INTRODUCTION: Ion mobility-mass spectrometry (IM-MS) has emerged over the last decade as a powerful discovery tool in the biomedical -omics. Despite this emergence, IM-MS has yet to see routine implementation in the clinical lab due to challenges in reproducibility, resolving power, and quantitation. However, recent advances in chemical/sample preparation, high-resolution instrumentation, and software/data processing tools have brought IM-MS closer to potential clinical applications.
OBJECTIVES: The primary objective of this study was to demonstrate how recent advances in IM-MS methods and technology have led to significant improvements in isomer resolution and quantitation for targeted steroid applications.
METHODS: A targeted LC-IM-MS/MS method was developed for detection and targeted quantitation of steroids in relevant biological matrixes (e.g., urine). Novel sample preparation steps involved structural specific derivatization reactions simultaneously targeting hydroxyl and carbonyl chemistry; these reactions were simple and relatively fast (<30 minutes per sample). IM measurements were made using multiplexing (i.e., multiple packet injections within the same IM experiment). All data was subsequently processed using demultiplexing software to reduce noise and improve resolving power. MS/MS was used for identification of derivatization products based on known neutral loss(es). Quantitation was performed over a clinically relevant concentration range for the analytes of interest.
RESULTS: Although many commercial IM-MS platforms struggle with resolution of isomers (especially stereoisomers) and quantitation, we demonstrated that a combination of simple sample preparation, methodological improvements, and data processing strategies could simultaneously yield benefits on all fronts. First, structurally selective derivatization reactions targeting hydroxyl and carbonyl chemistry provided: (a) structure/class determination based on relative number of functional groups; (b) improved ionization efficiency by introduction of fixed charge (quaternary amine) derivatives; (c) improved IM resolution by amplification of minor stereospecific differences; and (d) identification by characteristic MS/MS neutral losses. Second, we implemented a multiplexing method that allowed for our duty cycle to improve from <10% to >50%; this increase allowed for improvement of limits of detection by nearly an order of magnitude. Third, we employed a high-resolution demultiplexing software tool that improved resolving power to further increase separation between stereoisomers in complex mixtures. Overall, confidence in identification was improved for targeted steroid applications and limits of detection in the low pg/mL concentration range were achieved.
CONCLUSION: IM-MS is a powerful combination with significant promise in the future of the clinical lab. Targeted applications can be developed for biomolecular classes of interest, such as the steroids shown in this current study. Challenges in separation and quantitation can be overcome using creative combinations of sample preparation, instrument methods, and data processing strategies. We hope to expand this workflow to other molecular classes relevant to the clinical lab, including opioids and other drugs of abuse.