Identification and semiquantitative estimation of antibiotics added to complete feeds, premixes, and concentrates

Classical microbiological methods for determining antimicrobial compounds in feeds are nonspecific. Thus, there is a need to identify biological activity, and bioautography is used for this purpose. A routine method for detecting the following antimicrobial substances in feeds is described: avilamycin, avoparcin, Zn-bacitracin, erythromycin, flavomycin, furazolidone, lasalocid, monensin, narasin, penicillin, salinomycin, spiramycin, tetracycline, tylosin, and virginiamycin. Carbides can be detected by UV light examination of the plates prior to bioautography. Semiquantitative estimations of antibiotic content are compared with quantitative determinations of the above mentioned substances in feeds, except erythromycin, penicillin, and tetracyclines. Detection limits range from 0.1 mg/kg (chlortetracycline) to 20 mg/kg (lasalocid). The method involves agar diffusion of buffered samples, a neutral extraction of polyether antibiotics followed by thin layer chromatography (TLC), and an acid extraction for other antibiotics followed by TLC. Five test bacteria were used for the main detection by agar diffusion: Micrococcus luteus ATCC 9341, Staphylococcus aureus ATCC 6538P, Corynebacterium xerosis NCTC 9755, Bacillus cereus ATCC 11778, and B. subtilis ATCC 6633. Identification after TLC was achieved by bioautography with the most sensitive microorganism(s). This method allows one laboratory technician to analyze up to 30 feed samples within 2.5 working days, provided that feeds of the same category are analyzed in the same run, and that labels of additives are available. Qualitative and semiquantitative information are valuable when performing a quantitative antibiotic determination and it provides proof that the activity determined is due to the tested substance. This last feature is essential from the perspective of quality assurance of results.