down the candidateadministered steroid for the doping tests in racehorses.
Introduction Anabolic steroids have been developed primarily for therapeutic purposes; however, they have been illegally used to improve physical performance in human sports and horseracing. Accordingly, their use is now forbidden in athletes and race* Author to whom correspondenceshould be addressed. E-mail:
[email protected].
horses, and doping test laboratories have been requested to develop methods for the detection of possibly misused anabolic steroids. We previously reported the urinary metabolites of 17c~-methyltestosterone (MTS) and mestanolone (MSL), two compounds having very similar chemical structures, and established doping tests for racehorses (1). As a result, we confirmed that 17cz-methyl-5a-androstan-31~,171~-diol (I), 17c~-hydroxymethyl-5cz-androstan-313,1713-diol (II), 17(zmethyl-5a-androstan-31~,16a,171~-triol(Ill), and 17c~-methyl5~-androstan-31~,161~,171~-triol (IV) were mainly excreted as common metabolites in horse urine after the administration of MTS and MSL. 17c~-Methyl-51~-androstan-3~,161~,171~-triol(V), one of the major metabolites of MTS, was not detected in horse urine after the administration of MSL. Furthermore, quantification of these metabolites in horse urine samples after administration revealed that IV had the highest concentration and was detected for the longest time, compared with the other metabolites. Therefore, IV may be a very useful screening target for MTS and MSL in the doping tests for racehorses. However, some human metabolism studies have indicated that structurally related anabolic steroids with 17c~methyl and 17[B-hydroxy groups; namely, methandienone (MDI), methandriol (MDO), and oxymetholone (OXM)yielded metabolites common to those of MTS and MSL (2,3). For this reason, when those main metabolites are detected in doping tests of MTS and MSL in racehorses, the suspect administered drug should include not only MTS and MSL but also MDI, MDO, and OXM.It is stipulated in Japanese regulations for the doping tests in racehorses that when metabolites are detected in urine or plasma, the administered drug should be specified. Therefore, knowledge of metabolites common to different drugs would be useful for the doping tests in racehorses. Regarding MDI, urinary metabolites common to those of MTS and MSL were suggested in a previous report (4). However, as far as we know, there are no detailed reports of the metabolism of MDO in horses, and it is not known whether findings in human experiments apply to horse. Our objective was to compare the metabolism of MDI, MDO, and OXMwith that of MTS
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Journal of Analytical Toxicology, Vol. 32, June 2008
and MSL, focusing on the metabolites common to them. Furthermore, metabolites that have not yet been reported in horses were detected, which could serve as target compounds for the doping tests in racehorses.
Material and Methods Steroids and chemicals
MDI was obtained from Gedeon Richter (Budapest, Hungary); MDO,from Diosynth (Oss, The Netherlands); and OXM, from Edmond Pharma (Paderno Dugnano, MI, Italy). I was purchased from Steraloids (Newport,RI). II, IIl, IV, and Vwere synthesized in our laboratory according to our previous report (1). I~-Glucuronidasederived from Pomaceacanaliculata, ~pe A-I, 22,000 Fishman units/mL, was purchased from Nihon giotest (Tokyo, Japan), and 1.0 mol/L hydrogen chloride-methanol solution was obtained from Kokusan Kagaku (Tokyo,Japan). All other reagents and solvents were of analytical or HPLC grade (purchased from Wako Pure Chemical Industries, Tokyo, Japan or Kanto Kagaku Reagent Division, Tokyo, Japan). Drug administration and sample collection We used four gelding experimental horses (8-14 years old, 450-590 kg), and each drug was administered to three of the four horses. Each drug in the form of an aqueous suspension was administered orally to each horse at 1.0 mg/kg through a nasogastric tube. The interval between administrations of each drug to the same horse was more than I month. Urine samples were collected pre-administration and at 3, 6, 9, 12, 24, 48, and 72 h post-administration from each horse. Urine samples were stored at temperatures below -40~ until analysis. All experimental procedures in this study were approved by Animal Care Committee of the Equine Research Institute, Japan Racing Association. Sample preparation for detection of urinary metabolites For detection of urinary metabolites after the administration of MDI, MDO,and OXM,extraction of unconjugated and conjugated metabolites was carried out and the extracts were divided into three fractions; namely, unconjugated steroid, glucuronide, and sulfate fractions. Sample preparation methods were the same as those used for MTS and MSL investigation (1). Briefly,the urine sample was eluted through a
Sep-Pak Plus C18 cartridge, and the eluate was subjected to hydrolysis with 15-glucuronidase derived from Pomacea canaliculata or methanolysis with 1.0 mol/L hydrogen chloride-methanol solution. After trimethylsilyl (TMS)derivatization was performed in each sample fraction, the products of the reactions were analyzed by gas chromatography-mass spectrometry (GC-MS). GC-MS analysis GC-MS was performed on an HP 5973 Mass Selective Detector (Agilent Technologies, Tokyo, Japan) connected to an HP 6890 series GC. The fused-silica capillary columns used were DB-I, DB-5ms, and DB-17 (15 m x 0.25-mm i.d., 0.25-1Jm film thickness, J&W Scientific, Folsom, CA). Helium was used as carrier gas (1.0 mL/min), and column oven temperature was programmed to increase from 150~ to 210~ at 20~ then to 300~ at 10~ and maintained at 300~ for i rain. Injector temperature was set at 250~ MS transfer line temperature at 275~ and ionization energy at 70 eV. Detection of metabolites was carried out in the full-scan mode, and the capillary column used was DB-1. Structural determination of urinary metabolites by comparison with synthesized reference standards was carried out in the full-scan mode, and the capillary columns used were DB-I, DB-5ms, and DB-17.
