LAUSR

Abstract Disclosure: T. Skodova: None. L. Kolatorova: None. J. Heracek: None. J. Vitku: None. Background: Androgens, along with other sex steroids, play a role in the initiation as well as the progression of prostate cancer (PCa). In recent years, 11-oxygenated androgens—11-hydroxy or 11-keto derivatives of classical androgens such as testosterone, dihydrotestosterone, and androstenedione—have gained attention due to their high androgenic potential, which is comparable to that of classic androgens. Therefore, 11-oxygenated androgens may contribute to the pathogenesis of PCa. However, a sensitive analytical method for the determination of 11-oxygenated androgens in the prostate is lacking. The aim of this study was to develop and validate a sensitive, specific, and rapid LC-MS/MS method for simultaneous quantification of five 11-oxygenated androgens in prostate tissue. Methods: After evaluating various additives, mobile phases, gradients, and chromatographic columns, optimal separation was achieved using a Kinetex Biphenyl column (50 mm × 3 mm, 2.6 μm) with a water-methanol mobile phase system containing 0.1% formic acid. The chromatographic conditions were set at 35°C with a flow rate of 0.7 mL/min. Detection was performed using a QTRAP6500+ mass spectrometer with an electrospray ionization (ESI) probe operating in positive mode. The method reached peak performance when approximately 45 mg of prostate tissue was homogenized and extracted using 2 mL of a hexane-ethyl acetate (3:2) solution. Results: The method enables simultaneous analysis of five 11-oxygenated androgens within a 6-minute runtime: 11β-hydroxyandrostenedione, 11-ketoandrostenedione, 11-ketotestosterone, 11β-hydroxytestosterone, and 11β-hydroxydihydrotestosterone. The assay demonstrated high precision, with both intra- and inter-day coefficients of variation not exceeding 15%. Accuracy ranged from 96% to 103% of nominal concentrations across all analytes. The method achieved sensitive detection with lower limits of quantification ranging from 0.2 to 1.6 pg/mg of prostate tissue. Conclusion: The method was successfully validated according to FDA guidelines and will be further used to analyze prostate tissue samples from PCa patients to evaluate their role in pathogenesis of disease. Acknowledgement: Supported by NU21J-01-00040 of Czech Ministry of Health. Presentation: Saturday, July 12, 2025