The comprehensive characterization of adrenocortical steroidogenesis using two-dimensional ultra-performance liquid chromatography - electrospray ionization tandem mass spectrometry.

The perturbation of the homeostasis of adrenocortical steroids plays a fundamental role in several pathological conditions. Currently, only a few of the substances involved in steroidogenesis are routinely analysed in clinical laboratories for the diagnosis of these conditions. Recently, interest has grown over the development of clinical assays of endogenous steroids using liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, no approaches have assessed the adrenocortical steroidogenesis comprehensively. Here, a novel LC-MS/MS assay is presented for evaluating the serum levels of all respective major substances (aldosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dihydrotestosterone, 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, corticosterone, cortisol, cortisone, pregnenolone, progesterone and testosterone). The analysis time was 5.5 min following highly efficient solid phase extraction conducted on a novel polymer phase with N-polyvinylpyrrolidine branches. The method was validated in accordance with the respective guideline of the European Medicines Agency. The cross-validation of 8 analytes with immunoassays was also accomplished. Two-dimensional chromatography allowed the elution of the 16 analytes between 2.3-4.6 min and with a sufficient resolution of isobaric compounds. Quantitation was performed throughout the clinically relevant concentration ranges. Within-run accuracy was 87.1-115%, 90.0-109%, 87.2-111% and 87.6-107% at spiking levels 1 thru 4, while the precision was 4.7-27.9%, 2.9-17.7%, 5.6-13.9% and 1.9-15.0%, respectively. Between-run accuracy was 81.0-119.5, 85.2-113, 87.4-113 and 93.1-113%, respectively, while the precision was 3.4-13.5%, 2.0-10.2%, 2.1-15.0%, and 1.5-6.6%, respectively. In cross-validation studies, the mean percentage differences ranged between -51.4% (dehydroepiandrosterone sulfate) and 17.5% (dehydroepiandrosterone). The approach allows the comprehensive characterization of the adrenocortical steroid homeostasis in clinical diagnostics.