A robust and validated LC–MS/MS method for the quantification of ramucirumab in rat and human serum using direct enzymatic digestion without immunoassay

•A robust, validated, and efficient LC–MS/MS method without immunopurification for quantification of ramucirumab in serum.•The oxidation and deamidation of unstable peptides under enzymatic conditions were investigated.•The linearity of ramucirumab was at a concentration range of 1–1000 μg/mL.•The validated method was applied for rat PK study. A new liquid chromatography tandem mass spectrometry (LC–MS/MS) method was established to quantify the anti-gastric cancer fully human monoclonal antibody (ramucirumab) in rat and human serum. The surrogate peptide (GPSVLPLAPSSK) for ramucirumab was generated by trypsin hydrolysis and quantified using the isotopically labeled peptide GPSVLPLAPSSK[13C6, 15N2]ST containing two more amino acids at the carboxyl end as an internal standard to correct for variations introduced during the enzymatic hydrolysis process and any mass spectrometry changes. Additionally, the oxidation and deamidation of unstable peptides (VVSVLTVLHQDWLNGK and NSLYLQMNSLR) were detected. The quantitative range of the proposed method was 1–1000 μg/mL, and complete methodological validation was performed. The precision, accuracy, matrix effect, sensitivity, stability, selectivity, carryover, and interference of the measurements met the required standards. The validated LC-MS/MS method was applied to pharmacokinetic studies in rats administered ramucirumab at 15 mg/kg intravenously. Overall, a robust, efficient, and cost-effective LC–MS/MS method was successfully developed for quantifying ramucirumab in rat and human serum.