EP1359 - ECE_1615 - Serum multi-steroid profiling analysis in Polycystic Ovary Syndrome (PCOS) and healthy individuals

Abstract

Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder in women. The defining feature of PCOS is androgen excess, which contributes to several symptoms such as hirsutism, acne and irregular periods. The adrenal-derived 11-oxygenated androgens were shown to significantly contribute to androgen excess in PCOS, however the clinical relevance and biological variation of 11-oxygenated androgens in PCOS and healthy population remain insufficiently understood. Here we developed a high-throughput serum multi-steroid profiling assay with low sample volume requirements for the investigation of steroid metabolome patterns and related clinical phenotypes and characterisation of the roles of classic vs 11-oxygenated androgens in women with PCOS. We developed a multi-steroid profiling assay for ultra-high performance liquid chromatography—tandem mass spectrometry (UHPLC-MS/MS) that included 18 steroids encompassing central precursors as well as mineralocorticoids, glucocorticoids, and androgens, including both classic and 11-oxygenated androgens, eg, testosterone, androstenedione, DHEA, 11-ketotestosterone, 11-ketoandrostenedione, 11β-hydroxyandrostenedione, and 11β-hydroxytestosterone. Steroid extraction was carried out by supported liquid extraction (SLE) and full validation of the assay was undertaken. We recruited 5 healthy individuals who donated blood samples weekly for 10 consecutive weeks to study intraindividual variation of steroid secretion in healthy individuals. Steroids were purified from 100 µL of serum using SLE with ethyl acetate elution. Steroids were separated on the C18 column in the gradient elution of water and methanol both containing 0.01% formic acid. Post-column infusion of 2 mmol/L ammonium fluoride was employed to enhance signal sensitivity. Lower limits of quantification were between 0.04 and 2.1 nmol/L, matrix effects were between 95% and 101%, and absolute recoveries were between 100% and 125%. Accuracy ranged from 90% to 111% and imprecision was below 7.2% and 12% for within-run and between-run analyses, respectively. Data on healthy individuals and a large PCOS cohort will be presented. We successfully developed and validated a UHPLC-MS/MS assay for multi-steroid profiling including comprehensive androgen analysis in serum of women with PCOS and healthy individuals. This assay will be applied to the UK & Ireland DAISy-PCOS Phenome cohort (n = 892). This research was supported by the Medical Research Council (MC_UP_1605/15) and the Wellcome Trust (209492/Z/17/Z).