LC-MS/MS analysis of 5 steroids in plasma in a clinical study of Congenital Adrenal Hyperplasia v2

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders that affects adrenal steroidogenesis, resulting in deficiency of the glucocorticoid cortisol and in many cases the mineralocorticoid aldosterone1. This resulting lack of glucocorticoid (and mineralocorticoid) activates the hypothalamic-pituitary-adrenal (HPA) axis causing excessive release of adrenocorticotrophic hormone (ACTH) and excess adrenal androgen synthesis. Diagnosing and treating CAH requires reliable methods for steroid analysis. Tandem mass spectrometry methods coupled with chromatographic separation are considered the gold standard analytical technique for steroid analysis2 with the added benefit of enabling simultaneous analysis of multiple steroids. There are a range of methods that have been developed to measure multiple steroids in CAH3. Here we have developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for application to a clinical study that specifically explores the administration of d8-corticosterone as an alternative to hydrocortisone for CAH treatment. Plasma samples (200 µL) were enriched with isotopically labelled steroids, diluted with water (0.1% formic acid v/v) and extracted alongside a (0.0025 - 400 ng) calibration curve, by automated 96-well supported liquid extraction (SLE), using dichloromethane and isopropanol as an organic solvent, on a Biotage Extrahera automated sample handler. Extracted steroids were separated on a Shimadzu Nexera uHPLC with gradient elution on a Kinetex C18 column (150 x 3 mm; 2.6 µm) and a mobile phase of methanol and water (0.1% formic acid in water and methanol). The run time was 16 minutes, followed by mass spectral analysis on an AB Sciex 6500+ tandem quadrupole mass spectrometer operated in multiple reaction mode, positive ionisation. The method measures five steroids - hydrocortisone (cortisol) and d8-corticosterone - combined with markers of CAH - androgens and the intermediate 17α-hydroxyprogesterone, alongside the internal standards - in plasma. Validation demonstrated that this method is sensitive, specific, and reliable.