Application of High-Performance Thin-Layer Chromatography and Bioautography Techniques for Determination of Antibiotic Residues in Mastitic Cow Milk Following Treatment

Abstract

A method to identify and quantify multiple antibiotic residues like ceftriaxone and enrofloxacin in mastitic cow’s milk by high-performance thin-layer chromatography coupled with bioautography was developed. The antibiotic residues were extracted from antibiotic treated cow’s milk suffering from mastitis with the help of acetonitrile by eliminating fat using petroleum ether and finally isolated with dichloromethane. The chromatogram of peaks of antibiotic residue confirmed the presence of antibiotics in the milk samples with presence of 0.108±0.011mg/ml (ceftriaxone) and 0.173±0.015mg/ml (enrofloxacin) in the milk sample, collected 10 days post-administration of drug with initial dose of 15g of ceftriaxone and 9g of enrofloxacin, respectively. In the present study, the presence of antibiotic residue beyond 10 days is contradictory from the withdrawal period given by the pharmaceutical company for 5 days. Bioautography confirmed the presence of active antibiotic residue in milk sample-1, 2, 3, 4, 5 and 6 with zone of inhibition of 6.5±0.407mm, 6.0±0.495mm, 5.0±0.666mm, 5.2±0.4mm, 5.1±0.432mm and 6.0±0.401mm, respectively. The test microorganism used for bioautography was Streptococcus spp.