Method Development and Validation of LC-MS/MS-Based Assay for the Simultaneous Quantitation of Trastuzumab and Pertuzumab in Cynomolgus Monkey Serum and its Application in Pharmacokinetic Study.

: We present a simple and robust LC-MS/MS assay for the simultaneous quantitation of an antibody cocktail of Trastuzumab and Pertuzumab in monkey serum. The LC-MS/MS method saved costs, decreased the analysis time and reduced quantitative times relative to the traditional ligand-binding assays. The serum samples were digested with trypsin at 50 °C for 60 min after methanol precipitation, ammonium bicarbonate denaturation, dithiothreitol reduction, and iodoacetamide alkylation. The tryptic peptides were chromatographically separated with a C18 column (2.1 x 50 mm, 2.6 μm) with mobile phases of 0.1% formic acid in water and acetonitrile. The other mAb, Infliximab, was used as internal standards to minimize the variability during sample processing and detection. A unique peptide for each mAb were simultaneously quantified by LC-MS/MS in the multiple reaction-monitoring mode (MRM). Calibration curves were linear from 2.0 to 400 μg/ml. The intra- and interassay precision (%CV) was within 8.9% and 7.4% (except 10.4% and 15.1% for LLOQ), respectively, and the accuracy (%Dev) was within ± 13.1%. The other validation parameters were evaluated, and all results met the acceptance criteria of International guiding principles. Finally, the method was successfully applied to a pharmacokinetics study following a single-dose intravenous drip administration to cynomolgus monkeys.