Bioautographic method for evaluation of glycopeptide actaplanin in milk

A bioautographic method is described that allows the identification and semiquantitation of residues of the glycopeptide antibiotic actaplanin in cow's milk at 0.01 ppm. The milk sample is precipitated with acetonitrile and the resultant pellet is extracted by a buffer. This extract is defatted, then chromatographed on an Amberlite resin column. Actaplanin is eluted with methanol-HCl. Purified extract is then chromatographed on a cellulose thin layer, developed in a methanol-chloroform-ammonium hydroxide mixture. This thin layer chromatography increased the sensitivity of the determination by concentrating the actaplanin components in a single spot. The antibiotic was then detected by bioautography, using Bacillus subtilis as a test organism. Parameters influencing the diffusion of the antibiotic (namely, the nature and concentration of the agar-agar), and the sensitivity of the test strain (namely, the nutritional value of the medium, and the addition of a synergistic inhibitor) have been optimized.