Liquid chromatography-ion mobility spectrometry-mass spectrometry analysis of multiple classes of steroid hormone isomers in a mixture

Methods for the analysis of steroids have long been of interest due to the multiple uses for such methods in medical applications, sports monitoring, and environmental science. The analysis of steroids involves inherent analytical hurdles due to their low biological concentrations, poor ionization efficiencies, and frequent occurrence of isomerism. One analytical technique that has been recently applied to steroid analysis is ion mobility spectrometry (IMS). While previous work has focused on the use of metal adduction and multimer formation to enhance separation through IMS analysis coupled to mass spectrometry (MS), this work furthers this approach by coupling IMS-MS with liquid chromatography (LC). Three different LC methods with varying tradeoffs between chromatographic resolution and run time were developed, with one of these achieving a resolution above 1.5 for all steroid isomers. These results also indicate that the coupling of LC to IMS-MS can increase the overall resolution of steroid isomers relative to what can be achieved by either LC or IMS alone. Furthermore, the use of LC and IMS in concert can allow for a more rapid analysis of steroid isomers than can be achieved by LC-MS alone. Finally, the IMS dimension provided for measurements of ion-neutral collision cross sections (CCSs), which were found to be in good agreement with previously reported measurements. Thus, this approach provides three complementary quantitative parameters (retention time, CCS, and mass-to-charge ratio) that can contribute the identification of analytes. Overall, the work presented here demonstrates the potential of coupling LC, IMS, and MS for the analysis of isomeric steroid hormones.