Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti, 1, 00197, Rome, Italy.
Dipartimento di Medicina Sperimentale, "Sapienza" Università di Roma, Viale Regina Elena 324, 00161, Rome, Italy.
Drug Test Anal. 2019 Nov;11(11-12):1737-1746. doi: 10.1002/dta.2702. Epub 2019 Nov 20.
An analytical procedure based on ultra-performance liquid chromatography-mass spectrometry was developed to screen and to confirm dutasteride and its metabolites in human urine. Sample preparation included an enzymatic hydrolysis followed by solid-phase extraction using the strong cation exchange cartridges OASIS MCX. The chromatographic separation was carried out on C18 column, employing as mobile phases ultra purified water and acetonitrile, both containing 0.1% formic acid. Detection was achieved using a triple quadrupole as a mass spectrometric analyzer, with positive ion electrospray ionization and multiple reaction monitoring as acquisition mode. The analytical procedure developed was validated according to ISO 17025 and World Anti-Doping Agency guidelines. The extraction efficiency was estimated to be greater than 75% for both dutasteride and its hydroxylated metabolites. Detection capability was determined in the range of 0.1-0.4 ng/mL. Specificity and repeatability of the relative retention times (CV% < 0.5) and of the relative abundances of the characteristic ion transitions selected (CV% < 10) were confirmed to be fit for purpose to ensure the unambiguous identification of dutasteride and its metabolites in human urine. The developed method was used to characterize the urinary excretion profile of dutasteride after both chronic and acute administration of therapeutic doses. After chronic administration, dutasteride and its hydroxylated metabolites were easily detected and confirmed. After acute administration, instead, only the two hydroxylated metabolites were detected for 3-4 days.
建立了一种基于超高效液相色谱-质谱联用的分析方法，用于筛查和确证人尿中的度他雄胺及其代谢物。样品前处理包括酶解，然后使用强阳离子交换固相萃取小柱 Oasis MCX 进行固相萃取。采用 C18 柱，以超纯水和乙腈为流动相，均含 0.1%甲酸，进行色谱分离。采用三重四极杆作为质谱分析仪，正离子电喷雾电离和多反应监测作为采集模式进行检测。所建立的分析方法按照 ISO 17025 和世界反兴奋剂机构指南进行了验证。对于度他雄胺及其羟化代谢物，提取效率均大于 75%。检测能力在 0.1-0.4ng/mL 范围内。选择的特征离子跃迁的相对保留时间（CV%<0.5）和相对丰度（CV%<10）的特异性和重复性均得到确认，适用于确保在人尿中明确鉴定度他雄胺及其代谢物。所建立的方法用于表征治疗剂量的慢性和急性给药后度他雄胺的尿排泄特征。慢性给药后，很容易检测和确认度他雄胺及其羟化代谢物。相反，急性给药后，在 3-4 天内仅检测到两种羟化代谢物。
