CEEPC/IPM/CMSC - Abstrakt prezentace

(CEEPC/IPM/CMSC 2022 - FrP-06)
Optimisation of middle-up HPLC-MS and CE-DAD approaches for infliximab quantification in therapeutic drug monitoring

Jana Havlíková 1 *, Katarína Maráková 1, Juraj Piešťanský 1, Peter Mikuš 1

  1. Univerzita Komenského v Bratislave


Infliximab (IFX) is a chimeric mouse-human monoclonal antibody (mAb) commonly used in treatment of inflammatory bowel disease (IBD). During the therapy, some patients completely lack response to IFX treatment, while for other patients the treatment becomes ineffective over time. Therapeutic drug monitoring is therefore of high importance. This work focuses on optimisation of middle-up workflow for IFX quantification by using high performance liquid chromatography mass spectrometry (HPLC-MS) and capillary electrophoresis coupled to diode-array detector (CE-DAD). Stock solutions of IFX reference standard (Sigma Aldrich, Missouri, US) were prepared. IFX disulfide bonds were reduced by using tris(2-carboxyethyl)phosphine hydrochloride (TCEP), dithiothreitol (DTT) and DTT combined with iodoacetamide. Samples containing reduced IFX were analysed by HPLC system coupled to an 6520 qTOF mass spectrometer and CE 7100 system with DAD detection (all Agilent). The three reducing conditions (producing two different mAb subunits – heavy and light chains) were tested by HPLC-MS. So far, only a partial chromatographic separation of heavy and light chains was achieved. Subsequent deconvolution of the acquired mass spectra showed that the observed peaks correspond to IFX light and heavy chain subunits. CE-DAD experiments were carried out to confirm the presence of both heavy and light chains in the reduced IFX samples. For each reducing agent used, two separate peaks were detected in the electropherograms of the reduced IFX sample. CE-DAD was shown as a promising approach for middle-up analysis of reduced IFX with the option of coupling to MS detection in future experiments.

* Korespondující autor: jana.havlikova@uniba.sk


  1. S. L. Wang et al. Journal of Immunological Methods, vol. 382, no. 1–2, pp. 177–188, 2012.
  2. M. A. v. Willrich, E. Lazar-Molnar, M. R. Snyder, and J. C. Delgado. J Appl Lab Med, vol. 2, no. 6, pp. 893–903, 2018.


This project was funded by VEGA 1/0514/22, VEGA 1/0483/20 and FaF/36/2022. The whole work was carried out in Toxicology and Antidoping Centre.

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