Endocrine Abstracts

Background: Development of multi-steroid profiling allows comprehensive investigation into the different branches of steroid metabolism. Immunoassays only allow analysis of a single steroid per assay and suffer from problems with specificity due to cross reactivity of similar steroids. Liquid-chromatography mass spectrometry has the specificity to analyse multiple steroids in a single experiment and the dynamic range to quantify steroids at high concentrations such as those observed for cortisol (50-600nM) and at low concentration such as DHT (0.07-2.5nM). Here, we present the optimisation, validation and application of an ultra-high performance liquid chromatography-tandem mass spectrometry assay for the profiling of 25 steroids.

Methods and Results: Sensitivity in mass spectrometry can be enhanced by addition of mobile phase additives which aid ionisation. Typically for steroids this is an acidic additive such as formic acid. Ammonium fluoride significantly enhanced ionisation in a steroid structure-dependent fashion compared to the use of formic acid, with increases in average peak area ranging between 100% and 1280%. Therefore, we validated our method with ammonium fluoride as the additive. Quantification was performed on a Waters Xevo TQ-XS mass spectrometer using electrospray ionisation in positive ion mode. Steroids were extracted via liquid-liquid extraction (using 1ml tert-methyl butyl ether) from 200μL of serum after addition of an isotopically labelled internal standard mixture. Steroids were chromatographically separated using a Phenomenex Luna Omega C18 column (1.6μm, 100Å, 2.1 x 50mm) with a water-methanol gradient. To extend column lifetime ammonium fluoride was introduced via post-column infusion (6mmol/L at 5μL/min). This method was then clinically validated and applied to serum from a healthy control cohort (167 females and 125 males, aged 21-95 years) to obtain a reference range, 18 of the 25 steroids were quantifiable in serum. Lower limits of quantification ranged from 0.28 to 3.42nM. Extraction efficiencies ranged from 90-122% and matrix effects were -19 to + 19%. Imprecision and bias at four concentrations did not exceed 15% for the majority of analytes.

Conclusions: Ammonium fluoride used as a mobile phase additive significantly enhanced sensitivity for steroid multiplex analysis. Thus, allowing quantitative analysis of 25 steroids from glucocorticoid, mineralocorticoid and androgen biosynthetic pathways in a single assay- a comprehensive assessment of the steroid metabolome. This method can be applied to a variety of biological fluids, including serum, cell and tissue culture extracts.