MSACL 2023 Abstract

INTRODUCTION:
Identification and quantification of steroid metabolites can be confounded by significant isomeric heterogeneity, compounded by other analytical challenges associated with steroid analysis (poor ionization efficiency, wide polarity range, etc.). Ion mobility-mass spectrometry (IM-MS) has shown promise as a rapid approach (acquisition times <100 ms) that can easily be coupled with existing chromatographic methods. Herein we will demonstrate a targeted LC-IM-MS/MS method for the quantification of androgen metabolites in human urine.

OBJECTIVES:
The primary objective of this study was to demonstrate simultaneous quantification of nearly forty androgen metabolites in human urine, with a reduced emphasis on chromatographic separation of isomers.

METHODS:
A targeted LC-IM-MS/MS method was developed for targeted quantitation of androgen metabolites in human urine. A simple 2-minute chromatographic gradient was chosen to highlight the rapid separation of isobaric/isomeric species with IM. Urine samples were extracted by SPE and injected onto the LC-IM-MS/MS system, where data was collected in multiplexed acquisition mode. All data was processed using a streamlined workflow software workflow culminating in quantification with Skyline. Confirmation for each metabolite was provided by retention time, collision cross section (CCS), and exact mass. Quantitation was performed over a clinically relevant concentration range for the analytes of interest. All data were acquired using either Agilent 6560 or MOBILion Systems MOBIE HRIM IM-MS platforms.

RESULTS:
Traditional GC- and LC-MS/MS methods for quantification of steroid metabolites rely on chromatographic approaches for separation of isobaric/isomeric species. Herein, we demonstrated that a fast RP-LC method (2 mins), which in many cases did not provide adequate separation, could be easily coupled with IM-MS/MS to provide rapid gas-phase separation by ion mobility. Nearly 40 androgens and their metabolites were first analyzed individually to populate a target library with retention time, CCS, and exact mass for each ion adduct identified. CCS values ranged from 166-190 Å2 for protonated species and 193-207 Å2 for sodiated species; these values were highly reproducible (RSD ≤ 0.5%), allowing improved confidence of identification. Human urine was then analyzed to detect and quantify these metabolites and determine limits of detection and sensitivity over a concentration range from 1 pg/mL to 10 ng/mL; preliminary results (Velosa et al. JASMS, 2022) demonstrated an LOD of ~500 pg/mL for testosterone.

CONCLUSION:
IM-MS/MS was demonstrated as a rapid method for detection and quantification of androgen metabolites in human urine. A fast chromatographic gradient was coupled with the developed IM-MS/MS method, such that isobaric/isomeric resolution was not reliant upon chromatographic differentiation but instead by mobility. Skyline was employed as a streamlined data processing approach for quantification. We hope to expand this workflow to other molecular classes relevant to the clinical lab, including opioids and other drugs of abuse.