Development of a High‐Throughput LC–MS/MS Method for Simultaneous Quantification of Four Therapeutic Monoclonal Antibodies in Human Serum: Application in Clinical Therapeutic Drug Monitoring

ABSTRACT Monoclonal antibody (mAb) therapies have revolutionized cancer treatment, significantly improving patient outcomes. However, the pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs exhibit considerable variability due to nonlinear kinetics and individual differences, highlighting the need for therapeutic drug monitoring (TDM). Therefore, this study aimed to develop and validate a reliable LC–MS/MS method for the simultaneous quantification of bevacizumab, trastuzumab, rituximab, and pertuzumab in human serum and evaluate its clinical applicability. Characteristic peptides were identified using Skyline. Serum samples underwent Protein G purification and trypsin digestion. Separation used a C18 column with 0.1% FA and acetonitrile, and detection employed multiple reaction monitoring with cadonilimab as the internal standard. The method demonstrated excellent linearity (1–200 μg/mL), precision (CV < 8.9%), and accuracy (±9.8%). With a runtime of 12 min, the validated method requires only 10 μL of serum per sample and meets international validation standards, supporting the clinical monitoring of these therapies. A robust, cost‐effective, and high‐throughput LC–MS/MS method was successfully developed for the simultaneous quantification of four therapeutic mAbs. The method significantly reduces sample volume and analysis time while maintaining high accuracy and reproducibility, making it well‐suited for routine TDM and broader clinical applications.