A quantitative analysis of total and free 11-oxygenated androgens and its application to human serum and plasma specimens using liquid-chromatography tandem mass spectrometry

• Published in: Journal of Chromatography A 2021-08, Vol.1650, p.462228, Article 462228
• Main Authors: Caron, Patrick, Turcotte, Véronique, Guillemette, Chantal
• Format: Article
• Language: English
• Subjects: 11-oxygenated androgens; Adrenal androgens; Mass spectrometry; Validation
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2021.462228

•Validated LC-MS/MS method with improved sensitivity and specificity achieved with hydroxylamine derivatization.•Concurrent analysis of 7 analytes including the abundant adrenal precursor 11OHA4, the most potent androgenic 11KT and 11KDHT and their metabolites 11OHAST and 11KAST.•Applicability was demonstrated in men and women donors using a small volume of 200 μL.•Flexibility for studying serum or plasma and free and total steroids.•Sex-differences were observed with higher levels in women than men. Bioactive 11-oxygenated C19 adrenal-derived steroids (11-oxy C19) are potentially relevant in diverse endocrine and metabolic contexts. We report the development and validation of a liquid chromatography electrospray ionization tandem mass spectrometric method (LC-ESI-MS/MS) for the simultaneous quantification of seven 11-oxy C19 using 200 µL of plasma or serum. Sample preparation involved chemical derivatization using hydroxylamine after liquid–liquid extraction to improve specificity and sensitivity. The method allowed the quantitation of total 11-oxy C19 (free + sulfate and glucuronide conjugates) following enzymatic hydrolysis. This included the abundant precursor 11-hydroxyandrostenedione (11OHA4) and the most potent androgenic derivatives 11-keto-testosterone (11KT) and 11-keto-dihydrotestosterone (11KDHT), their abundant metabolites 11-hydroxyandrosterone (11OHAST) and 11-keto-androsterone (11KAST) potentially feeding back into the pool of potent androgens, in addition to 11-keto-androstenedione (11KA4) and 11-hydroxytestosterone (11OHT). Stable isotopes were used as internal standards, and calibrators and quality controls were prepared in the same matrix as the study samples. Performance was validated against the Food and Drug Administration Criteria. The method was sensitive with lower limit of quantification (LLOQ) values of 10 and 20 pg/mL for free and total 11-oxy C19, respectively. The applicability was demonstrated in men and women adult donors that showed sex-differences. All steroids were quantified well above LLOQ, except 11KDHT that remained undetectable suggesting interfering endogenous molecules present in non-derivatized samples in which a peak was observed. By providing accurate and reliable quantitative data, this method will permit to evaluate how profiling of 11-oxy C19 will be most informative as diagnostic, prognostic and/or theranostic tools.