Abstract: 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%.

Key words: high performance liquid chromatography, aquatic products, malachite green, methylene blue, crystal violet, metabolite