Multidimensional Separations of Intact Phase II Steroid Metabolites Utilizing LC–Ion Mobility–HRMS

Abstract

The detection and unambiguous identification of anabolic-androgenic steroid metabolites are essential in clinical, forensic, and antidoping analyses. Recently, sulfate phase II steroid metabolites have received increased attention in steroid metabolism and drug testing. In large part, this is because phase II steroid metabolites are excreted for an extended time, making them a potential long-term chemical marker of choice for tracking steroid misuse in sports. Comprehensive analytical methods, such as liquid chromatography–tandem mass spectrometry (LC–MS/MS), have been used to detect and identify glucuronide and sulfate steroids in human urine with high sensitivity and reliability. However, LC–MS/MS identification strategies can be hindered by the fact that phase II steroid metabolites generate nonselective ion fragments across the different metabolite markers, limiting the confidence in metabolite identifications that rely on exact mass measurement and MS/MS information. Additionally, liquid chromatography–high-resolution mass spectrometry (LC–HRMS) is sometimes insufficient at fully resolving the analyte peaks from the sample matrix (commonly urine) chemical noise, further complicating accurate identification efforts. Therefore, we developed a liquid chromatography–ion mobility–high resolution mass spectrometry (LC–IM–HRMS) method to increase the peak capacity and utilize the IM-derived collision cross section (CCS) values as an additional molecular descriptor for increased selectivity and to improve identifications of intact steroid analyses at low concentrations.

Comparability of Steroid Collision Cross Sections Using Three Different IM-HRMS Technologies: An Interplatform Study

“…Due to their role in regulating metabolism, growth, reproductive function and immune response, steroids are analyzed across a wide range of research fields 1,2 . Furthermore, the use of exogenous anabolic androgenic steroids (AASs) to enhance growth in livestock production was banned by the European Union (EU) 4 and various natural and synthetic steroids are prohibited doping agents in competitive sports [5][6][7] . Within this context, steroids are analyzed in diverse biological matrices including serum, brain tissue, or urine 1,6,8 .…”

“…Steroid phase II metabolites are of major interest when analyzing urine (e.g. for drug testing, in chemical food safety or doping control) due to a high degree of metabolization and secretion 5,6 . The combination of complex matrices, low concentrations and large number of possible steroid isomers 8 demands for high-performance methods in terms of selectivity and sensitivity 6 .…”

“…Therefore, mass spectrometry (MS) coupled to front-end chromatographic separation such as liquid chromatography (LC) has become one of the key technologies for the analysis of both steroids and their phase II metabolites 2 . More recently, ion mobility-mass spectrometry (IM-MS) has also gained significant attention for potential application to separate and identify isomeric and isobaric steroids 5,9,10 . As a standalone technology, IM-MS has been applied for rapid steroid analysis, but its application is limited for the most demanding applications due to the high complexity of biological matrices and by the limited resolution of current IM technology 1,11 .…”

“…However, in combination with LC (i.e. LC-IM-MS), the benefits of both analytical platforms can be exploited 5 . For example, signal-to-noise ratios were improved when IM-filtering is used for mass spectra clean-up 6 .…”

“…For example, signal-to-noise ratios were improved when IM-filtering is used for mass spectra clean-up 6 . In addition to increased peak capacity and cleaner fragment spectra 12 , IM-derived collision cross sections (CCSs) have been extensively discussed as a potential ion species-specific descriptor for identity confirmation 6,13,14 and some notable studies focused on the potential analysis of steroids using (LC-)IM-MS have been published recently 5,6,9,10,[14][15][16] . Application of CCS as additional identification parameter is of special interest for small molecules that exhibit limited fragmentation or formation of only unspecific fragments 17,18 .…”