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 intro-duced 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 & mu;g/mL to300 & mu;g/mL, with an LLOQ of 0.2 & mu;g/ 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.

Automated summary

We aimed to establish an LC-MS/MS method for quantifying bevacizumab content in biological fluids using a surrogate peptide generated through trypsin hydrolysis and an isotopically labeled peptide as an internal standard. The method involved a series of pre-treatment processes and had a total duration of approximately 2.5-3 h. The validated method showed a linear range of 0.2 μg/mL to 300 μg/mL in different matrices, with satisfactory precision and accuracy.