Infliximab is a monoclonal antibody therapy used to treat several chronic immune-mediated diseases, including Crohn’s disease, ulcerative colitis, and rheumatoid arthritis. Infliximab acts by binding to tumor necrosis factor and, thus, inhibiting the inflammatory cascade. While it is a highly effective therapy, a subset of patients on infliximab will develop a loss of response to therapy. In these circumstances, therapeutic drug monitoring of infliximab offers a rational approach to clinical decision making and is associated with improved outcomes. While infliximab has most commonly been measured by immunometric approaches, mass spectrometric approaches offer the opportunity to improve test accuracy and reduce test costs. Herein, we describe a simple, bottom-up high performance liquid chromatography tandem mass spectrometry (LC–MS/MS) approach for quantitation of infliximab in serum. Method development included pre-digestion and digestion experiments to determine critical sample preparation steps, optimization of the workflow and selection of rapidly produced proteolytic peptide(s) for quantitation. The workflow was further improved by automating all sample preparation steps on a robotic liquid handler, facilitating implementation in routine clinical use. A method comparison was performed against a Health Canada and US Food and Drug Administration licensed enzyme-linked immunosorbent assay. Our LC–MS/MS assay accurately reported concentrations based on drug manufacturer targets and demonstrated no interference from endogenous antibodies to infliximab; immunoassay methods did not share these performance characteristics. This LC–MS/MS method provides a workflow amenable to implementation in a clinical laboratory and desired performance characteristics for guiding clinical decision making.

英夫利昔单抗是一种单克隆抗体疗法，用于治疗多种慢性免疫介导的疾病，包括克罗恩病、溃疡性结肠炎和类风湿性关节炎。英夫利昔单抗通过结合肿瘤坏死因子发挥作用，从而抑制炎症级联反应。虽然它是一种非常有效的疗法，但一部分使用英夫利昔单抗的患者会失去对治疗的反应。在这些情况下，英夫利昔单抗的治疗药物监测为临床决策提供了一种合理的方法，并与改善的结果相关。虽然英夫利昔单抗最常通过免疫测定方法测量，但质谱方法提供了提高测试准确性和降低测试成本的机会。在这里，我们描述一个简单的，自下而上的高效液相色谱串联质谱 (LC-MS/MS) 方法用于血清中英夫利昔单抗的定量。方法开发包括预消化和消化实验以确定关键的样品制备步骤、优化工作流程和选择快速生成的蛋白水解肽进行定量。通过在机器人液体处理机上自动化所有样品制备步骤，进一步改进了工作流程，促进在常规临床使用中的实施。与加拿大卫生部和美国食品和药物管理局许可的酶联免疫吸附试验进行了方法比较。我们的 LC-MS/MS 分析准确报告了基于药物制造商目标的浓度，并证明内源性抗体对英夫利昔单抗没有干扰；免疫测定方法不具有这些性能特征。这种 LC-MS/MS 方法提供了适合在临床实验室中实施的工作流程和用于指导临床决策的所需性能特征。