| Abstract: | A method for the detection and estimation of theobromine in compounded horse feed by high-performance liquid chromatography (h.p.l.c.) is presented. The sample is extracted by refluxing with distilled water and clarified by the addition of Carrez reagents. The level of co-extractives is reduced by precipitation by the addition of 4 vols of acetone, and a measured volume of the filtrate transferred on to a silica column for clean-up. The theobromine is eluted with chloroform (85%)-propan-2-ol (15%) and the eluate evaporated to dryness. The residue is dissolved in water and analysed by h.p.l.c. (using a reverse-phase column and ultraviolet detection at 272 nm). The method has been tested on mixed horse feeds and their ingredients. Theobromine recoveries were in the range 78–86% and concentrations down to 2 mg kg?1 could be measured. Three ways to confirm positive results are presented. |
