Toripalimab, a humanized IgG4 monoclonal antibody (mAb) against programmed death 
receptor-1, is being extensively studied to treat various malignancies. At 
present, there is no complete methodology reported for quantifying toripalimab, 
except for an electrochemiluminescence immunoassay (ECLIA) mentioned in several 
clinical studies. Therefore, a sensitive and robust ultra-high performance 
liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to 
accurately detect toripalimab levels, compared with the ECLIA. Plasma samples 
were pretreated by a five-step process, encompassing denaturation, reduction, 
alkylation, enzymatic hydrolysis and quenching. And a unique, sensitive and 
stable enzymatic peptide (ASGYTFTDYEMHWVR) selected as surrogate of toripalimab 
was eluted and monitored by UPLC-MS/MS system with the linear range of 
5.0375-201.5 μg/mL. After fully validated, the UPLC-MS/MS method was applied to 
determine 77 plasma samples from 29 patients in a phase I clinical trial, and 
compared with ECLIA based on 56 samples. Wilcoxon paired samples test showed 
toripalimab levels by UPLC-MS/MS were significantly higher than that by ECLIA 
(p < 0.001), though a strong correlation was observed (r = 0.96). Moreover, 
Passing-Bablok regression analysis exhibited constant and proportional biases: 
UPLC-MS/MS = 2.25 + 1.21 * ECLIA. This discrepancy could be mainly attributed to 
different forms determined: total mAb for UPLC-MS/MS and free mAb for ECLIA, 
respectively. As a result, this UPLC-MS/MS method may be complementary to ECLIA 
to monitor different forms of toripalimab. Beyond that, it can be easily 
modified to simultaneously quantitate multiple-analyte with a small volume of 
plasma.