Determination of Neomycin in Milk by Reversed Phase Ion-Pairing Liquid Chromatography

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Abstract
A fluorometric high performance liquid chromatographic (HPLC) method has been developed for the determination of neomycin in milk. Whole or shelf milk was defatted by initial centrifugation at 4°C. The resulting skim milk was deproteinated with trichloroacetic acid and centrifuged again. The neomycin was determined directly in the supernate by HPLC. The HPLC conditions consisted of an ion-pairing mobile phase, a reversed-phase column, post-column derivatization with o-phthalaldehyde (OPA) reagent and fluorescence detection. The overall recovery of neomycin was 94% (coefficient of variation 6.5%), in whole milk spiked at 0.15–10 ppm and 99% (coefficient of variation 6.4%) in shelf milk spiked at 0.15–5 ppm. The method was used to detect neomycin in milk obtained from cows dosed intramuscularly with neomycin (10 mg/kg). The neomycin concentrations in milk at 8 and 24 h after dosing were 0.3 and 0.2 ug/ml, respectively.