Enhancement of specificity of aldosterone measurement in human serum and plasma using 2D-LC–MS/MS and comparison with commercial immunoassays

• Sensitive and specific 2D-LC–MS/MS method for serum and plasma aldosterone.
• Faster than published LC–MS/MS methods with no compromise to specificity.
• Low sample volume requirement.
• Immunoassays found to be overestimating aldosterone concentrations between 22% and 37%.
• Linearity of the method will allow testing of urine and adrenal venous samples.

BackgroundAccurate measurement of aldosterone is important for the standardized testing of primary hyperaldosteronism. Commercial immunoassays show substantial between-method variations resulting in significant clinical consequences. We developed a specific two dimensional (2D)-LC–MS/MS method for measuring aldosterone in human serum and plasma and compared it with three commercial immunoassays and an LC–MS/MS method.Methods250 μL samples, controls and calibrators spiked with d4-aldosterone were subjected to liquid–liquid extraction. The samples were analyzed using negative mode electrospray and 2D-LC followed by MS detection using an ABSciex 5500 mass spectrometer and compared with immunoassays of Siemens (Coat-A-Count), DiaSorin (CLIA-LIAISON), and IBL (ELISA). Data was acquired using multiple reaction-monitoring mode.ResultsLOQ and LOD of the method were 0.04 and 0.02 nmol/L respectively. The assay was linear up to 166 nmol/L. Inter and intra-assay imprecision at 0.13, 1.38 and 8.30 nmol/L were <10%. Interferences were absent and no differences were observed between serum and plasma matrices. Method recovery ranged from 95% to 113%. Ion suppression was not observed. Evaluated immunoassays showed positive biases ranging between 22% and 37% when compared with the developed method.ConclusionsWe developed and validated an accurate method for measurement of aldosterone in human serum and plasma using 2D-LC–MS/MS which is suitable for clinical purposes. The method is faster than previously published LC–MS/MS methods, uses less sample, has adequate sensitivity while being able to preserve high specificity in a cost effective manner. Linearity of the assay makes it promising for urine and adrenal venous samples. Comparison with three commercial immunoassays demonstrates the advantages of the developed method.