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

Highlights

• A new RPIC method for the determination of antazoline hydrochloride is developed.

• Concentration of antazoline hydrochloride in biological sample is determined.

• The pharmacokinetic characteristic of antazoline hydrochloride in dog is studied.

• t_1/2 result shows higher therapy compliance of our drug for further study in patient.

Abstract

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.

Introduction

Antazoline hydrochloride (N-benzyl-N-(4, 5-dihydro-1H-imidazol-2-ylmethyl)aniline hydrochloride (1:1)) is an ethylenediamines histamine receptor antagonist. At first it was used as antihistamines and anticholinergic drug. The chemical structure of antazoline hydrochloride is shown in Fig. 1A. Now, it is used as a new type of medicine for heart protection which can lower the atrial, ventricular prematurebeat and nonparoxysmal nodal tachycardia due to digitalis excess. It belongs to I_A class anti-arrhythmia drug [1], [2]. There is widely known sound randomized clinical studies conducted to evaluate the efficacy and safety of antazoline [3], [4] and determine the drug concentration in preparation by spectroscopy and chromatography [5], [6]. To our knowledge, the determination of antazoline in dog plasma for pharmacokinetics evaluation has not been reported. The present study about its mechanism of action is mainly on the level of molecular and physiological, such as how to interfere with the penetration of potassium and sodium ion from myocardial cell membrane and slow the conduction velocity between the myocardial fibers [7], but as an anti-arrhythmia drug with obvious effects, a better understanding of the pharmacokinetics of antazoline is very important for explaining its mechanism of action and therapeutic effect. Thus, RPIC (reversed phase ion pair chromatography) [8], [9], [10], [11] with liquid–liquid extraction technique is developed to determine antazoline in biological samples with good specificity and reproducibility. We developed a method that was efficient in analyzing trace amount of drug in plasma obtained from pharmacokinetics study after intravenous infusion of antazoline hydrochloride. The validated method was used for the pharmacokinetics characterization of antazoline in Beagle dog.