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 chicken feed was developed utilizing reversed-phase high-performance liquid chromatography (HPLC) after sample clean-up of a methanolic extract by solid-phase extraction using CN cartridges. HPLC was done with benzocaine as internal standard on a C-8 column with methanol-water 40:60, containing octanesulfonic acid, triethylamine and acetic acid, as mobile phase. Eluate was monitored at 274 nm. Baseline separation was achieved with retention times of ≈7.5, 9.4, and 10.4 min, for amprolium, benzocaine, and ethopabate respectively. Feed constituents 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 feed, gave percent recoveries (± SD) of 99.4 ± 1.4% for amprolium and 100.5 ± 2.6% for ethopabate. Applying the method to two different batches of commercial feed gave results which were comparable to those obtained by the AOAC spectrofluorometric methods.