Endocrine Abstracts

Comprehensive multi-steroid profiling offers a powerful tool for the investigation, diagnosis and management of steroidogenic disorders by simultaneously quantifying multiple steroids from several pathways of steroid biosynthesis and metabolism. Difficulties can arise when optimising chromatography and mass spectrometry conditions for many analytes in a single method. Low concentrations of ammonium fluoride have previously been shown to enhance the sensitivity of select analytes when used as an LC mobile phase additive; however, its impact on multi-steroid profiling has yet to be investigated. Here, we present the optimisation, validation and application of an ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the profiling of 25 steroids in serum. Samples were mixed with isotopically labelled internal standards and extracted by liquid-liquid extraction. Steroids were chromatographically separated in 5.5 minutes using a Phenomenex Luna Omega C18 column (1.6 μm, 100 Å, 2.1 x 50 mm) and a water-methanol gradient. Quantification was performed on a Waters Xevo TQ-XS mass spectrometer using electrospray ionisation in positive ion mode. Ammonium fluoride (6 mmol/l, post-column infusion (PCI)) and formic acid (0.1 % (v/v), mobile phase additive) were compared as ionisation additives. PCI of ammonium fluoride significantly enhanced ionisation in a structure-dependent fashion compared to the use of formic acid as mobile phase additive, with the exception of androstanediol that showed reduced ionisation efficiency with NH₄F. Using PCI NH₄F, accuracy was acceptable for 23/25 analytes (bias range 14.0% to 11.9%). Imprecision for serum and spiked surrogate matrix samples ranged from 2.3% to 23.9% and was <15% for 18/25 analytes. PCI of the mobile phase additive increased both ionisation of the steroids and column lifetime. PCI enabled the simultaneous, sensitive profiling of 25 steroids from glucocorticoid, mineralocorticoid and androgen biosynthesis pathways allowing for a comprehensive assessment of the steroid metabolome in serum.