Development and validation of a UHPLC-MS/MS bioanalytical method to quantify in plasma the analgesic candidate PT-31 following a preliminary pharmacokinetic study in rats.

A selective and sensitive UHPLC-MS/MS bioanalytical method to determine PT-31, an analgesic drug candidate, in rat plasma was developed and validated. Analyses were performed using a UHPLC-MS/MS system equipped with an electrospray ionization interface operating in the positive ionization mode using a C18 reversed-phase column with a mobile phase of water:acetonitrile (68:31, v/v) containing 0.1% acetic acid eluting in a gradient mode with a flow rate of 0.3 mL/min. Plasma samples were deproteinized with cold acetonitrile containing 0.01% TFA (1:2, v/v) and 50 μL of the supernatant were injected into the system. PT-31 and phenytoin (internal standard) retention times were roughly 1.0 and 1.5 min, respectively. Linear standard curves were plotted for the 0.01-10 µg/mL concentration range, with a coefficient of determination > 0.99. The method's precision was over 88%. Maximum intra- and inter-day relative standard deviations were 14.6% and 11.6%, respectively. Interfering substances were not detected in the chromatogram, indicating that the method was specific. PT-31 stability was assessed under different temperature and storage settings. The method was used to characterize PT-31 plasma pharmacokinetics following administration of 5 mg/kg i.v. to Wistar rats. Therefore, the method described is sensitive, linear, precise and specific enough to determine PT-31 in preclinical pharmacokinetic investigations.