Determination of penicillin G in milk by high-performance liquid chromatography with automated liquid chromatographic cleanup

Abstract

Specific confirmatory tests are needed to identify and quantify β-lactam antibiotic residues detected in milk at levels of <10 parts per 10 9 (ppb) by screening tests. A liquid chromatographic method for penicillin G was developed using the liquid chromatography system for cleanup as well as analysis. Milk was deproteinized with two volumes of acetonitrile. The acetonitrile was extracted with hexane-methylene chloride (1:1) and the remaining water layer was concentrated by evaporation. The water layer (2 ml = 5 ml milk) was injected onto a Polymer Laboratories PLRP-S column using a WISP autosampler with the solvent, 0.01 M pH 7.0 phosphate buffer (A). Penicillin G was eluted with acetonitrile (B) gradient A−B (100:0) (0–3 min)–(40:60) (25 min). Penicillin G eluted as a narrow band in <0.5 min. A narrow fraction containing penicillin G was collected and rechromatographed on the same type of column at low pH (1.96). This effectively separated penicillin G from interferences. Recoveries were 92±9% with a sensitivity limit near 2 ppb. The approach used is applicable to determination of other β-lactam antibiotics but specific conditions for analysis must be determined for each one. The cleanup procedure can be automated using an autosampler, gradient controller, and fraction collector.