Simultaneous quantification of rituximab and eculizumab in human plasma by liquid chromatography-tandem mass spectrometry and comparison with rituximab ELISA kits |
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| Affiliation: | 1. Laboratoire de Pharmacologie, Pharmacogénétique et Toxicologie, CHU Grenoble Alpes, France;2. Laboratoire d''Immunologie, Hôpital l''Archet, CHU de Nice, Université de Nice-Côte d’Azur, 06202 Nice cedex 3, France;3. UMR2CA, université de Nice Côte d’Azur, 06202 Nice cedex 3, France;4. Service de Néphrologie, Hémodialyse, Aphérèses et Transplantation, Centre Hospitalier Universitaire Grenoble-Alpes, Grenoble, France;5. Univ. Grenoble Alpes, INSERM, CHU Grenoble Alpes, HP2, 38000 Grenoble, France;1. The Department of Cardiology, The First Affiliated Hospital of Harbin Medical University, Harbin, China;2. The Department of Medical Administration, The First Affiliated Hospital of Harbin Medical University, Harbin, China;1. Biomarker Exploring Program, Shanghai Xuhui Central Hospital/Zhongshan-Xuhui Hospital/Shanghai Clinical Research Center, Chinese Academy of Sciences, 966 Middle Huaihai Road, Shanghai 200031, China;2. Department of Cardiology, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, 725 South Wanping Road, Shanghai 200032, China;3. Department of Sport Rehabilitation, Shanghai University of Sport, 650 Qingyuanhuan Road, Shanghai 200438, China;1. Department of Pharmaceutics, School of Pharmacy, Virginia Commonwealth University, Richmond, VA, USA;2. PPD Laboratories, Richmond, VA, USA;3. Department of Analytical Chemistry, Faculty of Pharmacy, Zagazig University, Egypt;1. Department of Analytical Chemistry/Biohealth Research Institute (ibs.GRANADA), University of Granada, E-18071 Granada, Spain;2. UGC Intercentro Interniveles Farmacia Granada, “San Cecilio Hospital”, Biohealth Research Institute(ibs.GRANADA), University Hospital San Cecilio, E-18012 Granada, Spain |
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| Abstract: | ObjectivesSpecific 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.MethodsHere, 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®).ResultsCalibration 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).ConclusionsThis new LC-MS/MS method allows the simultaneous quantification of RTX and ECU in human samples and could be used for therapeutic drug monitoring. |
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| Keywords: | Rituximab Eculizumab Liquid-chromatography tandem mass spectrometry Therapeutic drug monitoring ELISA |
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