HPLC Method for Simultaneous Identification and Quantification of Enrofloxacin, Bromhexine*HCl and Their Related Substances/Degradation Products, in Veterinary Pharmaceutical Product, Bromflovet

Abstract

This study aims to develop a chromatographic analytical method, for separation, identification and quantification of degradation products, in veterinary finished product with two active substances enrofloxacin and bromhexine hydrochloride. Analytical method used an Agilent Eclipse Plus C18 column (150 × 4.6 mm, 5 µm particle size) and a mobile phase A consisted of 2.5% (v/v) phosphoric acid adjusted to pH 2.3 with triethylamine and mobile phase B was acetonitrile. Elution was in a gradient mode with total chromatographic time being 21 min. Detection was performed at 248 nm for bromhexine hydrochloride and at 272 nm for enrofloxacin. The method was developed to assure the separation of impurities of enrofloxacin and bromhexine hydrochloride. Specificity in relation with degradation products revealed up to 20 impurities for enrofloxacin and 7 impurities related with bromhexine hydrochloride. The spectra of impurities were chosen among the compounds found in forced degradation studies. Method validation was performed according to VICH GL 2—validation of analytical procedures and included selectivity/specificity, linearity, accuracy, precision, limit of quantification, limit of detection, robustness, and system suitability. Linearity was between 0.015 and 0.06 mg/mL for enrofloxacin and 0.001125–0.005625 mg/mL for bromhexine hydrochloride. Limit of quantification was 0.00292 mg/mL for enrofloxacin and 0.001103 mg/mL for bromhexine hydrochloride. These limits assured method performance, because they are under the reporting threshold of 0.3% as stated in VICH GL11 Impurities in new veterinary medicinal products which is 0.30%. Recovery was calculated on three concentrations for every compound and was 102.99% for enrofloxacin and 102.91% for bromhexine hydrochloride. In terms of intermediate precision, a relative maximum deviation of 2.50% was obtained for area and retention time using two analysts in two different days of application.