Determination of aminoglycoside antibiotics in animal feed by high performance liquid chromatography using evaporative light scattering detector

Abstract

This study was carried out to develop a method for the simultaneous analysis of 5-aminoglycosides such as streptomycin (STR), neomycin (NEO), kanamycin (KAN), spectinomycin (SPE), and amikacin (AMI) in animal feedstuffs using the (HPLC-ELSD) high performance liquid chromatography with Evaporative Light Scattering Detector detection. The 5-aminoglycoside antibiotics in animal feed samples were extracted with a solvent containing 20% ACN, 1% HCl (v/v). The extract was purified by solid-phase extraction (SPE) with the stationary phase of PCX, at pH 5 which was adjusted by NH3 solution. The 5-aminoglycosides were eluted from the SPR column with 10% NH3 solution. The eluted sample was evaporated to dryness and the residue was dissolved in 20 mM hepta fluorobutyric acid (HFBA) solution followed by filtering through a 0.22 µm membrane prior to analyze by HPLC – ELSD. The 5-aminoglycosides were separated on an Eclipse Plus C18, 1.8 µm, 4.6×100 mm column, an aqueous/acetonitrile containing 15 mM HFBA in gradient mode with ratio of H2O/ACN: 0-1 min fix at 76:25, 1-6 min reduce 55:45, 6-7 min fix at 55-45, 7-8 min increase to 75:25. A carrier gas velocity of the ELSD detector was 1 L/min and the misting temperature was 40oC. This method achieved a detection limit (MDL) of 4 mg/kg and a recovery yield of 87-92% for 5-aminoglycosides in feed sample with the HPLC – ELSD system.