Determination of testosterone and epitestosterone glucuronides in urine by ultra performance liquid chromatography-ion mobility-mass spectrometry

Abstract

UPLC-ion mobility spectrometry separations combined with mass spectrometry (UPLC-IM-MS) and tandem mass spectrometry (UPLC-IM-MS/MS) have been investigated for the simultaneous determination of testosterone and epitestosterone glucuronides in urine. The glucuronide epimers of testosterone and epitestosterone were separated by ion mobility spectrometry prior to mass analysis on the basis of differences in their collision cross sections, which have been measured in nitrogen. Combining ion mobility separation with UPLC/MS enhances the analysis of these low-abundance steroids in urine by selective interrogation of specific retention time, mass-to-charge and mobility regions. Detection limits for the UPLC-IM-MS/MS analysis of TG and ETG were 9.9 ng mL⁻¹ and 98 ng mL⁻¹ respectively, equivalent to 0.7 ng mL⁻¹ and 7.4 ng mL⁻¹ in urine, with linear dynamic ranges corresponding to 0.7–108 ng mL⁻¹ and 7.4–147 ng mL⁻¹ in urine. Repeatability (%RSD) for urine extracts was 0.64% and 2.31% for TG and ETG respectively.