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

Abstract

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. 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 d(7)-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. The linear range was 69.4-5548.0 pmol/L (2.5-200 ng/dL) (r(2) > 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). 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.