Abstract

Background: A HPLC-MS/MS assay can outperform immunoassays in TDM due to its increased accuracy (no cross-reactivity with target analyte congeners), sensitivity, and precision. Sudden and unpredictable ion yield attenuation, often only known as "ion suppression effect" can be considered as one central analytical problem of using MS/MS as detector in bio-analysis. Since pathological patient specimen compositions or co-medications are major causes for these effects, measures have to be taken to evaluate these interferences prior to transferring an HPLC-MS/MS assay to routine use.
Material/Methods: A fully validated online SPE-HPLC-MS/MS assay setup (MS: API4000QTrap, SPE column: Oasis HLB, HPLC column: XDB-C-18, 1:20 sample dilution, cycle time 3.5 min) designed for fast routine immunosuppressant TDM was used. Post column infusion or drug spiking experiments were performed. A total of 54 drugs as well as icteric, haemolytic, and lipaemic blank matrices have been screened.
Results: Infusion experiments showed only little influence of the spiked drugs on the analyte ion profiles, whereas spiking experiments unveiled strong signal suppression or enhancement in many cases. For cyclosporine A/tacrolimus the internal standards cyclosporine D/ascomycin balanced the effects, whereas some significant changes were observed for sirolimus and everolimus using either 32-desmethoxyrapamycin or ascomycin as co-eluting internal standards. Pathologically altered matrices did not distinctly influence quantitative results
Conclusions: Even if using highly diluted samples and state of the art 2D chromatography, co-medication can cause ion yield attenuation effects in fast routine TDM. Optimal internal standards are needed to balance such effects.

Keywords: immunosuppressant, Cyclosporine, Tacrolimus