A validated LC-MS/MS method for the quantification of bevacizumab in rat, cynomolgus monkey, and human serum

Bevacizumab is a humanized monoclonal antibody used in the treatment of advanced colorectal and non-small cell lung cancer. Our main aim was to establish a simple, economical, and high efficiency liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantifying the content of bevacizumab in various biological fluids (rat, cynomolgus monkey, and human serum). A surrogate peptide of bevacizumab, specifically FTFSLDTSK, was generated through trypsin hydrolysis, and quantified using an isotopically labeled peptide containing two amino acids, FTFSLDTSK[¹³C₆, ¹⁵N₂]ST, as an internal standard to correct for variations introduced during the enzymatic hydrolysis process and any mass spectrometry variabilities. The pre-treatment process included denaturation, disulfide bond reduction and alkylation, trypsin hydrolysis, and termination of the reaction, with a total duration of approximately 2.5-3 h. The results of the methodological validation showed that the linear range in three different biological matrices was 0.2 µg/mL to300 µg/mL, with an LLOQ of 0.2 µg/mL. The precision and accuracy of the measurements met the required standards. The validated LC-MS/MS method was used to conduct pharmacokinetic analysis in rats administered bevacizumab at a dose of 10 mg/kg intravenously.