TY - JOUR
T1 - Optimized sample pre-treatment procedure for the simultaneous UPLC-MS/MS quantification of ipilimumab, nivolumab, and pembrolizumab in human serum
AU - de Jong, Karen A.M.
AU - Rosing, Hilde
AU - Huitema, Alwin D.R.
AU - Beijnen, Jos H.
N1 - Publisher Copyright:
© 2022
PY - 2022/4/30
Y1 - 2022/4/30
N2 - 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 - 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.
AB - 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 - 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.
KW - Bottom-up sample pre-treatment
KW - Human serum
KW - Ipilimumab
KW - Nivolumab
KW - Pembrolizumab
KW - UPLC-MS/MS
UR - http://www.scopus.com/inward/record.url?scp=85125783192&partnerID=8YFLogxK
U2 - 10.1016/j.jchromb.2022.123215
DO - 10.1016/j.jchromb.2022.123215
M3 - Article
C2 - 35276512
AN - SCOPUS:85125783192
SN - 1570-0232
VL - 1196
JO - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
M1 - 123215
ER -