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.

中文翻译：

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通过液相色谱-质谱法同时测量人和小鼠血清中的 18 种类固醇，无需衍生化，以分析雄激素合成和代谢的经典途径和替代途径

背景

最近发现的 DHT 合成替代途径或后门途径提供了有关经典途径之外的雄激素生物合成的重要新信息。我们报告了一种快速、通用的液相色谱-串联质谱 (LC-MS/MS) 方法，可同时准确地定量人或小鼠血清中参与经典或后门雄激素合成途径的关键类固醇。

方法

用同位素标记内标强化的血清 (200 µL) 使用 MTBE 进行液-液萃取 (LLE)，并在 LC-MS/MS 上分析提取物。用于定量的目标类固醇是睾酮（T）、二氢睾酮（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)、别孕酮 (Allo-P5) 和二氢孕酮 (DHP)。

结果

E2 和 E1 的定量限 (LOQ) 为 5 pg/mL，T 为 25 pg/mL，A4 为 50 pg/mL，DHT、17OHP5、P4、P5、AD、Adiol、DHEA、 AlloP5 和 17OHP4、3α 二醇、3β 二醇、DHP 为 0.20 ng/mL，B 为 0.25 ng/mL，F 为 1 ng/mL。准确度、精密度、重现性和回收率均在生物分析方法验证可接受的限度内。该方法在人类和小鼠、男性和女性血清中进行了说明。

结论

该方法具有足够的敏感性、特异性和可重复性，能够满足常规实验室应用的质量标准，用于准确定量人类或小鼠的雄性和雌性血清中的 18 种类固醇浓度，以分析雄激素合成和代谢途径。