Matabosch Xavier, Pozo Oscar J, Pérez-Mañá Clara, Papaseit Esther, Segura Jordi, Ventura Rosa
Bioanalysis Research Group, IMIM, Institut Hospital del Mar d'Investigacions Mèdiques, Doctor Aiguader 88, 08003, Barcelona, Spain.
J Mass Spectrom. 2015 Mar;50(3):633-42. doi: 10.1002/jms.3571.
Glucocorticosteroids are prohibited in sports when used by systemic administrations (e.g. oral), whereas they are allowed using other administration ways. Strategies to discriminate between administrations routes have to be developed by doping control laboratories. For this reason, the metabolism of prednisolone (PRED) was studied using liquid chromatography coupled to tandem mass spectrometry. A single oral (10 mg) dose of PRED was administered to two healthy male volunteers. Urine samples were collected up to 6 days after administration. Samples were hydrolyzed with β-glucuronidase and subjected to liquid-liquid extraction with ethyl acetate in alkaline conditions. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry. Precursor ion scan methods (m/z 77, 91, 105, 121, 147 and 171) in positive ionization and neutral loss scan methods (76 and 94 Da) in negative ionization modes were applied for the open detection of PRED metabolites. Using these methods, PRED parent compound plus 20 metabolites were detected. PRED and 11 metabolites were characterized by comparison with standards of the compounds (PRED, prednisone, 20β-dihydro-PRED and 20α-dihydro-PRED, 20β-dihydro-prednisone and 20α-dihydro-prednisone, 6β-hydroxy-PRED and 6α-hydroxy-PRED, 20β isomers and 20α isomers of 6β,11β,17α,20,21-pentahydroxypregnan-1,4-diene-3-one, 6α,11β,17α,20β,21-pentahydroxypregnan-1,4-diene-3-one and Δ(6) -PRED). Using mass spectrometric data, feasible structures were proposed for seven of the remaining nine detected metabolites, including several 6-hydroxy-metabolites. Eleven of the characterized metabolites have not been previously described. Maximum excretion rates for PRED metabolites were achieved in first 24 h; however, most of the metabolites were still detectable in the last collected samples (day 6).
全身性给药（如口服）时，糖皮质激素在体育赛事中被禁用，而其他给药方式则是允许的。反兴奋剂检测实验室必须制定区分给药途径的策略。因此，使用液相色谱-串联质谱法研究了泼尼松龙（PRED）的代谢情况。向两名健康男性志愿者口服单剂量（10毫克）的PRED。给药后长达6天收集尿液样本。样本用β-葡萄糖醛酸酶水解，并在碱性条件下用乙酸乙酯进行液-液萃取。提取物通过液相色谱-串联质谱法进行分析。在正离子模式下采用前体离子扫描方法（m/z 77、91、105、121、147和171），在负离子模式下采用中性丢失扫描方法（76和94 Da）对PRED代谢物进行开放检测。使用这些方法，检测到PRED母体化合物加20种代谢物。通过与化合物标准品（PRED、泼尼松、20β-二氢-PRED和20α-二氢-PRED、20β-二氢泼尼松和20α-二氢泼尼松、6β-羟基-PRED和6α-羟基-PRED、6β,11β,17α,20,21-五羟基孕烷-1,4-二烯-3-酮的20β异构体和20α异构体、6α,11β,17α,20β,21-五羟基孕烷-1,4-二烯-3-酮和Δ(6)-PRED）比较，对PRED和11种代谢物进行了表征。利用质谱数据，为其余9种检测到的代谢物中的7种提出了可能的结构，包括几种6-羟基代谢物。11种已表征的代谢物此前未曾被描述过。PRED代谢物的最大排泄率在最初24小时内达到；然而，在最后收集的样本（第6天）中，大多数代谢物仍可检测到。
