Optimized sample pre-treatment procedure for the simultaneous UPLC-MS/MS quantification of ipilimumab, nivolumab, and pembrolizumab in human serum
de Jong, Karen A M; Rosing, Hilde; Huitema, Alwin D R; Beijnen, Jos H
(2022) Journal of Chromatography B, volume 1196, pp. 1 - 9
(Article)
Abstract
Ipilimumab, nivolumab, and pembrolizumab are immune checkpoint inhibiting monoclonal antibodies. Their efficacy has been proven to be correlated with clearance, and hence, bioanalytical assays to study their pharmacokinetics are of pivotal importance. We present the first kit-free sample pre-treatment procedure of only three hours for the Ultra-Performance Liquid Chromatography -
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tandem Mass Spectrometry (UPLC-MS/MS) simultaneous quantification of ipilimumab, nivolumab, and pembrolizumab in human serum. The conventional bottom-up sample pre-treatment steps for protein MS bioanalysis including pre-digestion purification, denaturation, reduction, alkylation, and digestion were optimized in terms of sensitivity and reproducibility. In the final, optimal sample pre-treatment procedure, samples were purified by protein precipitation with saturated ammonium sulfate solution, reduced with dithiothreitol, denatured with methanol, and digested with trypsin. The method was then validated according to European Medicines Agency (EMA) and the United States Food and Drug Administration (FDA) guidelines for bioanalytical method validation, and 4-6-20 acceptance criteria were applied. This method was selective, accurate, and precise within the range of 3-200 µg/mL for all analytes. The validated developed assay was applied to determine ipilimumab, nivolumab, and pembrolizumab concentrations in patient serum, and the results were compared to enzyme-linked immunosorbent assay (ELISA) results.
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Keywords: Antibodies, Monoclonal, Humanized, Chromatography, High Pressure Liquid, Chromatography, Liquid/methods, Humans, Ipilimumab, Nivolumab/therapeutic use, Reproducibility of Results, Tandem Mass Spectrometry/methods, United States, Human serum, Pembrolizumab, Bottom-up sample pre-treatment, Nivolumab, UPLC-MS/MS, Analytical Chemistry, Biochemistry, Clinical Biochemistry, Cell Biology, Journal Article
ISSN: 1570-0232
Publisher: Elsevier
Note: Funding Information: The authors thank Ignace Rooseboom, Michel Hillebrand, and Dick Pluim for the practical support. This research was not funded by any specific grant, nor were there any conflicts of interest related to this study. Publisher Copyright: © 2022
(Peer reviewed)