LC-MS Analysis of Dimethyl Fumarate in Rat plasma with Measurement Uncertainty Estimation

Abstract

Dimethyl fumarate (DMF) is the methyl ester of  fumaric acid initially recognized as a very  effective hypoxic cell radio sensitizer. Phase III  clinical trials found that DMF successfully  reduced relapse rate and increased time to  progression of disability in multiple sclerosis.  Small molecules like dimethyl fumarate pose  particular difficulties when analyzing biological  samples due to the increased possibility of matrix  effects and in this scenario DMF immediately  converts to its active metabolite MMF  (Monomethyl fumarate) by oral route. It is for  this reason that a sensitive liquid  chromatography–tandem mass spectrometry  method has now been developed for the analysis  of DMF and for studying the pharmacokinetic  profile in rats. Sample preparation was by rapid  protein precipitation with acetonitrile. Analyte  separation was achieved on a reversed-phase  XTerra MS C18 column (100 x 3.9 mm, 3.5µ) with  0.01M ammonium formate and acetonitrile in  gradient mode as the mobile phase at a flow rate  of 1.0 mL/min and analyzed by a hybrid triple quadrupole linear ion trap mass spectrometer in  positive electrospray ionization mode for both  DMF and MMF. Limits of detection, and quantification were 20 and 50 ng/mL, for DMF  and 1 and 10 ng/mL for MMF respectively.  Calibration curve showed excellent linearity  within the 50–2500 ng/mL range for DMF and  10–500 ng/mL range (r2 > 0.999) for MMF. Intra and inter-day precision defined by coefficient of  variation was <10% and accuracy (bias %) was  within 90–110%. Measurement uncertainty  estimation was 8.6% for DMF and 11.6% for  MMF. The method has been successfully used in  the analysis of DMF and MMF in rat plasma  following its administration to male wistar rats  for pharmacokinetic studies.