Multiplexed steroid profiling of gluco- and mineralocorticoids pathways using a liquid chromatography tandem mass...

Serum steroid assays are major tools in the clinical evaluation of adrenal disorders. The main adrenal steroids are routinely measured with immunoassays. However, chromatographic methods are known to offer better specificity. We report a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of 15 adrenal steroids targeting the mineralo- and gluco-corticosteroid pathways. Serum steroids combined with deuterated internal standards were extracted using successive protein precipitation and solid phase extraction steps. Cortisol, cortisone, 11-deoxycortisol, 17-hydroxyprogesterone, 21-deoxycortisol, progesterone, 11-deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, 18-hydroxycorticosterone, 18-hydroxy-11-deoxycorticosterone, aldosterone, dehydroepiandrosterone sulfate, testosterone and androstenedione were resolved in fourteen minutes using a BEH C18 column coupled to a methanol-ammonium formate gradient. Detection was performed using multiple reaction monitoring quantitation. Routinely determined steroid levels by immunoassays were compared to those measured by LC-MS/MS. This method was applied to assess steroid profiles in congenital adrenal hyperplasia (CAH) patients with 21-hydroxylase deficiency. Low quantification limits depending on each steroid (ranging from 0.015ng/mL for aldosterone to 20ng/mL for DHEAS) are adapted to the clinical use. Recoveries of steroids range from 64% for 21-deoxycortisol to 101% for cortisol and are fully corrected by internal standards. A good linearity with R>0.989 is obtained for each compound. The inter-day variation coefficients ranged from 4.7% for cortisol to 16.3% for 11-deoxycorticosterone. The immunoassay for cortisol (Immulite 2000, Siemens) showed acceptable agreement with LC-MS/MS (bias +7.2%). However, Bland-Altman plots revealed large negative bias for aldosterone (-33.4%, AldoCT, CisBio international), for 17-hydroxyprogesterone at concentrations below 2ng/mL (-74.1%, OHP-CT MP Biomedical), for androstenedione (-80.3%, RIA D4, Beckman Coulter) and for 11-deoxycortisol (-125.3%, Diasource Immunoassays). Finally, the analysis of samples from 21-hydroxylase defective patients demonstrated the potential usefulness of multiplexed steroid profiling for the diagnosis and/or monitoring of different forms of congenital adrenal hyperplasia. This LC-MS/MS method provides highly sensitive and specific assessments of mineralo- and glucocorticoids pathways from a small volume sample and is therefore a promising potent tool for clinical and experimental endocrine studies.