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Urinary aldosterone and tetrahydroaldosterone by LC-MS/MS with enzymatic hydrolysis: validation and age-stratified reference intervals
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DOI：10.1515/cclm-2025-1679.png)
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Abstract
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Objectives Primary aldosteronism (PA) is a common cause of secondary hypertension. To address the specificity limits of immunoassays, we developed and validated an LC-MS/MS method for urinary aldosterone and tetrahydroaldosterone and established method-matched reference intervals for excretion in 24 h urine.Methods Urinary aldosterone, tetrahydroaldosterone and their glucuronidated metabolites were extracted in the presence of internal standards using offline solid-phase extraction (SPE), followed by enzymatic hydrolysis to release the glucuronidated fraction. Subsequently, aldosterone and tetrahydroaldosterone were analyzed by online SPE in combination with LC-MS/MS. Reference intervals were established based on 24 h urine samples from 265 individuals participating in the Lifelines Cohort study.Results Intra- and inter-assay imprecision ranged from 2.0-12.3 % for aldosterone, and 1.3-6.3 % for tetrahydroaldosterone. The lower limits of quantification were 0.44 nmol/L and 0.10 nmol/L, respectively. Recoveries ranged from 97-106 %, calibration was linear, with correlation coefficients greater than 0.999, and no carry-over was observed. Total aldosterone concentrations measured by LC-MS/MS were consistently higher than those obtained by radioimmunoassay. In the reference population, 24 h urinary excretion ranged from 5.4-76.7 nmol/24 h for aldosterone and 21.4-269.9 nmol/24 h for tetrahydroaldosterone.Conclusions This validated LC-MS/MS assay, together with a method-matched normative dataset, enables standardized urinary aldosterone profiling and defines reference intervals that will help improve the interpretability of results in the biochemical diagnosis of PA.
Keywords:
aldosterone
tetrahydroaldosterone
LC-MS/MS
enzymatic hydrolysis
primary aldosteronism
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