Simultaneous Quantification of Vedolizumab, Ustekinumab, and Infliximab in Human Serum by LC-MS/MS: Application to Therapeutic Drug Monitoring in Inflammatory Bowel Disease.

Abstract

Purpose: With the expanding use of therapeutic monoclonal antibodies (mAbs) in inflammatory bowel disease (IBD), there is a critical need for robust, multiplex analytical tools to support therapeutic drug monitoring (TDM) and pharmacokinetic studies. This study aimed to develop and validate a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of infliximab (IFX), vedolizumab (VDZ), and ustekinumab (UST) in human serum.

Patients and methods: A universal immunocapture strategy utilizing Protein G magnetic beads was used to enrich total IgG from human serum. Samples were subjected to tryptic digestion and analyzed in multiple reaction monitoring (MRM) mode, with rituximab as a full-process internal standard. Method validation was performed in accordance with regulatory guidelines, evaluating linearity, precision, accuracy, selectivity, recovery, matrix effect, and stability. The validated method was subsequently applied to residual serum samples from IBD patients undergoing treatment.

Results: The method demonstrated excellent linearity (R²>0.99) over the concentration ranges of 0.5-100 μg/mL for IFX and UST, and 1-200 μg/mL for VDZ. Total precision ranged from 1.1% to 14.9%, and accuracy ranged from -12.1% to 14.5%. Validation parameters, including selectivity, carryover, recovery, matrix effects, dilution integrity, and stability, were all within acceptable criteria. Analysis of the 37 clinical samples revealed marked inter-individual variability in drug concentrations. However, the absence of key covariates (eg, body weight and dosing history) precluded a formal pharmacokinetic analysis or attribution of this variability to specific clinical factors.

Conclusion: The developed LC-MS/MS method provides a practical, specific, and reproducible tool for the multiplex quantification of therapeutic mAbs. Based on a preliminary exploratory study of 37 patient samples, this method demonstrates analytical feasibility and serves as a promising tool for pharmacokinetic research; however, its definitive clinical applicability and readiness for routine TDM require further confirmation through cross-platform comparison, incurred sample reanalysis, and external validation in larger cohorts.