Simultaneous measurement of 18 steroids in human and mouse serum by liquid chromatography-mass spectrometry without derivatization to profile the classical and alternate pathways of androgen synthesis and metabolism

Reena Desai; D Tim Harwood; David J Handelsman

doi:10.1016/j.clinms.2018.12.003

Simultaneous measurement of 18 steroids in human and mouse serum by liquid chromatography-mass spectrometry without derivatization to profile the classical and alternate pathways of androgen synthesis and metabolism

Clin Mass Spectrom. 2019 Jan 6:11:42-51. doi: 10.1016/j.clinms.2018.12.003. eCollection 2019 Jan.

Authors

Reena Desai¹, D Tim Harwood², David J Handelsman¹

Affiliations

¹ ANZAC Research Institute, University of Sydney, Sydney, NSW 2139, Australia.
² Cawthron Institute, Nelson, New Zealand.

Abstract

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.

Keywords: 17OHP4, 17-hydroxyprogesterone; 17OHP5, 17hydroxypregnenolone; 3α diol, 5α-androstane-3α17β-diol; 3β diol, 5α-androstane-3β17β-diol; A4, androstenedione; AD, androsterone; APPI, atmospheric pressure photoionization; Adiol, androstenediol; AlloP5, allopregnanolone; Androgen; B, corticosterone; CSP, Charcoal Stripped Plasma; DHEA, dehydroepiandrosterone; DHP, dihydroprogesterone; DHT, dihydrotestosterone; Dihydrotestosterone; E1, estrone; E2, estradiol; F, cortisol; IS, internal standard; LOD, lower limit of detection; LOQ, lower limit of quantification; Liquid chromatography–mass spectrometry; ME, matrix effect; MTBE, methyl tert-butyl ether; NMI, National Measurement Institute; P4, progesterone; P5, pregnenolone; S/N, signal-to-noise ratio; Steroidogenesis; T, testosterone; Testosterone.