MSACL 2025 Abstract

Screening of Classic and 11-Oxygenated Androgens in Polycystic Ovary Syndrome (PCOS): Method Development

Vera Dosedelova (1), Karl-Heinz Storbeck (2), Wiebke Arlt (1,3)
(1) MRC Laboratory of Medical Sciences, London, United Kingdom, (2) Stellenbosch University, Stellenbosch, South Africa, (3) Imperial College London, London, United Kingdom

Vera Dosedelova, PhD (Presenter) MRC Laboratory of Medical Sciences

Presenter Bio: Vera was awarded PhD in Life Sciences at the Masaryk University, Brno, Czech Republic. She focused on the analysis of small molecules in saliva and exhaled breath condensate by capillary electrophoresis and LC-MS. In 2024, she joined the Steroids and Metabolism research group led by Wiebke Arlt as a postdoctoral researcher where she works on the LC-MS analysis of steroids in serum and urine samples from women with PCOS.

Relevant Financial Disclosures (within past 24 months, reported on Jul 07, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder affecting approximately 10% of women. The defining feature of PCOS is androgen excess, which contributes to several symptoms such as hirsutism, acne and irregular periods. Androgen excess has also been linked to metabolic dysfunction in women with PCOS. The international PCOS guidelines recommend the measurement of testosterone (T) and, if normal, also of androstenedione (A4) and DHEA sulfate. However, the adrenal-derived 11-oxygenated androgens were shown to significantly contribute to androgen excess in PCOS (1). 11-ketotestosterone (11KT) binds and activates the androgen receptor with similar potency as T and circulates in similar concentrations as T in healthy women. However, the clinical relevance and ranges of 11-oxygenated androgens in PCOS remain insufficiently understood.

OBJECTIVES:
To develop and optimize a UPLC-MS/MS assay for the quantification of 18 steroids in serum samples from women with PCOS.

METHODS:
The steroid panel included 18 steroids encompassing precursors and active glucocorticoids, mineralocorticoids and androgens, both classic, e.g., A4, T, DHEA, and 11-oxygenated, e.g., 11KT, 11β-hydroxyandrostenedione (11OHA4), and 11-ketoandrostenedione. Steroids were extracted from 100 µL of serum using supported liquid extraction (SLE) with ethyl acetate and analysed by UPLC-MS/MS. The assay was applied to a preliminary screen of 109 serum samples from women with PCOS.

RESULTS:
Optimization of UPLC-MS/MS focused mostly on the chromatographic separation and ionization efficiency. Comparing post-column infusion of ammonium fluoride and formic acid in the mobile phase revealed that ammonium fluoride enhanced the signal intensity of most steroids up to fourfold. However, steroids with a saturated A ring (e.g., DHEA, 5α-androsterone) ionized better with formic acid. To balance this, a timed post-column infusion of 2 mmol/L ammonium fluoride was employed during chromatographic separation. The mobile phase consisted of water and methanol with 0.01% formic acid. Different extraction solvents were tested, i.e., ethyl acetate, MTBE, dichloromethane, and MTBE : ethyl acetate (1:1, 4:1). Ethyl acetate provided the best recovery and reproducibility. Preliminary profiling results revealed DHEA, A4 and 11OHA4 as the predominant androgens in the PCOS serum samples.

CONCLUSION:
The UPLC-MS/MS assay was developed for comprehensive steroid profiling and is currently undergoing full validation. The assay will be applied to the large-scale prospective DAISy-PCOS cohort study to investigate steroid metabolome patterns and related clinical phenotypes and characterize the roles of classic vs. 11-oxygenated androgens in women with PCOS.

REFERENCES:
1. O´Reilly, M.W., et al., 11-Oxygenated C19 steroids are the predominant androgens in Polycystic Ovary Syndrome. J Clin Endocrinol Metab, 2017. 102(3): p. 840-848.

ACKNOWLEDGMENTS:
This research was supported by the Medical Research Council (MC_UP_1605/15) and the Wellcome Trust (209492/Z/17/Z).