Simultaneous quantification of rituximab and eculizumab in human plasma by liquid chromatography-tandem mass spectrometry and comparison with rituximab ELISA kits.

OBJECTIVES: Specific and sensitive analytical techniques to quantify therapeutic monoclonal antibodies (mAbs) are required for therapeutic drug monitoring. The quantification of mAbs has been historically performed using enzyme-linked immunosorbent assays (ELISAs), for which the limitations in terms of specificity have led to the development of alternative analytical strategies. METHODS: Here, we describe the validation of a liquid chromatography tandem mass-spectrometry (LC-MS/MS) method for the simultaneous quantification of rituximab (RTX - anti-CD20) and eculizumab (ECU - anti-C5). Sample preparation was based on our previously published method, using protein G purification and trypsin digestion. A new specific peptide for RTX, containing an N-terminal pyroglutamine and a trypsin miss-cleavage, enables better sensitivity, while peptide of ECU was chosen thanks to an in silico trypsin digestion and the Skyline® software. Full-length stable-isotope-labeled adalimumab was added to plasma samples as an internal standard. RTX in 50 human serum samples was quantified by LC-MS/MS and the concentrations obtained compared to those obtained with two commercial ELISA kits (Lisa Tracker® and Promonitor®). RESULTS: Calibration curves were linear from 1 to 200 µg.mL-1 for RTX and 5 to 200 µg.mL-1 for ECU, and within-day and between-day accuracy and precision fulfilled Food and Drug Administration validation criteria. Comparison of the LC-MS/MS method with ELISA showed a negligible bias with the Lisa Tracker® kit (4%), but significant bias with the Promonitor® assay (mean underestimation of 69% for the Promonitor® assay). CONCLUSIONS: This new LC-MS/MS method allows the simultaneous quantification of RTX and ECU in human samples and could be used for therapeutic drug monitoring.