Development and validation of an LC-MS/MS assay for serum 5α-androstane-3α, 17β-diol 17-glucuronide with enhanced interference resolution.

5α-androstane-3α,17β-diol 17-glucuronide (3α-diol G) is a testosterone metabolite and an important biomarker of hormonal dysregulation, including polycystic ovary syndrome (PCOS) and virilizing syndromes in women such as idiopathic hirsutism. While immunoassay kits are commonly used for its quantification in clinical laboratories, these methods are prone to interference due to the structural similarity among steroidal compounds. In this study, we developed and validated a robust LC-MS/MS method for the quantification of 3α-diol G in human serum. Extraction was performed using a μelution SPE approach, allowing the achievement of a suitable LLOQ (0.140 ng/mL) with a low sample volume (100 μL). The chromatographic method successfully separated major known interferences, including glucuronidated androsterone (ADT-G) and etiocholanolone (Etio-G), and resolved unknown coeluting compounds that could otherwise lead to overestimation. Additionally, monitoring the transitions of the most common phospholipid showed no interference from these species. Method validation followed CLSI recommendations, assessing imprecision, accuracy, linearity, limit of quantification, carryover, and stability. The routine analysis incorporated a system suitability protocol based on ion ratio monitoring to detect potential coeluting interferences prior to batch analysis. This work highlights the importance of chromatographic separation and ion ratio monitoring in steroid analysis and provides a reliable alternative to immunoassay-based quantification of 3α-diol G.