a European Union Reference Laboratory , RIKILT - Institute of Food Safety , Wageningen , the Netherlands.
Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2013;30(9):1517-26. doi: 10.1080/19440049.2013.810346. Epub 2013 Jul 24.
For future targeted screening in National Residue Control Programmes, the metabolism of seven SARMs, from the arylpropionamide and the quinolinone classes, was studied in vitro using S9 bovine liver enzymes. Metabolites were detected and identified with ultra-performance liquid chromatography (UPLC) coupled to time-of-flight mass spectrometry (ToF-MS) and triple quadrupole mass spectrometry (QqQ-MS). Several metabolites were identified and results were compared with literature data on metabolism using a human cell line. Monohydroxylation, nitro-reduction, dephenylation and demethylation were the main S9 in vitro metabolic routes established. Next, an in vivo study was performed by oral administration of the arylpropionamide ostarine to a male calf and urine samples were analysed with UPLC-QToF-MS. Apart from two metabolites resulting from hydroxylation and dephenylation that were also observed in the in vitro study, the bovine in vivo metabolites of ostarine resulted in glucuronidation, sulfation and carboxylation, combined with either a hydroxylation or a dephenylation step. As the intact mother compounds of all SARMs tested are the main compounds present after in vitro incubations, and ostarine is still clearly present in the urine after the in vivo metabolism study in veal calves, the intact mother molecules were selected as the indicator to reveal treatment. The analytical UPLC-QqQ-MS/MS procedure was validated for three commercially available arylpropionamides according to European Union criteria (Commission Decision 2002/657/EC), and resulted in decision limits ranging from 0.025 to 0.05 µg l⁻¹ and a detection capability of 0.025 µg l⁻¹ in all cases. Adequate precision and intra-laboratory reproducibility (relative standard deviation below 20%) were obtained for all SARMs and the linearity was 0.999 for all compounds. This newly developed method is sensitive and robust, and therefore useful for confirmation and quantification of SARMs in bovine urine samples for residue control programmes and research purposes.
为了未来在国家残留控制计划中进行有针对性的筛选，使用 S9 牛肝酶在体外研究了七种 SARMs（芳基丙酰胺类和喹啉酮类）的代谢情况。使用超高效液相色谱 (UPLC) 与飞行时间质谱 (ToF-MS) 和三重四极杆质谱 (QqQ-MS) 检测和鉴定代谢物。鉴定出几种代谢物，并将结果与使用人细胞系进行的代谢文献数据进行比较。单羟基化、硝基还原、脱芳基化和去甲基化是建立的主要 S9 体外代谢途径。接下来，通过口服芳基丙酰胺奥沙他汀给雄性小牛进行体内研究，并使用 UPLC-QToF-MS 分析尿液样本。除了在体外研究中也观察到的两种羟基化和脱芳基化的代谢物外，牛体内奥沙他汀的代谢物还导致葡萄糖醛酸化、硫酸化和羧化，与羟基化或脱芳基化步骤相结合。由于所有测试的 SARMs 的完整母体化合物是体外孵育后存在的主要化合物，并且在小牛体内代谢研究后奥沙他汀仍明显存在于尿液中，因此选择完整的母体分子作为指示物来揭示治疗情况。根据欧盟标准（欧盟委员会 2002/657/EC 号决定），对三种市售芳基丙酰胺进行了 UPLC-QqQ-MS/MS 分析方法验证，结果表明决策限值范围为 0.025 至 0.05 µg l⁻¹，在所有情况下检测能力均为 0.025 µg l⁻¹。所有 SARMs 的精密度和实验室内部重现性（相对标准偏差低于 20%）均足够，所有化合物的线性度均为 0.999。该新开发的方法灵敏且稳健，因此可用于牛尿液样品中 SARMs 的残留控制计划和研究目的的确认和定量。
