Development of a Rapid, Targeted LC-IM-MS Method for Anabolic Steroids

Anabolic steroids are of high biological interest due to their involvement in human development and disease progression. Additionally, they are banned in sport due to their performance-enhancing characteristics. Analytical challenges associated with their measurement stem from structural heterogeneity, poor ionization efficiency, and low natural abundance. Their importance in a variety of clinically relevant assays has prompted the consideration of integrating ion mobility spectrometry (IMS) into existing LC-MS assays, due primarily to its speed and structure-based separation capability. Herein we have optimized a rapid (2 min) targeted LC-IM-MS method for the detection and quantification of 40 anabolic steroids and their metabolites. First, a steroid-specific calibrant mixture was developed to cover the full range of retention time, mobility, and accurate mass. Importantly, this use of this calibrant mixture provided robust and reproducible measurements based on collision cross section (CCS) with interday reproducibility of <0.5%. Furthermore, the combined separation power of LC coupled to IM provided comprehensive differentiation of isomers/isobars within 6 different isobaric groups. Multiplexed IM acquisition also provided improved limits of detection, which were well below 1 ng/mL in almost all compounds measured. This method was also capable of steroid profiling, providing quantitative ratios (e.g., testosterone/epitestosterone, androsterone/etiocholanolone, etc.). Lastly, phase II steroid metabolites were probed in lieu of hydrolysis to demonstrate the ability to separate those analytes and provide information beyond total steroid concentration. This method has tremendous potential for rapid analysis of steroid profiles in human urine spanning a variety of applications from developmental disorders to doping in sport.