Determination of isoxazolylpenicillins residues in milk by ion-pair reversed-phase high-performance liquid chromatography after precolumn derivatization.

A high-performance liquid chromatographic method has been developed for the determination of isoxazolylpenicillins (oxacillin, cloxacillin and dicloxacillin) residues in milk. This method involves extraction of the penicillins from milk with phosphate buffer pH 8, deproteinization by acidification with sulfuric acid followed by cleanup and concentration on a C18 solid-phase extraction column and reaction with 1,2,4-triazole and mercury(II) chloride solution pH 9.0 at 65 degrees C. The derivatized compound is eluted on a C8 column with a mobile phase containing acetonitrile, methanol and phosphate buffer (pH 6.5, 0.1 mol l(-1)) loaded with sodium thiosulfate and ion-pairing tetrabutylammonium hydrogenosulphate. The detection limit of the method is 2 microg l(-1) for oxacillin, 3 microg l(-1) for cloxacillin and 5 microg l(-1) for dicloxacillin in milk and the three penicillins have been quantified down to 15 microg l(-1) in line with the EU criteria of the directive No. 93/256/EEC.