Determination of antazoline hydrochloride in Beagle dog plasma by HPLC–UV and its application to pharmacokinetics

In order to evaluate the pharmacokinetics characteristic of antazoline hydrochloride in Beagle dogs, a sensitive and specific HPLC method was developed and validated using phenacetin as the internal standard (IS). The analyte and the IS were extracted from dog plasma by ethyl acetate under the basic condition. The analyte was separated by a C18 column and detected with a variable wavelength UV-detector. The mobile phase consisted of methanol–5 mmol L−1 tetrabutyl ammonium bromide (45:55, v/v) containing 0.5% glacial acetic acid in a flow rate of 1.0 mL min−1. Standard calibration graph for antazoline was linear over a curve range of 20–1600 ng mL−1 (R > 0.99) and the lower limit of quantification was 20 ng mL−1 using a plasma sample of 500 μL. The intra- and inter-day precision values were less than 14.3% relative standard deviation (RSD). The intra-day assay accuracy was in the range of 98.1–100.6% and the inter-day assay accuracy in the range of 99.2–101.1%. The extraction recoveries were on the average of 88.4% for antazoline and 76.8% for IS. Plasma samples were stable at least for 1 month at −20 °C. This method was successfully applied to pharmacokinetics study of antazoline after intravenous administration to Beagle dogs.