Determination of Residual Avoparcin in Chicken Muscle by High Performance Liquid Chromatography

Abstract

An analytical method was developed for residual avoparcin in chicken muscle by measuring α- and β-avoparcin, major components of the pharmaceutical preparation of avoparcin, using high performance liquid chromatography (HPLC). Alpha- and β-avoparcin were used as authentic samples after isolation by preparative HPLC. The analytical HPLC was achieved by using a Cosmosil 5C18-AR column (4.6mm×25cm), a mobile phase of 2.5% acetic acid, 0.01M sodium heptane sulfonic acid-acetonitrile (88.5:11.5) (pH 4.0) with UV detection at 280nm. Since the values obtained by HPLC as the sum of α- and β-avoparcin were highly correlated to those obtained by bioassay (r=0.95, n=24), it was suggested that the proposed HPLC method may substitute for the time-consuming bioassay.Avoparcin was extracted from chicken muscle by homogenization in methanol -0.2M sulfuric acid (6:4) followed by centrifugation after pH adjustment to 3 by 1N sodium hydroxide. The supernatant was evaporated to dryness, and the residue was dissolved in water. The aqueous layer was adjusted to pH 3 by adding 1N sodium hydroxide. Then it was purified on a Sep-pak C18 cartridge. The cartridge was washed with water, and retained substances were eluted with 50% methanol. The eluate was evaporated to dryness under reduced pressure. The residue was dissolved in water and determined by HPLC. Recoveries for avoparcin spiked in chicken muscle were 104-99.5% at 2-16μg/g. The detection limit is 0.5μg/g.