Determination of phenylbutazone residues in bovine milk by liquid chromatography with UV detection.

A published liquid chromatographic (LC) method was modified for quantitation of phenylbutazone (PBZ) residues in the range of 25-300 ng/mL in bovine milk. Milk samples (1 mL) were diluted with absolute ethanol and 25% NH4OH. Diethyl ether and petroleum ether were added sequentially to the milk extract and the mixture was agitated on a Vortex mixer to partition out milk fat. The organic phase was removed and discarded. Tetrahydrofuran-hexane (1 + 4) was added to the aqueous phase and the extract was acidified with 3M HCl. The samples were mixed on a Vortex mixer for 30 min, and centrifuged. The organic layer, containing PBZ, was transferred to a clean test tube. The organic solvents were evaporated to dryness under a stream of N2 at room temperature. The resulting extract was dissolved in 1 mL mobile phase and filtered before injection. The chromatographic system was a C18 reversed-phase column connected to a UV detector set at 264 nm. Recoveries of PBZ from raw bovine milk fortified at 25-300 ng/mL ranged from 79 to 84%; relative standard deviations (RSDs) ranged from 6 to 7%. The RSDs for incurred PBZ quantitated from 34 to 229 ng/mL ranged from 1 to 4%.