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<p>This study describes the development and validation of a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantification of the monoclonal antibodies trastuzumab and bevacizumab in human serum. The method utilizes magnetic bead-based immuno-enrichment followed by protein denaturation, reduction, alkylation, and enzymatic digestion, requiring only 20 µL of serum. This streamlined process reduces sample preparation time to under 4&#xa0;h and shortens the LC–MS/MS analysis time per sample to 5.5&#xa0;min. The assay demonstrated a linear range of 1–500&#xa0;µg/mL for both analytes, with a lower limit of quantification (LLOQ) of 1&#xa0;µg/mL, meeting the requirements for clinical therapeutic drug monitoring. Validation confirmed the method’s performance for linearity, precision, accuracy, carry-over, specificity, and stability. The method employs SILuMab (ThermoFisher brand), a stable isotope-labeled universal protein internal standard sharing Fc region sequences with the target antibodies, eliminating the need for analyte-specific internal standards and reducing costs. Compared to existing methods, the developed approach offers a superior combination of minimal sample consumption (20 µL), rapid processing (&lt; 4&#xa0;h), and high-throughput analysis (5.5&#xa0;min/sample). The use of a universal SILuMab internal standard enhances cost-effectiveness. This makes it particularly suitable for timely therapeutic drug monitoring and pharmacokinetic studies in patients receiving combination antibody therapies.</p>

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Rapid quantification of trastuzumab and bevacizumab in serum using magnetic bead immuno-enrichment coupled with LC–MS/MS

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月7日

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  • Shuxia Zhao,
  • Yanna Ye,
  • Feng Du,
  • Xiaohui Zhang

摘要

This study describes the development and validation of a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantification of the monoclonal antibodies trastuzumab and bevacizumab in human serum. The method utilizes magnetic bead-based immuno-enrichment followed by protein denaturation, reduction, alkylation, and enzymatic digestion, requiring only 20 µL of serum. This streamlined process reduces sample preparation time to under 4 h and shortens the LC–MS/MS analysis time per sample to 5.5 min. The assay demonstrated a linear range of 1–500 µg/mL for both analytes, with a lower limit of quantification (LLOQ) of 1 µg/mL, meeting the requirements for clinical therapeutic drug monitoring. Validation confirmed the method’s performance for linearity, precision, accuracy, carry-over, specificity, and stability. The method employs SILuMab (ThermoFisher brand), a stable isotope-labeled universal protein internal standard sharing Fc region sequences with the target antibodies, eliminating the need for analyte-specific internal standards and reducing costs. Compared to existing methods, the developed approach offers a superior combination of minimal sample consumption (20 µL), rapid processing (< 4 h), and high-throughput analysis (5.5 min/sample). The use of a universal SILuMab internal standard enhances cost-effectiveness. This makes it particularly suitable for timely therapeutic drug monitoring and pharmacokinetic studies in patients receiving combination antibody therapies.