Julie A Ray1, Mark M Kushnir2, John Palmer3, Seyed Sadjadi4, Alan L Rockwood5, A Wayne Meikle6. 1. ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, United States. Electronic address: julie.ray@aruplab.com. 2. ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, United States. 3. Agilent Technologies Inc., Santa Clara, CA 95051, United States. 4. Phenomenex, Torrance, CA 90501, United States. 5. ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, United States; Department of Pathology, University of Utah, Salt Lake City, UT 84108, United States. 6. ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, United States; Department of Pathology, University of Utah, Salt Lake City, UT 84108, United States; Department of Medicine, University of Utah, Salt Lake City, UT 84108, United States.
Abstract
BACKGROUND: Accurate 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. METHODS: 250 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. RESULTS: LOQ 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. CONCLUSIONS: We 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.
BACKGROUND: Accurate 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. METHODS: 250 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. RESULTS: LOQ 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. CONCLUSIONS: We 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.
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