Simultaneous Determination of Atezolizumab and Bevacizumab by LC–MS/MS in Rat Plasma and Its Application to a Pharmacokinetic Study

Abstract

A robust, rapid, and highly sensitive LC–MS/MS method was developed and validated for the simultaneous quantification of atezolizumab and bevacizumab in rat plasma, with potential applications in pharmacokinetic studies. The mobile phase comprised water with 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B) in an isocratic ratio of 30:70, providing optimal separation on an Agilent Eclipse XDB column (250 mm × 4.6 mm, 5 μm). The selected peptides for atezolizumab (IYPTNGYTR) and bevacizumab (LTVLHQDWLNGK) yielded parent ions at m/z 579.78 and 686.38, and daughter ions at 746.36 and 859.44, respectively. The internal standard, abciximab, used the peptide VQWLQAGK with parent and daughter ions at m/z 471.26 and 590.32, respectively. Atezolizumab and bevacizumab were validated over linear ranges of 3.00–120 ng/mL and 1.25–50.00 ng/mL, showing excellent correlation coefficients (r² = 0.999). Method accuracy for atezolizumab ranged from 96.76% to 99.55%, with precision (%CV) between 0.09% and 2.45% across quality controls, while bevacizumab demonstrated accuracy from 97.05% to 99.82% with precision (%CV) ranging from 0.09% to 8.82%. Matrix effects at low and high QC levels confirmed consistency, with accuracy and precision for atezolizumab and bevacizumab exceeding ICH guidelines. This optimized method offers a valuable tool for pharmacokinetic profiling of atezolizumab and bevacizumab in rat plasma, providing insights for therapeutic monitoring and drug development.