Endocrine Abstracts

Background: The diagnostic workup of primary aldosteronism (PA) is challenging and the current screening tool, aldosterone-renin ratio, is increasingly challenged. Due to interference with immunoassays and the general high variability of plasma aldosterone levels false-positive as well as false-negative results are probably more frequent than previously assumed. Therefore, we aimed at the establishment of a reliable LC-MS/MS method to analyze aldosterone and related steroids in human urine to facilitate diagnostic workup.

Methods: 450 µl urine were treated with β-glucuronidase/arylsulfatase by Helix pomatia and purified by off-line solid phase extraction before analysis. Chromatographic separation was carried out using an Agilent 1290 Infinity LC system equipped with binary pump, autosampler and thermostatted column compartment and a Waters Acquity® HSS PFP 1.8 µm (2.1×50 mm) column with an eluent consisting of 5 mM ammonium formate as phase A and methanol as phase B. A Sciex QTRAP 6500 mass spectrometer was used for detection of the steroids aldosterone (Aldo), tetrahydroaldosterone (THAldo), cortisol (F), 18-oxocortisol (18-OxoF), 18-hydroxycortisol (18-OHF) and 18-hydroxycorticosterone (18-OHB). Multiple reaction monitoring in positive ion mode and stable isotope labeled internal standards allowed for quantification. Complete validation will be performed according to FDA and EMA guidelines.

Results: Calibration ranges of 0.2-30 ng/ml (Aldo and 18-OHB), 1-150 ng/ml (THAldo), 2-300 ng/ml (F and 18-OHF), and 0.1-15 ng/ml (18-OxoF) have been established. First 24-h urine samples from 18 patients with PA were successfully included in the validation process and mean values were obtained as follows: cortisol 63.4 µg/24 h [IQR:27.7-95.5], Aldo 12.9 µg/24 h [IQR: 6.2-22.1], 18-OxoF 4.5 µg/24 h [IQR: 0.4-5.8], 18-OHF 123.4 µg/24 h [IQR: 21.2-138.0], 18-OHB 8.0 µg/24 h [IQR: 2.6-13.1] and THAldo 67.1 µg/24 h [IQR: 30.4-100.5].

Conclusions: We herein describe a liquid chromatography tandem mass spectrometry method for the simultaneous quantification of six steroids in human urine, which are currently validated according to FDA and EMA guidelines for application in patient care. Prospectively, the method may contribute to an optimization of the diagnostic workup of PA.