HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY OF AMPROLIUM AND ETHOPABATE IN CHICKEN FEED USING SOLID-PHASE EXTRACTION

A method for the assay of mixtures of amprolium and ethopabate in chic ken feed was developed utilizing reversed-phase high-performance liqui d chromatography (HPLC) after sample clean-up of a methanolic extract by solid-phase extraction using CN cartridges. HPLC was done with benz ocaine as internal standard on a C-8 column with methanol-water 40:60, containing octanesulfonic acid, triethylamine and acetic acid, as mob ile phase. Eluate was monitored at 274 nm. Baseline separation was ach ieved with retention times of approximate to 7.5, 9.4, and 10.4 min, f or amprolium, benzocaine, and ethopabate respectively. Feed constituen ts did not give peaks after 6.5 min. Peak area ratios were linear over 10-180 ng of amprolium, and 2-18 ng of ethopabate injected. Limits of quantitation at AUFS 0.05 were 0.5 and 0.3 ng respectively. Recovery studies from spiked feed (n = 9), covering +/- 30% of usual doses in f eed, gave percent recoveries (+/- SD) of 99.4 +/- 1.4% for amprolium a nd 100.5 +/- 2.6% for ethopabate. Applying the method to two different batches of commercial feed gave results which were comparable to thos e obtained by the AOAC spectrofluorometric methods.