Development and validation of a liquid chromatography-mass spectrometric method for the determination of DPC 423, an antithrombotic agent, in rat and dog plasma

摘要

A sensitive and selective LC-MS-MS method for the determination of DPC 423 (I), an antithrombotic agent, i described. This method used a solid-phase extraction from 0.1 ml plasma with an Isolute C_2 cartridge. HPLC separation was carried out on a YMC ODS-AQ C_(18) column (50 * 2 mm) at a flow-rate of 300 μl/min with an analysis time of 5 min. Compounds were eluted using a mobile phase of H_2O/CH_3CN/HCOOH: 66:34:0.1 (v/v/v), pH 4.0. A structural analogue of I was used as the internal standard to account for variations in recovery and instrument response. Mass spectrometric detection was carried out with a PE Sciex API III~+ triple quadrupole mass spectrometer equipped with a Turbo IonSpray~(TM) source as the LC-MS interface. Good intra-day and inter-day assay precision (<10% CV) and accuracy (<10% difference) were observed over a concentration range of 0.005-2.5 μM in plasma. The extraction recoveries were approximately 90% and the method was found to be linear for the assay (r~2 > 0.999). The method has been successfully applied to discovery and preclinical pharmacokinetic studies, including a dose range-finding study and toxicokinetic exposure studies in rat and dog.