Development and validation of a sensitive and rapid UHPLC-MS/MS method for the simultaneous quantification of CG-0255 and its active metabolite in human plasma and its application to Phase I studies

Abstract

CG-0255, a thiol prodrug of clopidogrel's active metabolite H4 (CG-0236), is a novel thienopyridine P2Y12 receptor antagonist under initial clinical development for the treatment of acute coronary syndromes. Unlike clopidogrel, CG-0255 is converted to the active thiol metabolite H4 (CG-0236) in a single hydrolytic step. Compared with clopidogrel, CG-0255 exhibits more efficient and consistent H4 formation in humans, which can be quantified in plasma following either intravenous or oral administration. In this study, we developed and validated a sensitive, rapid, and robust UHPLC-MS/MS method for the simultaneous quantification of CG-0255 and its derivatized active metabolite (MP-H4, CG-0261) in human plasma. After solid-phase extraction from 94.5 μL of plasma, analytes and isotope-labeled internal standards were separated on an ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) using isocratic elution with 0.1 % formic acid in water and acetonitrile (57:43, v/v) at a flow rate of 0.5 mL/min, followed by a 3.5 min column washing and re-equilibration, giving a total analytical run time of 7 min. Baseline separation of CG-0255, CG-0261, and their respective isomers was achieved. Detection was performed using positive electrospray ionization in multiple reaction monitoring mode on a Q-Trap 6500⁺ mass spectrometer. Calibration curves were linear over 0.05-25 ng/mL for both analytes, corresponding to 0.0353-17.65 ng/mL for H4 (CG-0236). Intra- and inter-day precision and accuracy were within ±15 % at all quality-control levels. The validated assay was successfully applied to two phase I clinical studies conducted at our center, characterizing the pharmacokinetics of CG-0255 following single-dose intravenous and multiple-dose oral administration. This UHPLC-MS/MS method provides a reliable platform for the quantitative evaluation of CG-0255 and its active metabolite in human plasma, and is well suited to support further global clinical development.

Keywords: Active metabolite; CG-0255; Clopidogrel prodrug; LC-MS/MS; Treated human plasma.