Simultaneous Determination of Malachite Green,Methylene Blue,Crystal Violet as well as Their Metabolites in Aquatic Products by High Performance Liquid Chromatography

A solid-phase extraction coupled with high performance liquid chromatography(SPE-HPLC) was presented for the simultaneous determination of malachite green,methylene blue,crystal violet as well as their metabolites(leucomalachite green for malachite green,azure A,azure B,azure C for methylene blue,and leucocrystal violet for crystal violet).Samples were extracted with a mixture of ammonium acetate buffer and acetonitrile,cleaned up with dichloromethane,purified on an MCAX solid phase extraction cartridge using a mobile phased made up of acetonitrile and 0.125 mol/L ammonium acetate buffer,and detected by a UV detector.The limits of quantification(LOQ) of the method were 20 μg/kg for malachite green,leucomalachite green,crystal violet,leucocrystal violet and azure B and 0.5 μg/kg for methylene blue,azure A and azure C,respectively.An average spike recovery above 70% was found for malachite green,leucomalachite green,leucocrystal violet,crystal violet and azure B at spike levels ranging from 2 to 40 μg/kg and for methylene blue,azure A and azure C at spike levels between 5μg/kg and 80 μg/kg,with a relative standard derivation(RSD) less than 15%.