An LC-MS/MS assay for simultaneous determination of 13 steroid hormones and two synthetic steroids in saliva: potential utility for paediatric population and beyond

Saliva samples offer the possibility to obtain stress-free non-invasive samples, also for home-testing, especially useful when blood collection is either undesirable or difficult. The aim of this work was to develop an LC-MS/MS method to determine clinically relevant steroid hormones cortisol, cortisone, 11-deoxycortisol, 21-deoxycortisol, 17OH-progesterone, aldosterone, corticosterone, deoxycorticosterone, testosterone, androstenedione, DHEAS, DHEA, 17OH-pregnenolone, betamethasone and dexamethasone. A special effort was made to adapt the method to neonatal population with respect to choice of saliva as matrix, low sample volumes, selection of analytes and multiplexing. Validation included selectivity, interferences, matrix effects, lower and upper limit of quantification, linearity of calibration, dilution of samples, trueness, within-run and total analytical repeatability, robustness, carry-over, stability and recovery from sample collection swab. Ten microliters were acceptable but 50 µL preferable sample volume, except for 21-deoxycortisol. Cortisol, cortisone, aldosterone, 11-deoxycortisol, deoxycorticosterone, dexamethasone, betamethasone, 17OH-pregnenolone and DHEAS could be determined in as little as 2-5µL saliva. Total analytical variation was <15%, except for 17OH-progesterone, deoxycorticosterone, 17OH-pregnenolone, 21-deoxycortisol, androstenedione, DHEA and betamethasone, that could only be determined semi-quantitatively or qualitatively. The minimum turnaround time was 3-4 h. Recovery from the best performing sample collection swab SalivaBio ranged from 83 to 127%. Aldosterone, cortisone and DHEA were more stable in saliva compared to serum when stored at ambient temperature for one week. Corticosterone and 17OH-progesterone needed immediate freezing. The non-invasiveness, small saliva volume requirement, on-swab stability and analytical performance make the method relevant for both research and diagnostics, above all in the setting of neonatal intensive care unit.