Simultaneous determination of a p38 MAP kinase inhibitor and its amide hydrolyzed metabolite in Cynomolgus monkey plasma by LC-MS/MS, and its application to a toxicokinetic study

A LC-MS/MS method was developed for the determination of a p38 MAP kinase inhibitor (Compound I) and its amide hydrolyzed metabolite (M7) in Cynomolgus monkey plasma over the concentration range of 1.00-1000 ng/mL Stable isotope labeled compounds (d(3)-Compound I and d(3)-M7) were used as internal standards (IS). Samples were prepared using protein precipitation in the 96-well format with a 30 mu L plasma sample volume. Chromatographic separation was performed with reversed-phase liquid chromatography on a Varian Monochrom C(18) (100MM X 2.00 mm, 5 mu m) analytical column. The mobile phases were 5 mM ammonium formate in acetonitrile/water (95/5, v/v) pH 7.0 and 5 mM ammonium formate in acetonitrile/water (5/95, v/v) pH 7.0. Gradient elution, at a flow rate of 550 mu L/min, was used to separate Compound I and M7. Positive atmospheric pressure chemical ionization was utilized with detection by multiple reaction monitoring (MRM). Total run time was about 3.2 min. This method was validated following the current Food and Drug Administration (FDA) guidance for bioanalytical method validation. The intra- and inter-day precision (% CV) and accuracy (% bias) at all concentrations tested were below 15% for both analytes. The mean recoveries for Compound I. M7, d(3)-Compound I, and d(3)-M7 were 106%, 107%, 108% and 105%, respectively. The method was successfully applied to support a GLP toxicokinetic study in Cynomolgus monkeys after oral administration of Compound I. A total of 48 samples (similar to 12.5% of the total number of samples) were selected for incurred sample reanalysis (ISR). The % difference between the reassay concentrations and the original concentrations were all less than 20% of their mean values and met the acceptance criteria for ISR.