MSACL 2019 EU Abstract

Simultaneous Measurement of 18 Steroids in Human or Mouse Serum by LC-MS/MS to Profile the Classical and Alternate Pathways of Androgen Synthesis

Reena Desai*, D. Tim Harwood^, David J. Handelsman*
*ANZAC Research Institute, University of Sydney, Sydney, NSW 2139, Australia ^Cawthron Institute, Nelson, New Zealand

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Reena Desai (Presenter) University of Sydney >> POSTER (PDF) >> ADDL INFO (PDF)

Presenter Bio: I, Ms Reena Desai is the in charge of LCMS laboratory at ANZAC Research Institute (2009) in Andrology Laboratory. I am an analytical chemist who has experience developing and validating new analytical methods, including high-performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC-MS) My expertise has involved research in basic, clinical research and Pharmaceutical industry. My main interest from past few years has been on steroid assays to study hormone cancers and many metabolic disorders to improve men and women’s health and fertility problems. Have been supervising, teaching and helping PhD students in their mass spec projects. Experience in staffing decisions, supervision, coaching and collaborating on multi-departmental projects and ddevelopment and validation of analytical methods.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction:
Steroid measurement by LC–MS/MS now widely accepted as the method of choice for quantifying endogenous steroids including bioactive androgens, as well as their precursors
and metabolites. The alternate, or backdoor, pathway of DHT synthesis has recently been recognized as proving a testosterone-independent route for DHT synthesis bypassing the classical pathway. We developed a rapid and versatile liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to simultaneously quantify key steroids in human or mouse serum involved in either the classical or backdoor androgen synthesis pathways.

Methods information:

The LC–MS/MS analysis is based on reversed-phase chromatographic separation of the injected sample followed by gradient elution and detection of the 18 targeted steroids using triple quadrupole mass spectrometric analysis.
The liquid chromatography system was a Shimadzu Nexera UHPLC (Shimadzu Scientific Instruments, Columbia, MD, USA) with a Restek Raptor biphenyl column (100 cm×2.1 mm, 2.7 μm; cat#9309A12) with Raptor biphenyl guard cartridge (cat# 9309A0252). The solvents used were; A: Milli-Q water, B: methanol, and C: toluene (dopant). An API-5000 triple-quadruple mass spectrometer (Applied Biosystems/MDS SCIEX, Ontario, Canada) equipped with an atmospheric
pressure photoionization (APPI) source was operated in both positive and negative ion modes for the analysis. The APPI system consisted of a 10 eV krypton discharge lamp with dopant (toluene).

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 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 or female serum from humans or mice for the comprehensive profiling androgen synthesis and metabolism pathways.