Reverse phase high pressure liquid chromatography and fluorescence detection of ethoxyquin in milk

A high pressure liquid chromatographic (HPLC) method has been developed for the determination of ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethyl-quinoline) in milk. Milk solids are precipitated by adding acetonitrile, and the water-acetonitrile supernate is washed with hexane to remove fat. Addition of sodium chloride causes the water-acetonitrile solution to separate into an aqueous phase and an acetonitrile phase, thus separating ethoxyquin from most water-soluble impurities. A large volume of water is then added to the acetonitrile layer and ethoxyquin is partitioned into hexane, which is removed at reduced pressure. The residue is dissolved in the mobile phase and analyzed on a 4.6 mm id X 250 mm Ultrasphere ODS column using fluorescence detection (excitation 230 nm; 418 nm cutoff filter). Water-acetonitrile with a diethylamine-acetic acid buffer is the mobile phase. Recoveries from samples fortified at 1, 5, and 10 ppb averaged 78% with a coefficient of variation of 5.0%. Low levels (less than 1 ppb) of apparent ethoxyquin were found in commercial milk samples that were analyzed by using the method.