背景:最近确定的替代方案,或者后门,DHT合成途径为雄激素生物合成提供了超越经典途径的重要新信息。我们报告了一种快速且通用的液相色谱-串联质谱(LC-MS/MS)方法,可同时准确地定量参与经典或后门雄激素合成途径的人或小鼠血清中的关键类固醇。
方法:用同位素标记的内标强化的血清(200μL)用MTBE进行液-液萃取(LLE),并在LC-MS/MS上分析提取物。定量的靶向类固醇是睾酮(T),双氢睾酮(DHT),5α-雄甾烷-3α,17β-二醇(3α二醇),5α-雄甾烷-3β,17β-二醇(3β二醇),脱氢表雄酮(DHEA),雄烯二酮(A4),雄酮(AD),雌二醇(E2),雌酮(E1),孕酮(P4),孕烯醇酮(P5),雄烯二醇(Adiol),17-羟基孕酮(17-OHP4)和17-羟基孕烯醇酮(17-OHP5),皮质酮(B),皮质醇(F),别孕烯醇酮(Allo-P5)和二氢孕酮(DHP)。
结果:E2和E1的定量限(LOQ)为5pg/mL,T为25pg/mL,A4为50pg/mL,DHT为0.10ng/mL,17OHP5,P4,P5,AD,Adiol,DHEA,AlloP5和0.20ng/mL的17OHP4,3α二醇,3β二醇,DHP,B为0.25ng/mL,F为1ng/mL。准确性,精度,重现性和回收率在生物分析方法验证的可接受范围内.该方法在人类和小鼠中进行了说明,男性和女性血清。
结论:所提出的方法足够灵敏,特异性和可重复性,以满足常规实验室应用的质量标准,用于准确定量人或小鼠的男性和女性血清中18种类固醇浓度,以分析雄激素合成和代谢途径。
BACKGROUND: The recently identified alternate, or backdoor, pathway of DHT synthesis provides important novel information on androgen biosynthesis beyond the classical pathway. We report a rapid and versatile liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously and accurately quantify key steroids in human or mouse serum involved in either the classical or backdoor androgen synthesis pathways.
METHODS: Serum (200 µL) fortified with isotopically labelled internal standards underwent liquid-liquid extraction (LLE) with MTBE and extracts were analysed on a LC-MS/MS. The targeted steroids for quantification were testosterone (T), dihydrotestosterone (DHT), 5α-androstane-3α,17β-diol (3α diol), 5α-androstane-3β,17β-diol (3β diol), dehydroepiandrosterone (DHEA), androstenedione (A4), androsterone (AD), estradiol (E2), estrone (E1), progesterone (P4), pregnenolone (P5), androstenediol (Adiol), 17-hydroxyprogesterone (17-OHP4) and 17-hydroxypregnenolone (17-OHP5), corticosterone (B), cortisol (F), allopregnanolone (Allo-P5) and dihydroprogesterone (DHP).
RESULTS: The limits of quantification (LOQ) were 5 pg/mL for E2 and E1, 25 pg/mL for T, 50 pg/mL for A4 and 0.10 ng/mL for DHT, 17OHP5, P4, P5, AD, Adiol, DHEA, AlloP5 and 0.20 ng/mL for 17OHP4, 3α diol, 3β diol, DHP, 0.25 ng/mL for B and 1 ng/mL for F. Accuracy, precision, reproducibility and recovery were within acceptable limits for bioanalytical method validation. The method is illustrated in human and mouse, male and female serum.
CONCLUSIONS: The presented method is sufficiently sensitive, specific and reproducible to meet the quality criteria for routine laboratory application for accurate quantitation of 18 steroid concentrations in male and female serum from humans or mice for the purpose of profiling androgen synthesis and metabolism pathways.