High-throughput analysis of 19 endogenous androgenic steroids by ultra-performance convergence chromatography tandem mass spectrometry

11-Oxygenated steroids such as 11-ketotestosterone and 11-ketodihydrotestosterone have recently been shown to play a putative role in the development and progression of castration resistant prostate cancer. In this study we report on the development of a high throughput ultra-performance convergence chromatography tandem mass spectrometry (UPC²-MS/MS) method for the analysis of thirteen 11-oxygenated and six canonical C19 steroids isolated from a cell culture matrix. Using an Acquity UPC² BEH 2-EP column we found that UPC² resulted in superior selectivity, increased chromatographic efficiency and a scattered elution order when compared to conventional reverse phase ultra-performance liquid chromatography (UPLC). Furthermore, there was a significant improvement in sensitivity (5–50 times). The lower limits of quantification ranged between 0.01–10 ng mL⁻¹, while the upper limit of quantification was 100 ng mL⁻¹ for all steroids. Accuracy, precision, intra-day variation, recovery, matrix effects and process efficiency were all evaluated and found to be within acceptable limits. Taken together we show that the increased power of UPC²-MS/MS allows the analyst to complete in vitro assays at biologically relevant concentrations for the first time and in so doing determine the routes of steroid metabolism which is vital for studies of androgen responsive cancers, such as prostate cancer, and could highlight new mechanisms of disease progression and new targets for cancer therapy.