Improved high‐performance liquid chromatography tandem mass spectrometry method for quantification of infliximab in pediatric plasma and its application in therapeutic drug monitoring

Abstract Rationale The application of infliximab (IFX) to immune‐mediated disease is limited by the significant individual variability and associated clinical nonresponse, emphasizing the importance of therapeutic drug monitoring (TDM). Because of the cross‐reactivity, limited linear range, and high costs, the clinical application of the previous reported methods was limited. Here, an improved high‐performance liquid chromatography tandem mass spectrometry (HPLC‐MS/MS) method was developed to address the issues. Methods This study developed an improved bioanalytical HPLC‐MS/MS method coupling nanosurface and molecular‐orientation limited proteolysis technology. The commercially available compound P14R was selected as the internal standard. This method was developed with fewer volume of reagents and was thoroughly validated. The validated method was applied to TDM in pediatric inflammatory bowel disease (IBD). Results Chromatography was performed using a Shim‐pack GISS‐HP C 18 metal‐free column (3 μm, 2.1 × 100 mm) with a gradient elution of 0.1% formic acid in water and acetonitrile at 0.4 mL/min. Detection and quantitation were performed using electrospray ionization (ESI) and multiple reaction monitoring in the positive ion mode. The method was validated to demonstrate its selectivity, linearity, accuracy, precision, recovery, matrix effect, and stability. The method exhibited a linear dynamic range of 0.3–100 μg/mL, with intra‐ and inter‐day precision and relative errors below 15%. The recovery and matrix effect were measured as 87.28%–89.72% and 41.98%–67.17%, respectively, which were effectively compensated by the internal standard. A total of 32 samples collected from 24 pediatric patients with IBD were analyzed using the validated method, and only 46.9% achieved the reported targeted trough level. Conclusion This study developed an improved HPLC‐MS/MS method for the quantitative determination of IFX concentration in human plasma. The accurate, reliable, and cost‐effective method was validated and utilized in the analysis of clinical samples. The results confirmed the importance of TDM on IFX and the clinical application prospects of the improved method.