Determination of motolimod concentration in rat plasma by liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study

[Display omitted] •No validated method exists for quantitative analysis of motolimod in plasma.•A reliable LC–MS/MS method was developed for quantifying motolimod in rat plasma.•Motolimod showed non-linear pharmacokinetics in rat.•Motolimod is unstable in rat liver microsomes. Motolimod (VTX-2337) is an agonist of toll-like receptor 8. In this study, a novel and sensitive liquid chromatography-tandem mass spectrometry method was developed for quantifying motolimod in rat plasma and subsequently used in a pharmacokinetic study. Proteins were precipitated from plasma samples using acetonitrile prior to the analysis. GS-9620 was used as an internal standard. High-performance liquid chromatography was performed using a Spursil C18-EP column (3 μm, 50 × 2.1 mm). Aqueous ammonium formate and acetonitrile were used as the mobile phase. Motolimod was detected using an electrospray ionization interface under multiple reaction monitoring conditions in the positive ion mode. The developed method produced a linear correlation over a concentration range of 1–1000 ng/mL (r = 0.9944). Intra- and inter-day precision values ranged from 4.8%–10.7% (the lower limit of quantification precision value was 16.3 %), whereas intra- and inter-day accuracy values ranged from 0.3%–9.1 %. The mean recovery of motolimod from rat plasma was 109.4 %. Additionally, motolimod was found to be stable under various conditions (three freeze-thaw cycles, 6-h storage at room temperature, short- and long-term stability tests, and processing). The developed method was successfully used in a pharmacokinetic study in rats. Motolimod showed non-linear pharmacokinetics following its intravenous administration to rats at 0.6–6 mg/kg. Additionally, very low exposure (