Bioanalysis of monomethyl fumarate in human plasma by a sensitive and rapid LC-MS/MS method and its pharmacokinetic application

- First LC-MS/MS report for determination of monomethyl fumarate in human plasma.
- Use of stable labeled isotope as IS for any variability during extraction and LC-MS/MS analysis.
- The method employs only 100 μL of plasma for processing using SPE.
- Extensive evaluation of matrix effect and stability of analyte in plasma.
- Method reproducibility was demonstrated by incurred sample reanalysis.

Dimethyl fumarate (DMF) is the methyl ester of fumaric acid, after oral administration completely converts to its active metabolite monomethyl fumarate (MMF). A simple, rapid and sensitive LC-MS/MS method was developed and validated for the quantification of MMF in human plasma. Monomethyl fumarate d3 was used as an internal standard (IS). The analyte and the IS were extracted from plasma using a selective solid phase extraction technique. The clean samples were chromatographed on a C18 column using formic acid and acetonitrile (25:75, v/v) as mobile phase. An API-4000 LC-MS/MS system equipped with turbo ion spray (TIS) source and operated in multiple reactions monitoring (MRM) mode was used for the study. The method was validated for linearity in the range of 5.03-2006.92 ng/mL. Also, a number of stability tests were conducted to evaluate the stability of analyte, IS in plasma samples and in neat samples, the results comply with recent bioanalytical guidelines. A shortest run time helped us to analyze more than 300 samples in a day. The method was applied to a pharmacokinetic study in ten healthy male Indian subjects and the study data was authenticated by conducting incurred sample reanalysis (ISR).