Liquid chromatography with fluorescence detection of amoxicillin and ampicillin in feeds using pre-column derivatization

Abstract

A rapid analytical procedure for the routine identification and quantification of two penicillins, amoxicillin (AMOX) and ampicillin (AMPI), in feeds by liquid chromatography (LC) was developed and tested. AMOX and AMPI are normally the only penicillins added to medicated feeds because of their good resistance to gastric juice. The ground feed samples were extracted using water–acetonitrile (75:25, v/v) and derivatized, without any clean-up, with a formaldehyde solution in acidic medium at 100 °C for 30 min. The fluorescent derivatives were analysed by reversed-phase on an ODS column ( 150 mm×4.6 mm; 5 μm) with a gradient between acetonitrile, methanol and phosphate buffer containing thiosulfate as mobile phase. Fluorescence detection was carried out at excitation and emission wavelengths of 358 and 440 nm, respectively. The recoveries of both penicillins from spiked samples were, in a concentration range of 200–500 mg kg −1, >80% with repeatabilities below 15% R.S.D. The limits of detection of AMOX and AMPI in feed, based on a detector signal-to-noise ratio of 3, were 5 mg kg −1.