Determination of AmpiciUin and Amoxicillin in Milk with an Automated Liquid Chromatographic Cleanup

A method is described for determination of am-piciitin and amoxicillin in milk by using an automated liquid chromatographic (LC) cleanup. Milk was deproteinated by adding tetraethylammonium chloride solution and acetonitrile. The filtrate was concentrated by evaporation, filtered, and loaded into a 4 mL autosampler vial. For LC cleanup, a Waters W1SP 712 autosampler, a Varian 9010 pump, a Supelcosil LC-18 column, and an ISCO FOXY fraction collector were used. The cleanup program, where A is 0.01 M KH2PO4 and B is acetonitrile, was 100A + OB for 0-3 min and then 70A + 30B for 24 min. Sample concentrate (2 mL) was loaded onto the column for cleanup. Fractions containing amoxicillin and ampicillin were collected, partially acidified, and concentrated to 1 mL. Analysis was done on the LC-18 column, with a mobile phase of 0.015M H3PO4–{0.0075M sodium dodecyl sulfate-acetonitrile (70 + 30)] for amoxicillin and 0.0067M H3PO4–0.0033M KH2PO4–[0.005M sodium dodecyl sulfate–acetonitrile (67 + 33)] for ampicillin. Recoveries were generally 80–90% at a concentration range of 1–0.01 ppm, with a detection limit of 2–5 ppb. By collecting appropriate fractions, the method can be applied to the determination of any amphoteric β-lactam.