Direct and Precise Measurement of Bevacizumab Levels in Human Plasma Based on Controlled Methionine Oxidation and Multiple Reaction Monitoring

Bevacizumab is a monoclonal antibody which targets vascular endothelial growth factor A (VEGF-A) and is used to treat various cancers and recently COVID-19. The dosage recommendations for bevacizumab are determined on the basis of body weight, and the drug is administered after defined time intervals, when it is presumed to still be above its minimum effective serum concentration. Interindividual and disease-stage-related variations in bevacizumab catabolism, however, can affect the proper dosing of patients, resulting in plasma concentrations which may not be within the optimal therapeutic window for the drug. Therapeutic drug monitoring (TDM) enables the assessment of patients' serum concentrations and allows personalized dosing which has the potential to improve efficacy and reduce side effects. While TMD is often performed using ligand-based assays, mass spectrometry (MS)-based TDM offers improved specificity. Here, we present a robust multiple reaction monitoring (MRM)-MS-based TDM method for the precise quantification of bevacizumab plasma concentrations, based on the controlled oxidation of the methionine-containing peptide, STAYLQMNSLR. The assay shows good linearity ( r 2 = 0.9951), robustness, and precision (CVs < 20%) for the quantification of bevacizumab, with a lower limit of quantification ( S / N > 10) of 1.8 μg/mL of plasma, without the need for enrichment and requiring less than 1 μL of plasma and less than 6 h from sampling to result.