Crossreactivity of Isolated Everolimus Metabolites with the Innofluor Certican Immunoassay for Therapeutic Drug Monitoring of Everolimus

Everolimus is an immunosuppressant used as rejection prophylaxis in patients undergoing transplants. It requires blood concentration-guided dosing and is extensively metabolized. It was the goal to assess the crossreactivity of the major everolimus metabolites in the blood of patients undergoing kidney graft with the Innofluor Certican Assay (Seradyn, Inc., Indianapolis, IN), a clinical assay used to quantify the concentrations of everolimus in patients' blood samples. The three main hydroxy metabolites of everolimus (46-, 24-, and 25-hydroxy everolimus) and all other minor hydroxylated and demethylated metabolites were generated using pooled human liver microsomes and purified using semipreparative high-performance liquid chromatography with ultraviolet detection. Structures were confirmed using liquid chromatography-mass spectrometry/ion trap mass spectrometry and analysis of the fragmentation patterns. Blank blood samples were spiked with the isolated metabolites to determine the specific crossreactivity with the immunoassay. Crossreactivity testing with the immunoassay showed 1% or less for 46-hydroxy and 24-hydroxy everolimus and 6% or less crossreactivity for 25-hydroxy everolimus at therapeutically relevant concentrations. Crossreactivity testing of the minor metabolites showed crossreactivities of 16.3% for 45-hydroxy, 33.0% for 12-hydroxy, 18.3% for 11-hydroxy, 15.3% for 14-hydroxy, 38.7% for OH-piperidine I, 46.3% for OH-piperidine II, 43% for 39-O-desmethyl, 142% for 27-O-desmethyl, and 68% for 40-O-desethylhydroxy everolimus (sirolimus).