Simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human serum using an immobilized trypsin

• Published in: Journal of pharmaceutical and biomedical analysis 2017-11, Vol.146, p.266-272
• Main Authors: Shibata, Kaito, Naito, Takafumi, Okamura, Jun, Hosokawa, Seiji, Mineta, Hiroyuki, Kawakami, Junichi
• Format: Article
• Language: English
• Subjects: Calibration; cetuximab; Cetuximab - blood; Cetuximab - therapeutic use; Chromatography, Liquid - methods; clinical application; Head and Neck Neoplasms - blood; Head and Neck Neoplasms - drug therapy; human serum; Humans; LC-MS/MS; Peptides - chemistry; proteomics; Proteomics - methods; Reproducibility of Results; Sensitivity and Specificity; Serum - chemistry; Solid Phase Extraction - methods; surrogate peptide; Tandem Mass Spectrometry - methods; Trypsin - chemistry
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2017.08.012

[Display omitted] •Serum cetuximab was determined using an LC-MS/MS method without immunopurification.•Fourier transform mass spectrometer selected the surrogate peptide of cetuximab CDR.•Immobilized trypsin achieved the rapid digestion of serum cetuximab.•Interindividual variability was observed in serum concentration of cetuximab.•The serum concentration of cetuximab in LC-MS/MS was similar to that in ELISA. Proteomic approaches using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) without an immunopurification technique have not been applied to the determination of serum cetuximab. This study developed a simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human serum and applied it to clinical settings. Surrogate peptides derived from cetuximab digests were selected using a Fourier transform mass spectrometer. Reduced-alkylated serum cetuximab without immunopurification was digested for 20minutes using immobilized trypsin, and the digestion products were purified by solid-phase extraction. The LC-MS/MS was run in positive ion multiple reaction monitoring mode. This method was applied to the determination of serum samples in head and neck cancer patients treated with cetuximab. The chromatographic run time was 10minutes and no peaks interfering with surrogate peptides in serum digestion products were observed. The calibration curve of absolute cetuximab in serum was linear over the concentration range of 4–200μg/mL. The lower limit of quantification of cetuximab in human serum was 4μg/mL. The intra-assay and inter-assay precision and accuracy were less than 13.2% and 88.0–100.7%, respectively. The serum concentration range of cetuximab was 19–140μg/mL in patients. The serum cetuximab concentrations in LC-MS/MS were correlated with those in ELISA (r=0.899, P