Methylstenbolone (2,17α-dimethyl-5α-androst-1-en-17β-ol-3-one) is a synthetic anabolic and androgenic steroid (AAS) sold as an oral ‘nutritional supplement’ under the brand names ‘Ultradrol’, ‘M-Sten’ and ‘Methyl-Sten’. Like other AASs, methylstenbolone is a prohibited substance in both human and equine sports. This paper describes the studies of the in vitro and in vivo metabolism of methylstenbolone in horses using LC/HRMS, GC/MS and GC/MS/MS. Phase I in vitro metabolic study of methylstenbolone was performed using homogenised horse liver. Hydroxylation was the only biotransformation observed. Six in vitro metabolites were detected including four mono-hydroxylated metabolites, namely 16α/β-hydroxymethylstenbolone (M1a, M1b), 20-hydroxymethylstenbolone (M1c), 6-hydroxymethylstenbolone (M1d), and two dihydroxylated methylstenbolone metabolites (M2c–M2d). An in vivo experiment was carried out using two retired thoroughbred geldings. Each horse was administered with 100 mg methylstenbolone supplement by stomach tubing daily for three consecutive days. Methylstenbolone and 14 metabolites were detected in the post-administration urine samples. The proposed in vivo metabolites included 16α/β-hydroxymethylstenbolone (M1a, M1b), 20-hydroxymethylstenbolone (M1c), two dihydroxylated methylstenbolone (M2a, M2b), 17-epi-methylstenbolone (M3), methasterone (M4), 2,17-dimethylandrostane-16,17-diol-3-one (M5), dihydroxylated and reduced methylstenbolone (M6), 2α,17α-dimethylandrostane-3α,17β-diol (M7), 2,17-dimethylandrostane-3,16,17-triol (M8a–M8c) and 2,17-dimethylandrostane-2,3,16,17-tetraol (M9), formed from hydroxylation, reduction and epimerisation. Methylstenbolone and ten of its metabolites could be detected in post-administration plasma samples. The highest concentration of methylstenbolone detected in urine was about 10–36 ng/mL at 3–4 h after the last administration, while the maximum concentration in plasma was about 0.4–0.7 ng/mL at 1 h after the last administration. For controlling the misuse of methylstenbolone, M8c and M9 gave the longest detection time in urine, while M4, M5 and M6 were the longest detecting analytes in plasma. They could be detected for up to 5 and 4.5 days respectively in urine and plasma. Apart from 16α/β-hydroxymethylstenbolone (M1a, M1b), the methylstenbolone metabolites reported herein have never been reported before.

甲基斯坦勃龙（2,17α-二甲基-5α-androst-1-en-17β-ol-3-one）是合成的合成代谢和雄激素类固醇（AAS），作为口服“营养补品”，商标名为“ Ultradrol”， “ M-Sten”和“ Methyl-Sten”。像其他AAS一样，甲基斯坦勃龙在人类和马运动中都是被禁止的物质。本文描述了的研究在体外和在体内在马methylstenbolone的代谢使用LC / HRMS，GC / MS和GC / MS / MS。使用均质化的马肝进行甲基Stenbolone的I期体外代谢研究。羟基化是唯一观察到的生物转化。六个体外检测到的代谢物包括四种单羟基化代谢物，即16α/β-羟甲基史泰龙（M1a，M1b），20-羟甲基史泰龙（M1c），6-羟甲基史泰龙（M1d）和两个二羟化甲基史泰龙代谢物（M2c–M2d）。一个在体内进行实验使用两个退休纯种阉。每天通过胃管向每匹马服用100 mg甲基斯坦勃龙补充剂，连续三天。给药后尿液样品中检出了甲基斯坦勃龙和14种代谢物。拟议的体内代谢产物包括16α/β-羟甲基史泰龙（M1a，M1b），20-羟甲基史泰龙（M1c），两个二羟基化甲基史泰龙（M2a，M2b），17-表位-甲基斯坦勃龙（M3），甲甾酮（M4），2,17-二甲基雄甾烷-16,17-二醇-3-酮（M5），二羟基化和还原的甲基雌酮（M6），2α，17α-二甲基雄烷-3α，17β-二醇（ M7），2,17-二甲基雄烷-3,16,17-三醇（M8a–M8c）和2,17-二甲基雄烷-2,3,16,17-四醇（M9），是由羟基化，还原和差向异构形成的。给药后血浆样品中可检测到甲基斯坦勃龙及其十种代谢产物。上次给药后3-4小时尿液中检测到的甲基斯坦勃龙的最高浓度约为10-36 ng / mL，而上次给药后1 h血浆中的最高浓度约为0.4-0.7 ng / mL。为了控制甲基斯坦酮的滥用，M8c和M9在尿液中的检测时间最长，而M4，M5和M6在血浆中的检测时间最长。在尿液和血浆中分别可以检测到长达5天和4.5天。除了16α/β-羟甲基斯坦酮（M1a，M1b），本文报道的甲基斯坦酮酮代谢物以前从未被报道过。