Results and Discussion Detection of I, II, III, IV, and V In order to detect [, I[, Ill, IV, and V from urine samples collected after administration of MD], MDO and OXM, we examined the ion chromatograms of m/z 435 (l, II) and mlz 538 (II, III, IV, V), which were the characteristic ions of those metabolites (Table I). Their ion chromatograms are shown in Figure 1. From the urine sampled after MDI and MDO administration, I, II, ]II, IV, and V were detected in the glucuronide fraction, and ], II, III, and IV were detected in the sulfate fraction. On the other hand, from the urine sampled after OXM administration, I, II, III, and IV were detected in both glucuronide and sulfate fractions, but no 51~-H structural metabo]ite V was detected from the fractions. Regarding the structure determination of metabolites, McKinney et al. (5) reported that all the isomers of I, II, III, IV, and V have different GC retention data when derivatized with a combination of
Table I. Partial Mass Fragments of Metabolites Common to 17a-Methyltestosterone, Mestanolone, Methandienone, Methandriol, and Oxymetholone Melabolite 17a-Methyl-5ot-androstan-3lB,17i~-diol,bis-TM5(I) ] 7ct-Hydroxymethyl-Sa-androstan-3[3,17~-diol,tris-TMS(11) 17a-Methyl-5a-androstan-3[3,16a,171~-triol,tris-TMS(111) 17o:-Methyl-Sa-androstan-313,1613,17~-triol, tris-TMS(1%') 17a-Methyl-S~-androstan-3a,16i3,17~-tdol,tris.TMS(V)
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M§
CharacleristicIon m/z
45O(3) 538 (29) 538 (40) 538 (50) 538 (46)
435 {3]) 360 (5) 143 (100)130 (20) 75 (33)73 (37) 523 (5) 435 (95)345 (13)255 (46) 147 (49)73 (100) 523 (6) 231 (40)218 (71) 147 (31) 117 (33)73 (100) 523 (7) 231 (46)218 (80) 147 (39) 117 (38)73 (100) 523 (4) 231 (46)218 (99) 147(40) 117(37)73 ('100)
Journalof Analytical Toxicology,Vol. 32, June2008
three kinds of derivatives, TMS, cyclic phenylboronate ester (PB), and tert-butyldimethylsilyl ether (TBS) (5). According to this report, the GC retention data of III and IV with tris-TMS derivatives were different from those of stereoisomers with tris-TMS derivatives. In the case of V, although the GC retention data of V and 5~-isomer of V with tris-TMS derivatives were very similar, their PB-TBS derivatives enabled easy differentiation. After the A detection of I, II, II1, IV, and V from horse urine, we compared their retention times with those of our synthesized reference standards, ~vT I and confirmed that they differed from the 20001 retention times of their isomers, in agreement r with the report of McKinney et al. (5). l~ The results of common metabolite detection showed that I, II, III, and IV, which were the main metabolites of MTS and MSL, '~ r were detected from the urine analyzed after [ ,~ MDI, MDO, and OXM administration. Hence, j I it is difficult to specify the administered ~ ~ ~ steroid only by the detection of I, II, III, and IV. However, the detection of other characA, o teristic urinary metabolites derived from these .... steroids would help us narrow down the choice of the administered steroid. II E ....
(103 mass units) from the molecular ion, and indicated oxidation of the 17-methyl functmn in the D ring (6). On the other hand, the mass spectrum of VIII agreed with those of III, IV, and V. Further, as VIII was not observed in urine after OXM administration, it was presumed to be a 5I~-H structural metabolite. McKinney et al. (5) identified 17(z-methyl-51~I~
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Detection of other urinary metabolites As shown in Figure 1, metabolites other than I, II, III, IV, and V were also detected in the ion chromatograms of m/z 435 and m/z 538. Peaks VI, VII, and IX had the same mass spectra as peak II; namely, the characteristic fragment ion was m/z 435 and the molecular ion was m/z 538. They were presumed to be isomers of I~because the fragment ion at m/z 435 is due to the initial loss of CH~-O-Si(CH3)3
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Figure 1. Ion chromatogramsof TMS derivatives from 6 h post-administrationhorse urine sample. Glucuronide fraction of methandienone (A), sulfate fraction of methandienone (B), glucuronide fraction of methandriol (C), sulfate fraction of methandriol (D), glucuronide fraction of oxymetholone (E), sulfate fraction of oxymetholone (F).
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