Quantitation of aldosterone in human plasma by ultra high performance liquid chromatography tandem mass spectrometry

Abstract

Aldosterone is a mineralocorticoid steroid hormone whose measurement in the clinical laboratory is principally performed for the investigation of primary hyperaldosteronism. Traditionally measurement of aldosterone has been performed by radioimmunoassay, however these assays lack specificity as they are prone to interference from structurally related steroid hormones. Herein, we have developed a novel, sensitive and specific method utilising solid phase extraction and quantitation of aldosterone from human plasma by UPLC–MS/MS. Standards, quality controls and samples (250μL) were extracted using Oasis® HLB 96-well plates. Extract (30μL) was injected onto a Krudcatcher UPLC In-Line Filter, 0.5μm guard column, coupled to a Kinetex PFP, 100mm×2.1mm, 2.6μm column with methanolic mobile phase gradient elution. Eluant was connected to a Waters® Xevo TQS tandem mass spectrometer operating in electrospray negative mode. We detected multiple reaction monitoring (MRM) transitions of m/z 359.0>189.1 for aldosterone and 366.0>194.1 for d7-aldosterone respectively, which co-eluted at 2.65min. Ion suppression was negligible. Mean recovery was 89.6%, limit of detection and lower limit of quantitation were 26pmol/L and 30pmol/L respectively. The assay was linear up to 3200pmol/L (r2=0.9999). Mean intra- and inter-assay imprecision and bias were all <10%. Comparison of the UPLC–MS/MS method with an immunoassay in routine clinical use in the UK yielded the equation UPLC–MS/MS=0.789(RIA)–41.7, linear regression r2=0.88, n=54. We have developed a sensitive and specific method for the extraction and measurement of aldosterone from human plasma. The method features a simple 96-well plate solid phase extraction procedure, highly selective column chemistry and short chromatographic run times.