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

Background 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 (uPLC-MS/MS) assay for the profiling of 25 steroids using post-column infusion of ammonium fluoride. Methods Following liquid-liquid extraction, steroids were chromatographically separated over 5 minutes using a Phenomenex Luna Omega C 18 column and a water (0.1 % formic acid) methanol gradient. Quantification was performed on a Waters Acquity uPLC 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. Results Post-column infusion (PCI) of ammonium fluoride (NH 4 F) 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 fully analytically validated PCI with NH 4 F. Lower limits of quantification ranged from 0.28 to 3.42 nmol/L; 23 of 25 analytes were quantifiable with acceptable accuracy (bias range −14% to 11.9%). 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 23.9% for all analytes and was <15% for 18/25 analytes. The serum multi-steroid profile of 10 healthy men and 10 healthy women was measured. Conclusions uPLC-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. Highlights This multi-steroid profiling assay quantifies 25 steroids in 5.5 minutes Post-column infusion of NH 4 F enhances the ionisation of 3-keto-Δ4 steroids The assay simultaneously quantifies steroids from several biosynthetic pathways We present analytical data validated for serum steroid profiling