Determination of Afatinib in Human Plasma by 2-Dimensional Liquid Chromatography

Abstract

Introduction: A simple, sensitive, rapid, and practical 2-dimensional liquid chromatography (2D-LC) method was developed and validated for the quantification of a 500-μL afatinib sample extracted from human plasma. Methods: The plasma samples were pretreated with acetonitrile for protein precipitation. The mobile phase consisted of a first-dimensional mobile phase (acetonitrile, methanol, and 25 mmol/L ammonium phosphate in a ratio of 25:25:50, V/V/V) and a second-dimensional mobile phase (acetonitrile and 10 mmol/L ammonium phosphate in a ratio of 25:75, V/V). The average recovery of the plasma samples was stable and reproducible (98.56%–100.02%). Results: The analyte was sufficiently stable for handling and analysis. The calibration curve was linear, ranging from 10.93 to 277.25 ng/mL with regression equation y = 804.60 x – 4,169.87 (R² = 0.999). The relative standard deviations for accuracy and precision studies were within ±2.30% and <3.41%, respectively (intra- and interday). Finally, the validated method was successfully employed to determine the drug levels in plasma from the patients treated with afatinib. In clinical assessment, the patients with gastric cancer were orally administered with 30 or 40 mg per day of afatinib, which resulted in large plasma concentrations, ranging from 5.52 to 45.16 ng/mL. Conclusion: The results indicated that this method was useful for the therapeutic drug monitoring of afatinib and suitable for the assessment of the risks and benefits of chemotherapy in patients with non-small cell lung cancer.