Study On Multi-residue Analysis Method For Determination Of Malachite Green, Gentian Violet And Their Leuco-metabolites In Aquatic Products By LC-MS/MS

Malachite green (MG) and gentian violet (GV) are both triphenylmethane dye which has been used worldwide as fungicide and ectoparasicide in aquaculture since early 1930s. MG and GV have good curative effect on proliferative kidney disease in fish. However, they are easily absorbed and deposited in fish fatty tissue during waterborne exposure and extensively metabolized to the reduced into colourless compounds, leuco-malachite green (LMG) and leuco-gentian violet (LMG). Due to their potential human and animal carcinogenicity, mutagenity and teratogenicity, MG and GV have been banned in some countries including China. Therefore, there is anobvious need for a simple and sensitive method for determination of MG, GV, LMG and LGV. In the study, an LC-MS/MS method, including LC-LITMS/MS and LC-QQQMS/MS has been developed for determination of MG, GV and their leuco-metabolites using isotope dilution without post-column oxidation in various aquatic products. The validation of accuracy, repeatability, linearity and reproducibility for the method has been determined, which could be used to check performance of the method with analysis actural sample using this developed method.Sample was extracted with McIlvaine buffer and acetonitrile, purified on an OASIS MCX SPE cartridge. The eluent was detected by LC-LITMS/MS with mode of select reaction monitoring and by LC-QQQMS/MS with mode of multi-reaction monitoring. With LC-LITMS/MS mode, decision limits (CCα, a=0.01) and detection capabilities (CCβ,β=0.05) were in the range of 0.02～0.09μg/kg and 0.04～0.13μg/kg for MG, GV, LMG and LGV in grasscarp, eel, salmon, shrimp and shellfish, respectively, recoveries of MG, GV, LMG and LGV spiked in samples were from 84.7% to 112.1%, inter-day relative standard derivations were 1.4%～19.8%. And with LC-QQQMS/MS mode, decision limits (CCα,α=0.01) and detection capabilities (CCβ,β＝0.05) were from 0.02 0.07μg/kg and 0.06～0.12μg/kg in the grasscarp, eel, salmon, shrimp and shellfish, respectively, recoveries of were 84.7%～112.1%, inter-day standard derivations were in the range of 1.5%～12.8%.MG, GV, LMG and LGV at concentration of 10μg/kg were spiked into samples to investigate the stability. The results indicated the compounds degradated obviously in room temperature, while little degradation in -20℃. And their leuco-metabolites are less stable than MG and GV. However, it is very effective to decrease their degradation when TMPD or ascorbic acid is added into the extraction process.The conditions of sample praparation and detection by LC-MS were optimized in this study. Therefore, sensitivity, accuracy and precision for the method were obvious increased. Three product ions were selected as qualitatitive and quantitative for MG, LMG, GV and LGV, respectively. The identity of residues could in all tested cases be determined with at least four identifications points (three product ion, 3×1.5IP) for MG, LMG, GV and LGV according to that described in Commission Decision 2002/657/EC. This developed method can provide technology guidance to identification and confirmation of MG, LMG, GV and LGV in dubious samples.