Simplified detection method for residual tetracyclines and sulfa drugs in honey by microbiological assay

A simplified method was developed for the detection of residual tetracyclines and sulfa drugs in honey by microbiological assay. A honey sample was homogenized in 0.01M di-Na EDTA McIlvaine buffer (pH 4.0) and centrifuged. The supernatant was mixed well with chloroform and centrifuged. The chloroform layer was evaporated to dryness and the residue was dissolved in phosphate buffer (pH 8.0) to prepare fraction A. The aqueous layer was passed through a Sep-pak C18 cartridge column. The Sep-pak C18 cartridge column was washed with water and absorbed antibacterial agents were eluted with methanol. The eluate was evaporated to dryness, and the residue was dissolved in phosphate buffer (pH 4.5) to prepare fraction B. Fractions A and B contained sulfa drugs and tetracyclines, respectively. The pulp disk method with Bacillus subtilis ATCC 6633 and Bacillus cereus var. mycoides ATCC 11778 as test organisms was employed for the assay of sulfa drugs and tetracyclines. As little as 0.05μg/g (oxytetracycline), 0.01μg/g (chlortetracycline) or 0.1μg/g (sulfa drugs) was detectable. Thin layer chromatographic identification was performed for positive test solutions. The test solution A was spotted on an aluminum oxide plate (Merck 5550) and a silica gel plate (Merck 5553), then developed in chloroform-methanol (7:3) and ethyl ether-chloroform-n-butanol (5:1:1), respectively. The test solution B was spotted on a cellulose plate (Merck 5552), which was developed in n-butanol saturated with water. Rf values were determined by bioautography with B. subtilis and B. cereus as the test organisms. Microbioautography was suitable for separation and identification of sulfa drugs and tetracyclines in honey. We used this method to identify sulfa drugs and tetracyclines detected in 22 samples out of 297 commercial honeys. It was found that 8 specimens contained oxytetracycline, 12 specimens chlortetracycline, 1 specimen sulfadimethoxine, and 1 specimen sulfisomidine.