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 using a C18 column (2.1 × 50 mm, 2.6 μm) with mobile phases of 0.1% formic acid in water and acetonitrile. The other monoclonal antibody, infliximab, was used as internal standards to minimize the variability during sample processing and detection. A unique peptide for each monoclonal antibody was simultaneously quantified using LC–MS/MS in the multiple reaction monitoring mode. Calibration curves were linear from 2.0 to 400 μg/mL. The intra‐ and inter‐assay precision (%CV) was within 8.9 and 7.4% (except 10.4 and 15.1% for lower limit of quantitation), respectively, and the accuracy (%Dev) was within ±13.1%. The other validation parameters were evaluated, and all results met the acceptance criteria of the international guiding principles. Finally, the method was successfully applied to a pharmacokinetics study after a single‐dose intravenous drip administration to cynomolgus monkeys.

中文翻译：

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同时定量猕猴血清中曲妥珠单抗和帕妥珠单抗的基于LC-MS / MS的测定方法的开发和验证及其在药代动力学研究中的应用。

我们提出了一种简单而强大的LC-MS / MS分析方法，用于同时定量曲妥珠单抗和帕妥珠单抗在猴子血清中的抗体混合物。与传统的配体结合测定法相比，LC-MS / MS方法可节省成本，缩短分析时间并减少定量时间。甲醇沉淀，碳酸氢铵变性，二硫苏糖醇还原和碘乙酰胺烷基化后，用胰蛋白酶在50°C将血清样品消化60分钟。使用C18色谱柱（2.1×50 mm，2.6μm）色谱分离胰蛋白酶消化的肽，流动相为0.1％甲酸的水溶液和乙腈溶液。另一种单克隆抗体英夫利昔单抗用作内标，以最大程度减少样品处理和检测过程中的变异性。使用多反应监测模式中的LC-MS / MS同时定量每种单克隆抗体的独特肽段。校准曲线在2.0至400μg/ mL之间呈线性关系。批内和批间精密度（％CV）分别在8.9和7.4％之内（定量下限除外10.4和15.1％），准确度（％Dev）在±13.1％之内。对其他验证参数进行了评估，所有结果均符合国际指导原则的接受标准。最终，该方法在对食蟹猴单剂量静脉滴注后成功地用于药代动力学研究。定量下限分别为1％）和准确度（％Dev）在±13.1％以内。对其他验证参数进行了评估，所有结果均符合国际指导原则的接受标准。最终，该方法在对食蟹猴单剂量静脉滴注后成功地用于药代动力学研究。定量下限分别为1％）和准确度（％Dev）在±13.1％以内。对其他验证参数进行了评估，所有结果均符合国际指导原则的接受标准。最终，该方法在对食蟹猴单剂量静脉滴注后成功地用于药代动力学研究。