High-performance liquid chromatographic method for determination of phenylbutazone in bovine milk with special reference to the fat content in milk
Martin, K.; Stridsberg, M.I.; Wiese, B.M.
Journal of Chromatography 276(1): 224-229
1983
ISSN/ISBN: 0021-9673 PMID: 6672018 Document Number: 197212
Phenylbutazone (PBZ) is used in Sweden for treating cows with arthritis and laminitis. The method described for measuring PBZ in cows' milk consists of extracting 1 ml milk with 1.1 ml of a mixture of ethanol and 25% ammonia (in the proportions 10:1) plus 2.4 ml diethyl ether. After mixing, 2.4 ml light petroleum (b.p. 40-60 deg C) are added, and 1 h later the ether phase is discarded. The aqueous phase is extracted with 5 ml n-hexane and 0.4 ml 3 M HCl. The residue, obtained by evaporating the organic phase in a stream of N2, is redissolved in 0.5 ml mobile phase (prepared from 550 ml 0.02 M phosphate buffer pH 7.0 + 450 ml methanol) and analysed by reversed-phase HPLC on a LiChrosorb RP-18 column. In whole milk samples spiked with PBZ at 1 mu g/ml, the PBZ concn. in the skim milk was half that in whole milk, indicating a 25-fold higher concn. of PBZ in milk fat. For this reason it is recommended that milk samples to be tested should be representative of the whole udder content. Milk fat contents ranging from 0.5 to 4.4% had no significant effect on the measurement of PBZ. The method enabled PBZ to be detected at 20 ng/ml milk. Recovery of PBZ added to milk at 100 ng/ml was 89%.