LC-MS/MS measurement of endogenous steroid hormones and phase II metabolites in blood volumetric absorptive microsampling (VAMS) for doping control purposes

•VAMS is a non-invasive, innovative sample collection technology.•First LC-MS/MS method for quantification of wide panel of steroids in VAMS was developed.•Method quantitative validation in compliance with ISO/IEC 17025:2017 and WADA requirements.•Steroids are stable in VAMS stored at different temperature conditions for up to 100 days. Volumetric Absorptive Microsampling (VAMS) is emerging as a valuable technique in the collection of dried biological specimens, offering a potential alternative to traditional sampling methods. The objective of this study was to assess the suitability of 30 μL VAMS for the measurement of endogenous steroid hormones. A novel LC-MS/MS method was developed for the quantification of 18 analytes in VAMS samples, including main endogenous free steroids and phase II metabolites of androgens. The method underwent validation in accordance with ISO/IEC 17025:2017 and World Anti-Doping Agency (WADA) requirements. Subsequently, it was applied to authentic VAMS samples obtained from 20 healthy volunteers to assess the stability of target analytes under varying storage conditions. The validation protocol assessed method’s selectivity, matrix effect, extraction recovery, quantitative performance, carry-over and robustness. The analysis of authentic samples demonstrated the satisfactory stability of monitored steroids in VAMS stored at room temperature, 4 °C, −20 °C and −80 °C for up to 100 days and subjected to up to 3 freezing-thawing cycles. The validated LC-MS/MS method demonstrated its suitability for the measurement of steroids in dried blood VAMS. The observed stability of steroidal compounds suggests promising prospects for future applications of VAMS, both in anti-doping contexts and clinical research.