A Sensitive and Robust Back-Flush Transfer 2D-LC-MS/MS Combined with Quaternary Ammonium Oxime Derivatization for the Quantification of 12 Steroid Hormones in Serum from Polycystic Ovary Syndrome Patients

Hyperandrogenemia is one of the key clinical features of polycystic ovary syndrome (PCOS). However, current research has largely focused on serum levels of classical androgens like testosterone, which does not fully capture the comprehensive profile of sex hormones in PCOS. To address this, we established a method based on quaternary ammonium oxime (QAO) derivatization coupled with back-flush transfer 2D-LC-MS/MS, enabling simultaneous quantification of 4 progestogens and 8 androgens (including 4 classical androgens and 4 11-oxygenated androgens) in serum samples from PCOS and Non-PCOS individuals. Benefiting from the online capture and purification effects of first dimension column (LC1), this method increased the injection volume from 20 to 40 μL while maintaining negligible matrix effects. The flow path was reconfigured by valve switching to back-flush the mobile phase into LC1, delivering the preconcentrated analytes as a narrow band into the second-dimension column (LC2) for refined separation and improved chromatographic performance. Without sample preconcentration, this approach enhanced detection sensitivity, with the lower limits of quantification (LLOQ) of 5-100 pg/mL for all steroid hormones. Following method validation, we analyzed clinical samples. Most steroid hormones were significantly elevated in the PCOS group compared to the Non-PCOS group, with the exception of 11-hydroxytestosterone and 11-ketotestosterone. These findings provide insights into the androgen profiles of PCOS patients. To our knowledge, the back-flush transfer 2D-LC-MS/MS technique holds immense potential for the exploration of biomarkers across a wide range of diseases in the future.