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

Anabolic steroidsare of high biological interest due to theirinvolvement 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 fromstructural heterogeneity, poor ionization efficiency, and low naturalabundance. Their importance in a variety of clinically relevant assayshas prompted the consideration of integrating ion mobility spectrometry(IMS) into existing LC-MS assays, due primarily to its speed and structure-basedseparation capability. Herein we have optimized a rapid (2 min) targetedLC-IM-MS method for the detection and quantification of 40 anabolicsteroids and their metabolites. First, a steroid-specific calibrantmixture was developed to cover the full range of retention time, mobility,and accurate mass. Importantly, this use of this calibrant mixtureprovided robust and reproducible measurements based on collision crosssection (CCS) with interday reproducibility of <0.5%. Furthermore,the combined separation power of LC coupled to IM provided comprehensivedifferentiation 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. Thismethod was also capable of steroid profiling, providing quantitativeratios (e.g., testosterone/epitestosterone, androsterone/etiocholanolone,etc.). Lastly, phase II steroid metabolites were probed in lieu ofhydrolysis to demonstrate the ability to separate those analytes andprovide information beyond total steroid concentration. This methodhas tremendous potential for rapid analysis of steroid profiles inhuman urine spanning a variety of applications from developmentaldisorders to doping in sport.