RESUMO
Mycophenolate mofetil (MMF) is a prodrug of mycophenolic acid (MPA) used to prevent rejection in organ transplant patients. The purpose of this study is to develop a sensitive LC-MS/MS method to simultaneously quantify MMF, MPA, and two major metabolites, mycophenolic acid-glucuronide (MPAG) and Acyl-mycophenolic acid-glucuronide (AcMPAG) and applied this method in a pharmacokinetic (PK) and tissue distribution study. A Shimadzu UHPLC system coupled to an AB Sciex QTrap 4000 mass spectrometer was used for the analysis. Protein precipitation with a mixture of methanol: acetonitrile (2:1, v:v) was used to process the plasma samples and tissue samples. Separation was achieved using an Ultra Biphenyl 5 µm column (100 × 2.1 mm) with 0.1% formic acid in water (A) and acetonitrile (B) as the mobile phases. Quantification analysis was performed under positive ionization mode using the multiple reaction monitoring (MRM) approach. The method was linear in the range of 1.22 - 1250.00 nM for all four analytes with correlation coefficient values > 0.99. The method was reproducible, with intra- and inter-day accuracy ranging from 85.0 ± 11.2-108.3 ± 6.50 for all analytes in both plasma, liver and intestine homogenates. The extraction recovery and matrix effect of plasma sample using a mixture methanol/acetonitrile (2:1, V:V) can achieve an acceptable range (<20%), but extraction recovery and matrix effect of AcMPAG decreased to 64.10 ± 15.42 in the liver and intestine homogenates. The analytes in plasma were found to be stable under bench-top, freeze-thaw, and storage conditions. The validated method was successfully applied to quantify MMF, MPA, MPAG, and AcMPAG in a rat PK study. The PK results showed MPA was the major form exposed in the plasma in rats after oral administration of MMF, but the major metabolites in the rat's tissue disposition were MPAG.