Artifactual elevations of 11β-hydroxyandrostenedione and 11-ketoandrostenedione in mass spectrometry assays.

Comprehensive steroid profiling by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) can be achieved using small biospecimen samples. LC-MS/MS assays offer superior accuracy to immunoassays, but they also introduce layers of complexity and opportunities for errors. Validation and harmonization studies are essential to ensure reliable results, as these assays are being increasingly incorporated in clinical laboratories. We developed an LC-MS/MS assay that simultaneously quantifies 18 Δ steroids, including mineralocorticoids, glucocorticoids, and androgens (both classic and 11-oxygenated androgens). Non-enzymatic conversion of cortisol and cortisone to 11β-hydroxyandrostenedione (11OHA4), and 11-ketoandrostenedione (11KA4), respectively, was assessed in dry and extracted human serum, and pure cortisol and cortisone solutions, at various temperatures and timepoints. We observed non-enzymatic conversion of cortisol and cortisone to 11OHA4 and 11KA4, respectively, in both human serum samples and pure cortisol and cortisone solutions at ambient temperature, and when incubated at 37 °C, but not at -20 °C. This phenomenon was amplified in dried steroids extracts, reaching 50-fold and 34-fold increase in 11OHA4 and 11KA4 peak areas, respectively. Non-enzymatic conversion of cortisol and cortisone to 11OHA4 and 11KA4 is a source of spurious LC-MS/MS results. Prompt steroid reconstitution on ice following solvent evaporation is required for accurate measurements of 11-oxyandrogens. Inter-laboratory harmonization of LC-MS/MS assays is needed to generate reliable results prior to clinical implementation.