Determination and stability of CP-31398 in plasma from experimental animals by LC-MS/MS

Abstract

A sensitive and accurate approach for the determination of CP-31398 (N-{2-[( E)-2-(4-methoxy-phenyl)-vinyl]-quinazolin-4-yl}-N′,N′-dimethyl-propane-1,3-diamine hydrochloride) in rat and dog plasma by LC-MS/MS was validated to support preclinical toxicological and pharmacological studies. Based on the results of stability experiments with diluted CP-31398 solutions using NMR, LC-MS/MS and LC-Q-TOF, all sample preparation and handling steps were performed under yellow light to avoid CP-31398 decomposition. CP-31398 was extracted by protein precipitation with acetonitrile and separated using a Phenomenex Luna 3 μm phenyl-hexyl, 100 Å, 30 × 2.0 mm column (rat plasma) or a Phenomenex Synergi 4μ Polar-RP, 80 Å, 30 × 2.0 mm column (dog plasma) at a flow rate of 0.30 mL/min. The mobile phase consisted of A: 1% formic acid in water and B: 1% formic acid in methanol or acetonitrile. Total run times for rat and dog samples were 7 and 8 min, respectively, with accompanying retention times of 1.8 for both columns. A turbo ion spray interface was used as the ion source operating in positive mode. Calibration curves were linear from 5 to 1000 ng/mL. Linearity was assessed using the external standard method. Within-run and between-run accuracy was 93–109% of the true value for all analytes with precision (SD) of 8% or less for all experiments. The validated method was applied to preclinical toxicology studies in rats and dogs after oral administration of CP-31398.