Abstract
A sensitive and specific liquid chromatography–tandem mass spectrometry assay was developed to quantify the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human plasma. The analytes (I, II) and their stable isotope-labeled analogues as internal standards were extracted on a C 18 solid-phase extraction cartridge using a Zymark RapidTrace™ automation system. The chromatographic separation was carried out on a narrow-bore reversed-phase Zorbax XDB-C 8 HPLC column with a mobile phase of acetonitrile/water (40:60, v/v) containing 10 m M ammonium acetate (pH 7.4). The analytes were ionized using negative-to-positive switch electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/ z 415→163 and m/ z 431→337 was used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10–2500 ng/ml of plasma for both I and II. The lower limit of quantification was 10 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A throughput of 80 human plasma standards and samples per run was achieved with run time of 5 min for each injection. The assay has been successfully used in analyses of human plasma samples to support clinical studies.
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