Modified liquid chromatographic method for determination of gentian violet in animal feed.

A modified liquid chromatographic method is described for the determination of Gentian Violet (GV) in animal feed. The reliable detection limit is 0.5 ng (reference standards), and 1 ppm GV was reliably determined in feed. The calibration curve was linear between 1 and 40 micrograms/mL. The method, developed in a study by the National Center for Toxicological Research, was modified to use methanol-water (9 + 1) instead of benzene-methanol as the eluting solution in the column cleanup. GV is extracted from feed with methanol-1N HCl (99 + 1), cleaned up on a Sephadex LH-20 column to remove any remaining interferences, separated on a Nova-Pak C18 column fitted with a precolumn filter, and determined at 588 nm. The identity of GV is confirmed by thin-layer chromatography (Rf = 0.47) by comparison with a reference standard. Average recoveries from 3 sets of 5 feed samples containing 2.5, 5.0, and 10.0 ppm GV were 115, 95, and 102%, respectively.