Determination of tacrolimus, three mono-demethylated metabolites and a M1 tautomer in human whole blood by liquid chromatography - tandem mass spectrometry

The immunosuppressant tacrolimus is the primary drug used in kidney transplantation to prevent organ rejection. A sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to measure tacrolimus and its three known mono-demethylated metabolites 13-O-desmethyl tacrolimus (M1), 31-O-desmethyl tacrolimus (M2), 15-O-desmethyl tacrolimus (M3). By generating the metabolites to use as standards after incubation of tacrolimus with rat liver microsomes, we discovered multiple M1 peaks which we identified as two tautomers of M1. The M1 tautomer II was also successfully validated in this method. The separation and purification of the metabolites and tautomers were performed by semi-preparative liquid chromatography with UV-detection, while confirmation was done by UPLC-MS/MS and Nuclear Magnetic Resonance. For quantification an easy sample preparation was performed with zinc sulfate and acetonitrile as cell lyses and precipitation. Detection was performed in positive electrospray ionization. By better characterization of the metabolites and the tautomers, we could possibly explain insight into the clinical condition and thus adjust the immunosuppressant therapy individually per patient. Calibration curves were linear for all compounds. Precision was assessed according to the NCCLS EP5-T guideline, being below 15 % and mean recoveries were between 93 and 110 % for tacrolimus, its three metabolites and the M1 tautomer II. The validated method was successfully applied in a cohort of 20 patients after kidney transplantation.

Jaar van publicatie:2021

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Authors from:Higher Education

Toegankelijkheid:Closed