The clinical implementation of liquid chromatography–tandem mass spectrometry (LC–MS/MS) for quantifying serum tocilizumab has been limited due to labor-intensive and expensive sample preparation procedures. This study sought to establish and validate an LC–MS/MS assay for serum tocilizumab measurement employing immobilized trypsin digestion without the need for immunoglobulin G (IgG) purification, and to assess its applicability in rheumatoid arthritis (RA) patients receiving either intravenous or subcutaneous tocilizumab therapy. A tocilizumab-specific signature peptide was identified using a nano-LC system coupled with a hybrid quadrupole-Orbitrap mass spectrometer. Serum samples underwent rapid enzymatic digestion with immobilized trypsin for 30 minutes. The signature peptide and its corresponding internal standard were chromatographically resolved from serum digests within a total analysis time of 15 minutes. The assay demonstrated linearity over a concentration range of 2–200 μg/mL. Intra- and inter-day accuracy ranged from 90.7% to 109.4%, with relative standard deviations below 10%. Measured serum tocilizumab concentrations ranged from 5.8 to 28.9 μg/mL in patients receiving intravenous administration and from 2.4 to 63.5 μg/mL in those receiving subcutaneous administration. Concentrations obtained using this LC–MS/MS method showed a positive correlation with values determined by enzyme-linked immunosorbent assay, although a consistent systematic bias between the two methods was observed. Overall, this study presents a validated LC–MS/MS approach with simplified sample preparation suitable for monitoring serum tocilizumab levels in patients with rheumatoid arthritis.
