Evstafev, I.; Krakstrom, M.; Saarinen-Aaltonen, N.; Hakkarainen, J.; Hakkinen, M. R.; Auriola, S.; Bostrom, P. J.; Poutanen, M.; Oresic, M.; Dickens, A. M.

Comprehensive detection of steroids, beyond the limited panels typically analyzed in clinical chemistry laboratories, has become increasingly important given their pivotal roles in diverse biological processes. However, steroid quantification poses several analytical challenges, including differences in ionization efficiency and structural similarities across the entire steroid metabolic network. To address these challenges, we developed a targeted ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay to analyze 21 steroids using reverse-phase chromatography combined with rapid polarity switching. Mass spectrometry (MS) analysis was performed in scheduled multiple reaction monitoring (sMRM) mode. Depending on the steroid and matrix, the validated lower limits of quantitation (LLOQ) ranged from 12.0 pM to 1216 pM in plasma and 41.1 pM to 384 pM in fecal sample homogenates. In adipose tissue, it was from 0.01 pmol/g to 9 pmol/g. Measured steroid concentrations obtained from the commercial control samples (MassTrak Steroid Serum QC Set 1 and the MassCheck Steroid Panel 1 Serum Control) showed close agreement with the reference values. As a proof of concept, the method was successfully applied to 469 plasma samples in several projects, 15 adipose tissue samples, and 332 fecal samples, demonstrating its applicability to large-scale studies. In conclusion, the method enables sensitive, derivatization-free quantification of an expanded steroid panel in plasma and complex biological matrices, including adipose tissue and fecal samples, representing a significant advancement in comprehensive steroid profiling.