Ultrafiltration-MSPE with in-situ quaternary aminooxy oximation for LC-MS/MS quantification of nine serum free androgens

Accurate quantification of circulating androgens, particularly the biologically active free fraction, is analytically challenging due to ultra-low concentrations and extensive protein binding in human serum. Here, we developed and validated an integrated ultrafiltration-LC-MS/MS workflow for simultaneous quantification of nine endogenous serum free androgens. Free fractions were isolated by phosphate-buffered saline dilution followed by temperature-controlled centrifugal ultrafiltration (37 °C) using pretreated regenerated-cellulose membranes to minimize non-specific adsorption. Ultrafiltrates were purified and enriched by magnetic solid-phase extraction (MSPE), and in-situ oximation derivatization with a quaternary aminooxy reagent was applied to enhance electrospray response. The validation data demonstrated pg/mL-level sensitivity with LLOQ of 0.5-10 pg/mL, good linearity (r > 0.99), acceptable matrix effects, and high recovery. The method was applied to serum samples from 86 female patients (21-50 years) undergoing infertility evaluation, showing higher free testosterone, 11β-hydroxyandrostenedione, and androstenedione in an AMH-enriched PCOS-suspicion subgroup versus a control group. Overall, this ultrafiltration-MSPE-in-situ derivatization LC-MS/MS method provides a practical and robust platform for multiplexed measurement of serum free androgens at pg/mL levels for clinical research applications.