Multi-steroid profiling by UHPLC-MS/MS with post-column infusion of ammonium fluoride

Multi-steroid profiling is a powerful analytical tool that simultaneously quantifies steroids from different biosynthetic pathways. Here we present an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the profiling of 23 steroids using post-column infusion of ammonium fluoride.

Following liquid-liquid extraction, steroids were chromatographically separated over 5 min using a Phenomenex Luna Omega C₁₈ column and a water (0.1 % formic acid) methanol gradient. Quantification was performed on a Waters Acquity UHPLC and Xevo® TQ-XS mass spectrometer. Ammonium fluoride (6 mmol/L, post-column infusion) and formic acid (0.1 % (vol/vol), mobile phase additive) were compared as additives to aid ionisation.

Post-column infusion of ammonium fluoride enhanced ionisation in a steroid structure-dependent fashion compared to formic acid (122-140 % for 3βOH-Δ5 steroids and 477-1274 % for 3-keto-Δ4 steroids). Therefore, we analytically validated post-column infusion of ammonium fluoride. Lower limits of quantification ranged from 0.3 to 3 nmol/L; All analytes were quantifiable with acceptable accuracy (bias range -14 % to 11.9 % for 21/23, -21 % to 11.9 % for all analytes). Average recovery ranged from 91.6 % to 113.6 % and average matrix effects from -29.9 % to 19.9 %. Imprecision ranged from 2.3 % to 23 % for all analytes and was < 15 % for 18/23 analytes. The serum multi-steroid profile of 10 healthy men and 10 healthy women was measured.

UHPLC-MS/MS with post-column infusion of ammonium fluoride enables comprehensive multi-steroid profiling through enhanced ionisation particularly benefiting the detection of 3-keto-Δ4 steroids.