Determination of colchicine residues in sheep serum and milk using high-performance liquid chromatography combined with electrospray ionization ion trap tandem mass spectrometry

Colchicine is a naturally occurring alkaloid used in human and veterinary medicine. It shows genotoxicity in in vitro and in vivo systems even at low concentrations. Therefore, no ADI has been established, and colchicine has been included in Annex IV of Council Regulation (EEC) No. 2377/90. No abuse of this drug in intensive livestock farming has yet been reported. However, there may be a natural route of entry for this compound into the food chain when Colchicum autumnale is consumed by animals kept outdoors. To address this concern, we developed and validated a highly sensitive and selective quantitative LC-ESI-MS-MS method for the detection of colchicine in sheep serum and milk. For sample pretreatment, all samples were liquid-liquid extracted with phosphate buffer (pH 8.0) and dichloromethane. LC separation was carried out on an RP C 18 column employing a 0.5% formic acid/acetonitrile gradient system. The recoveries in both matrixes at a concentration range from 0.0005 to 1 mg/L were > 80% with RSDs of < 10%. The applicability of the method was demonstrated in serum and the corresponding milk samples from Albanian sheep. After exposure to C. autumnale, the animals showed clinical signs of intoxication. Significant concentrations (up to 1 mg/L) of the drug were found in serum as well as in milk samples.