Liquid Chromatographic Determination of β-Lactam Antibiotics in Milk: A Multiresidue Approach

Abstract

In the United States, testing of all milk for residues of beta-lactam antibiotics is now mandatory. Although a number of screening tests for determination of beta-lactam antibiotic residues have been proposed, few reference methods of the required sensitivity (< 10 ppb) are available. Methods for determination of several beta-lactam antibiotics using an automated liquid chromatography (LC) cleanup have been described recently. This paper describes the integration of these methods into a single extraction and cleanup procedure. Milk was deproteinized with 0.2M Et4NCI and acetonitrile. The resulting filtrate was evaporated to about 1 mL, made to 4 mL, and filtered through a disposable filter cartridge. For cleanup, 2 mL filtrate was loaded onto a bonded C18 LC column in 0.01M KH2PO4 (A) and eluted with an acetonitrile (B) gradient using a program of 100 A:0 B(0-3 min) to 60 A:40 B (30 min). The beta-lactams were concentrated into narrow bands and separated from each other. A fraction corresponding to each compound of interest was collected and rechromatographed for analysis. The procedure has been applied successfully to determination of ampicillin, amoxicillin, cephapirin, penicillin G, penicillin V, ceftiofur, and cloxacillin. In principle it can be applied to the determination of any beta-lactam antibiotic or metabolite thereof by collecting the appropriate fractions.