Carbon Isotope Ratio Analysis of Urinary Steroids Following Extensive Cleanup and Formylation

Gas chromatography–combustion–isotope ratio mass spectrometry (GC‐C‐IRMS) is the method of choice to detect the abuse of synthetic forms of naturally occurring steroids for doping control purposes. GC‐C‐IRMS relies on a multistep sample cleanup to ensure each target analyte peak is chromatographically pure before combustion. To achieve that, liquid–liquid or solid phase extraction (SPE) is commonly used in combination with preparative liquid chromatography (LC). In this work, a procedure for isolation, purification, and analysis of endogenous steroids by GC‐C‐IRMS was developed and validated. The key elements were successive application of strong cation and strong anion exchange SPE with enzymatic hydrolysis in between to strip the ionic species from urine and decrease matrix complexity prior to LC cleanup; preparative two‐dimensional LC, where only the testosterone fraction required secondary purification (40 min total run time per sample); and derivatization of selected fractions with formic acid to yield formate esters, followed by GC‐C‐IRMS analysis. Formylation afforded excellent separation between 5α‐ and 5β‐androstanediols and simplified the detection of the endogenous reference compound pregnanetriol by converting it to a volatile artifact, tentatively identified as 3α,20‐diformoxy‐17‐methyl‐18‐nor‐5β,17α‐pregn‐13‐ene. The overall method performance benefited from the customization of the GC‐C‐IRMS combustion interface, which improved robustness and facilitated the transfer of sample components into the hot zone of the oxidation reactor, minimizing peak tailing. The former was achieved by keeping the oxygen flow through the reactor at all times, obviating the need for periodic oxidation, and the latter—by implementing a direct capillary‐in‐capillary connection of the chromatographic column to the oxidation reactor.