Validation of an LC-MS/MS method for simultaneous determination of icotinib and its four major circulating metabolites in human plasma and its application in a pharmacokinetic study.

ABSTRACT

In this study, a simple and sensitive LC-MS/MS method was developed and validated for simultaneous determination of icotinib and its four circulating metabolites in human plasma. The analytes were extracted with acetonitrile and separated on a C18 column using 2 mm ammonium acetate containing 0.2% formic acid and acetonitrile as mobile phase. The analytes were introduced into the mass spectrometer via an electrospray ionization source operated in positive ion mode. Precursor-to-product transitions were optimized to be m/z 392.2 → 304.1 for icotinib, m/z 424.1 → 278.2 for M1 and M2, m/z 408.2 → 320.1 for M3, m/z 410.2 → 322.1 for M4 and m/z 394.4 → 278.1 for IS. The assay showed good linearity over the concentration ranges of 0.1-600 ng/mL for icotinib and 0.1-200 ng/mL for metabolites, with correlation coefficients >0.994 (r > 0.994). The LLOQ was 0.1 ng/mL for each analyte. The intra- and inter-day precisions (RSD) were ≤12.98% while the accuracy (RE) ranged from -8.76 to 12.01%. No significant matrix effect was observed. The validated method was successfully applied for the pharmacokinetic study of icotinib and its four circulating metabolites in human plasma after oral administration of icotinib at a single dose of 125 mg.