Simultaneous determination of apatinib and its four major metabolites in human plasma using liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

► Simultaneously determine apatinib and its four major metabolites in human plasma. ► Apatinib and its metabolites have great different physicochemical properties. ► A simple protein precipitation and short chromatographic run time were achieved. ► The method shows advantages of high selectivity and reproducibility. ► The method was successfully applied to clinical study of apatinib mesylate. Apatinib, also known as YN968D1, is a novel antiangiogenic agent that selectively inhibits vascular endothelial growth factor receptor-2. Currently, apatinib is undergoing phase II/III clinical trials in China for the treatment of solid tumors. Apatinib is extensively metabolized in humans, and its major metabolites in circulation include cis-3-hydroxy-apatinib (M1-1), trans-3-hydroxy-apatinib (M1-2), apatinib-25-N-oxide (M1-6), and cis-3-hydroxy-apatinib-O-glucuronide (M9-2). To investigate the pharmacokinetics of apatinib and its four major metabolites in patients with advanced colorectal cancer, a sensitive and selective liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of apatinib, M1-1, M1-2, M1-6, and M9-2 in human plasma. After a simple protein precipitation using acetonitrile as the precipitation solvent, all the analytes and the internal standard vatalanib were separated on a Zorbax Eclipse XDB C18 column (50mm×4.6mm, 1.8μm, Agilent) using acetonitrile: 5mmol/L ammonium acetate with 0.1% formic acid as the mobile phase with gradient elution. A chromatographic total run time of 9min was achieved. Mass spectrometry detection was conducted through electrospray ionization in positive ion multiple reaction monitoring modes. The method was linear over the concentration range of 3.00–2000ng/mL for each analyte. The lower limit of quantification for each analyte was 3.00ng/mL. The intra-assay precision for all the analytes was less than 11.3%, the inter-assay precision was less than 13.8%, and the accuracy was between −5.8% and 3.3%. The validated method was successfully applied to a clinical pharmacokinetic study following oral administration of 500mg apatinib mesylate in patients with advanced colorectal cancer.