Determination of tilmicosin in ovine milk using high-performance liquid chromatography

Tilmicosin is a novel macrolide antibiotic with a wide range of therapeutic uses against gram positive (+ve) and gram negative (-ve) bacteria and mycoplasmae causing pneumonia and mastitis and can be used to treat these diseases in sheep. After its use there may be residues present in ovine milk that interfere with cheese making and processing of other milk products. It is important to monitor for the presence of tilmicosin in ovine milk and a method has been optimized and validated for its determination. Tilmicosin is extracted from milk into methanol. The methanol extract is acidified and non-polar co-extractives removed using hexane followed by carbon tetrachloride. The pH is adjusted to 9.0 and the tilmicosin partitioned into chloroform. The chloroform extract is evaporated to dryness and the residue resuspended in high-performance liquid chromatography (HPLC) mobile phase. Tilmicosin is determined using reversed-phase HPLC and ultraviolet (UV) detection at 280 nm. Recovery of tilmicosin from ovine milk fortified over the range 50 to 250 micrograms l-1 is in the range 84.3-104.8%, with a relative standard deviation ranging from 6.6 to 12.9%. The proposed procedure allows the determination of residues of tilmicosin in ovine milk at levels less that 50 micrograms l-1 and satisfies the quality criteria specified in European Commission Decision 93/526/EEC with the exception of reproducibility data from interlaboratory trials.