Quantification of active infliximab in human serum with liquid chromatography–tandem mass spectrometry using a tumor necrosis factor alpha -based pre-analytical sample purification and a stable isotopic labeled infliximab bio-similar as internal standard:

• A selective purification and quantification of Infliximab in human serum is proposed.
• The purification is based on immuno-affinity using biotinylated TNF-alpha captured onto a streptavidin coated 96 well plate.
• Quantification is performed using LC–MS/MS on one signature peptide after tryptic digestion.
• The method was validated according to EMA guidelines, has a lower limit of quantification of 0.5 μg/mL using 2 μL serum.
• Cross validation against reference ELISA method showed a strong agreement of r2 = 0.95.

The therapeutic monoclonal antibody Infliximab (IFX) is a widely used drug for the treatment of several inflammatory autoimmune diseases. However, approximately 10% of patients develop anti-infliximab antibodies (ATIs) rendering the treatment ineffective. Early detection of underexposure to unbound IFX would result in a timely switch of therapy which could aid in the treatment of this disease. Streptavidin coated 96 well plates were used to capture biotinylated-tumor necrosis factor -alpha (b-TNF-α), which in turn was used to selectively extract the active form of IFX in human serum. After elution, IFX was digested using trypsin and one signature peptide was selected for subsequent analysis on liquid chromatography − tandem mass spectrometry (LC–MS/MS). The internal standard used was a stable isotopic labeled IFX bio-similar. The assay was successfully validated according to European Medicines Agency (EMA) guidelines and was found to be linear in a range of 0.5–20 μg/mL (r2 = 0.994). Lower limit of quantification for the assay (<20% CV) was 0.5 μg/mL, requiring only 2 μL of sample. Cross-validation against enzyme-linked immunosorbent assay (ELISA) resulted in a high correlation between methods (r2 = 0.95 with a ρc = 0.83) and the accuracy was in line with previously published results. In conclusion, a sensitive, robust and cost-effective method was developed for the bio-analysis of IFX with LC–MS/MS by means of a target-based pre-analytical sample purification. Moreover, low volume and costs of consumables per sample promote its feasibility in (pre)clinical studies and in therapeutic drug monitoring. This method should be considered as first choice due to its accuracy and multiple degree of selectivity.