UPLC-MS/MS-Based Analysis of Trastuzumab in Plasma Samples: Application in Breast Cancer Patients Sample Monitoring

Trastuzumab is a target-based recombinant humanized IgG1 monoclonal antibody (mAbs), extensively employed for treatment of metastatic breast cancer with human epidermal growth receptor 2 (HER2) overexpression. Studies around the world have reported that mAbs have substantial inter-patient unpredictable absorption, distribution, metabolism, and excretion (ADME-pharmacokinetics) because of multiple elements manipulating the concentration of mAbs in plasma. Herein, we have established a bioanalytical technique using UPLC-MS/MS with an easy sample workup method and in-solution digestion protocol to assay the trastuzumab plasma samples from breast cancer patients in clinical studies. Surrogated proteolytic peptides were used for accurate quantification of trastuzumab (CanMab) with a trastuzumab signature peptide with [C-13(6), N-15(4)]-arginine and [C-13(6), N-15(2)]-lysine stable isotope-labeled (SIL) peptide. Experiments to validate the method were accurately carried out according to the guidelines mentioned in the bioanalytical method validation protocol. The evaluation established excellent linearity over a wide range of 5-500 mu g/mL. The experimental procedure was efficaciously performed in a pilot study of five breast cancer patients and residual concentrations of drugs from responding and non-responding subjects were compared. The receiver operating characteristic (ROC) examination displayed that 52.25 mu g/mL was the C min threshold predictive response with a satisfactory sensitivity of 88.58% and specificity of 79.25%.

Automated summary

Trastuzumab, a monoclonal antibody used for treating breast cancer with HER2 overexpression, shows unpredictable inter-patient variation in absorption, distribution, metabolism, and excretion. In this study, a bioanalytical method using UPLC-MS/MS was developed to accurately measure trastuzumab plasma concentrations in breast cancer patients. The method demonstrated excellent linearity over a wide concentration range, and a threshold concentration of 52.25 μg/mL was identified as predictive of response. This method provides a reliable tool for monitoring trastuzumab levels in clinical studies.