HPLC–DAD protein kinase inhibitor analysis in human serum

We here describe an HPLC–DAD method to analyse different protein kinase inhibitors. Potential applications of this method are pharmacokinetic studies and therapeutic drug monitoring. Optimised chromatography conditions resulted in a very good separation of seven inhibitors (vatalanib, bosutinib, canertinib, tandutinib, pazopanib, dasatinib – internal standard and erlotinib). The good sensitivity makes this method competitive with LC/MS/MS. The separation was performed with a Lichrospher 100-5 RP8, 250 mm × 4 mm column maintained at 30 ± 1 °C, and with a mobile phase of 0.05 M H3PO4/KH2PO4 (pH = 2.3)–acetonitrile (7:3, v/v) at a flow rate of 0.7 mL/min. A simple and fast sample preparation sequence with liquid–liquid extraction led to good recoveries (73–90%) of all analytes. The recovery hardly reached 50% only for pazopanib. This method can also be used for targeted protein kinase inhibitor quantification. A perfect linearity in the validated range (20–10,000 ng/mL) and an LOQ of 20 ng/mL were achieved. The relative standard deviations and accuracies of all examined drug concentrations gave values much lower than 15% both for between- and within-batch calculations. All analysed PKIs were stable for 6 months in a 1 mg/mL dimethyl sulfoxide stock solution. Vatalanib, bosutinib and erlotinib were also stable in human serum in the whole examined concentration range.