Development of a LC-MS/MS method for the determination of CKD-712 in rat plasma: Application to a pharmacokinetic study in rats

CKD-712 is a potential treatment for sepsis, as it exhibits protective effects against lipopolysaccharide-mediated platelet aggregation, inducible nitric oxide synthase expression, and cecum-ligation puncture-induced septic mortality in mice. In this study, we develop a rapid and sensitive LC-MS/MS method for determining CKD-712 in rat plasma. CKD-712 and papaverine hydrochloride (an internal standard) were analyzed using an LC-MS/MS system consisting of an Agilent HPLC system (HP-1100) equipped with an Atlantis HILIC Silica (2.1×50mm, 3μm) column and a API 4000 (Applied Biosystems/MDS Sciex, USA) in a positive ESI mode. We utilized multiple reaction monitoring (MRM) at m/z transitions of 306.2-164.0 to analyze CKD-712, and 340.3-202.1 m/z for IS, with a mobile phase of acetonitrile (0.025% trifluoroacetic acid):20mM ammonium acetate (94:6, v/v) at a flow rate of 0.25mL/min. The lower limit of quantification (LLOQ) was 5ng/mL, with a linearity ranging from 5 to 1000ng/mL (r>0.999). Validation parameters including specificity, precision, accuracy, matrix effect, recovery, dilution effect and stability results were well within acceptance criteria, and applied successfully on a pharmacokinetic study in rats.