A Simplified and Robust LC-MS/MS Method for Simultaneous Quantification of Six Steroid Hormones Associated With...

Polyendocrine metabolic ovarian syndrome (PMOS), formerly known as polycystic ovary syndrome (PCOS), is characterized by dysregulation of multiple steroid hormones. Accurate quantification of these analytes is essential for laboratory evaluation; however, currently may involve complex sample-preparation workflows or provide insufficient analytical coverage, particularly for dehydroepiandrosterone sulfate (DHEAS). A liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was established for the simultaneous measurement of six steroid hormones associated with PMOS. Sample preparation combined liquid-liquid extraction with protein precipitation. Chromatographic separation was completed within 6 min. Validation followed international recommendations and included linearity, sensitivity, precision, accuracy, matrix effects, selectivity, and specificity. All analytes showed excellent linearity (R = 0.9959-0.9995), with intra- and inter-day CVs ≤ 5.6%. Spike recovery ranged from 85.2% to 114.9%. Accuracy was further supported by third-party QC materials and successful participation in an external quality assessment program. The assay provided an expanded linear range for DHEAS (20-20,000 ng/mL). This validated LC-MS/MS assay provides rapid, sensitive, and reproducible quantification of six PCOS-related steroid hormones and is suitable for routine laboratory implementation.