Development and validation of a productive liquid chromatography-tandem mass spectrometry method for the analysis of androgens, estrogens, glucocorticoids and progestagens in human serum

Adrenal and gonadal disorders are very often coupled, due to common etiology or pathophysiology. We present the development, validation and application of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of androgens (androstenedione (A4), testosterone (T), dihydrotestosterone (DHT), and dehydroepiandrosterone sulfate (DHEA-S)), estrogens (estrone (E1), estradiol (E2), estriol (E3)), glucocorticoids (cortisol (F), cortisone (E), corticosterone (B), 11-deoxycortisol (S), 21-deoxycortisol (21DF), 11-deoxycorticosterone (11DB)), and progestagens (progesterone (P4), 17α-hydroxyprogesterone (17OHP4) and 17α-hydroxypregnenolone (17OHP5)) in human serum for clinical use. Samples (250 μL of matrix) spiked with isotopic labelled internal standards were extracted with tert-butylmethyl ether (TBME) prior to LC-MS/MS analysis. The chromatographic separation of the underivatized endogenous steroids was achieved on a reversed-phase column (C18 Zorbax Eclipse Plus) using a methanol-water gradient. The LC column was coupled to a triple quadrupole mass spectrometer equipped with an electrospray (ESI) source operating both in positive and in negative mode, with acquisition in multiple reaction mode. The method was validated using surrogated matrices and human serum samples. The proposed method was proven to be specific for all the considered steroids; and linearity was also assessed (R2 > 0.99) in the ranges of quantification investigated. The lower limits of quantification (LLOQs) were in the range of 10 - 400 pg/mL depending on the target steroid. Accuracy was in the range 80 - 120% for all the target compounds, the extraction recovery was higher than 65% for all the steroids considered and no remarkable matrix effect, expressed in terms of ion enhancement and ion suppression, was observed. To test the reliability of the developed and validated method, the analysis of serum samples collected from ten healthy subjects (5M/5F) was performed. In the clinical settings there is a growing need to develop accessible methods for full steroid hormone profiling. The dynamic link between steroidogenic glands and liver enzymatic processing (activation and clearance) attributes to the profile a much greater clinical meaning than a set of individually measured hormones. The presented method can be used to identify trajectories of deviation from the concentration normality ranges applied to disorders of the gonadal and adrenal axes.