A sensitive bioanalytical method development and validation of afatinib in human plasma by LC-ESI-MS/MS

Abstract

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of Afatinib in human plasma using Cabozantinib as an internal standard (IS). Chromatographic separation was performed on Water X Bridge c18 2.1x100 column with an isocratic mobile phase composed of acetonitrile and 0.2% Ammonia in water in the ratio of (70:30 v/v), at a flow rate of 0.250mL/min. Afatinib and Cabozantinib were detected with parent ions at m/z 486.36 to 370.90 and the daughter mass was found to be 381.45 to 304.93 in multiple reaction monitoring (MRM) positive mode respectively. The protein precipitation method was used to extract the drug and IS. The method was validated over a linear concentration range of 2.0-1000.0 ng/mL with a correlation coefficient (r2) ≥ 0. 9994. This method demonstrated Intra and inter-day Precision within 0.3 to 2.5 and 0.4 to 3.9 % and Accuracy from 96.55 to 105.45 and 95.26 to 110.6%. Afatinib was found to be stable throughout Long-term stability studies, benchtop, and postoperative stability studies.