Measurement of Aldosterone in Human Plasma by Semiautomated HPLC–Tandem Mass Spectrometry

Abstract

Abstract Background: Reliable measurement of aldosterone with less interlaboratory variation than RIA would help standardize testing for primary aldosteronism. We set out to validate a high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) method for aldosterone in human plasma. Methods: We prepared samples (EDTA plasma, lithium heparin plasma, and serum from separator and plain clot tubes) and measured aldosterone using online HPLC-MS/MS with d7-aldosterone as internal standard. We also analyzed EDTA plasma samples by immunoassay. We established a reference range for HPLC-MS/MS aldosterone by analyzing blood collected midmorning from 97 normotensive seated subjects. Results: The linear range was 69.4–5548.0 pmol/L (2.5–200 ng/dL) (r2 > 0.994, n = 14). Inter- and intraday analytical recovery and imprecision for quality control samples of 166.4, 1109.6, and 4161.0 pmol/L (6.0, 40.0, and 150.0 ng/dL) were 92.2%–102.0% and <6.3%, respectively (n = 5). The lower limit of quantification was 69.4 pmol/L (2.5 ng/dL), with inter- and intraday analytical recovery and imprecision of 91.4%–94.5% and <9.5% (n = 5). No interferences were observed in plasma from Addison’s disease patients (n = 5). Comparison of collection tubes, using EDTA as the reference, revealed similar aldosterone results. Comparison of HPLC-MS/MS with immunoassay gave an acceptable mean bias (0.83%) but wide range (−44.8% to 39.7%) of differences. HPLC-MS/MS aldosterone concentrations in normotensive subjects ranged from <69.4 to 635.2 pmol/L (<2.5 to 22.9 ng/dL). Conclusions: This first reported aldosterone method using online HPLC-MS/MS is precise across the clinically relevant range, not influenced by collection tube type, and offers semiautomated sample preparation and high throughput.