RAPID HPLC DETERMINATION OF TETRACYCLINE ANTIBIOTICS IN MILK

Authors: Moats, William - Bill; Harik Khan, Raida

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/16/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: Several years ago a new and sensitive screening test called the Charm II procedure was introduced and used to test milk from commercial sources for a number of antibiotics including those of the tetracycline group. These investigators reported widespread contamination of milk with tetracycline antibiotics based on this screening test. These results were widely reported in the popular press and on TV and were of major concern to the dairy industry. However, these findings could not be confirmed or disproven by the HPLC methods available at that time which were less sensitive than the screening tests. There is therefore an urgent need for HPLC methods of sensitivity comparable to screening tests. This paper reports a rapid and sensitive HPLC method for determination of several tetracycline antibiotics in milk. By use of a technique known as ion-pairing, the antibiotics are separated from interferences in milk extracts. There is therefore no need for lengthy sample preparation. The new procedure is therefore not only much faster, but more accurate than other reported methods. No antibiotics of the tetracycline group were detected in a number of cartons of milk purchased from local stores for use as controls.

Technical Abstract: A method described previously (White et al. 1993a) using on-line concentration and gradient elution was modified for more rapid isocratic analysis. Milk (5 mL) was extracted/deproteinized with 1 mL of 1N HCl and 14 mL of acetonitrile. The resulting filtrate (12 mL) was either (1) evaporated directly or (2) the water layer resulting from addition of hexane and methylene chloride was evaporated. The extract could be concentrated to about 1 mL without significant degradation of tetracyclines. The concentrates were filtered. For analysis, a Polymer Laboratories PLRP-S column was used with a mobile-phase of 0.02M H3PO4, 0.01M sodium decanesulfonate-acetonitrile, (72+28) or oxytetracycline and tetracycline and (68+32) for chlortetracycline. The injection volume was 200 uL with UV detection at 380 nm. Recoveries were 90-100 percent with detection limits of 2-4 ppb.