Potential of ion mobility-mass spectrometry for both targeted and non-targeted analysis of phase II steroid metabolites in urine

In recent years, the commercialization of hybrid ion mobility-mass spectrometers and their integration in traditional LC-MS workflows provide new opportunities to extend the current boundaries of targeted and non-targeted analyses. When coupled to LC-MS, ion mobility spectrometry (IMS) provides a novel characterization parameter, the so-called averaged collision cross section (CCS, Ω), as well as improves method selectivity and sensitivity by the separation of isobaric and isomeric molecules and the isolation of the analytes of interest from background noise. In this work, we have explored the potential and advantages of this technology for carrying out the determination of phase II steroid metabolites (i.e. androgen and estrogen conjugates, including glucuronide and sulfate compounds; n = 25) in urine samples. These molecules have been selected based on their relevance in the fields of chemical food safety and doping control, as well as in metabolomics studies. The influence of urine matrix on the CCS of steroid metabolites was evaluated in order to give more confidence to current CCS databases and support its use as complementary information to retention time (Rt) and mass spectra for compound identification. Samples were only diluted 10-fold with aqueous formic acid (0.1%, v/v) prior analysis. Only an almost insignificant effect of adult bovine urine matrix on the CCS of certain steroid metabolites was observed in comparison with calve urine matrix, which is a less complex sample. In addition, high accuracy was achieved for CCS measurements carried out over four months (ΔCCS < 1.3% for 99.8% of CCS measurements; n = 1806). Interestingly, it has been observed that signal-to-noise (S/N) ratio could be improved at least 2 or 7-fold when IMS is combined with LC-MS. In addition to the separation of isomeric steroid pairs (i.e. etiocholanolone glucuronide and epiandrosterone glucuronide, as well as 19-noretiocholanolone glucuronide and 19-norandrosterone glucuronide), steroid-based ions were also separated in the IMS dimension from co-eluting matrix compounds that presented similar mass-to-charge ratio (m/z). Finally, based on CCS measurements and as a proof of concept, 17α-boldenone glucuronide has been identified as one of the main metabolites resulted from boldione administration to calves.