High performance liquid chromatographic determination of colchicine-alkaloid from the tissues and biological fluid of bovine poisoned by the corm of autumn crocus (Colchicum autumnale L.)

The tissues and biological fluid of bovine that had apparently been poisoned by the corm of Colchicum autumnale L. were examined. The test solutions were prepared by the following steps: (1) colchicine-alkaloid extraction with acetone, (2) defatting of the extract with petroleum ether, (3) removal of protein from the extract with 5% zinc sulfate solution and 0.3N barium hydroxide, (4) extract of colchicine-alkaloid with chloroform. High performance liquid chromatography was performed with a LiChrosorb RP-18 column using acetonitrile-methanol-phosphate buffer (pH 6.0) (32:5:63) as the mobile phase with detection by absorbance measurement at 350nm. Colchicine (COL) and demecolcine (DEM) contents in the muscle were in the range of 0.05-0.12ppm and 0.03-0.05ppm, respectively. In the spleen, COL and DEM levels were 0.76-1.17ppm and 0.71-1.19ppm, respectively. The contents of COL and DEM in the spleen were higher than those in the liver and kidney. The determination limits of COL and DEM were 0.01 and 0.02ppm, respectively, in muscles. At the level of 5ppm, the recoveries of COL and DEM added to beef were 77.1% and 72.7%, and those of COL and DEM added to milk were 77.0% and 75.0%, respectively.