Determination Of Residues Of Triphenylmethane And Sulfonamides In Food By HPLC After Immunoaffinity Column Clean-up

The immunoaffinity column (IAC) method, which is based on the highly specificinteraction between antigen and antibody，is a good alternative method to clean upsamples in chromatographic separation and analysis. IAC is prepared by immobilizingthe antibodies onto a sol–gel matrix as the solid phase absorbent.The aim of this study was to develop a novel IAC-HPLC method to detectveterinary drug residues, such as malachite green, crystal violet and theirleuco-metabolites, and sulfonamides, in food samples after IAC cleanup procedure.Several important parameters affecting the IAC purification efficiency were studied.The major contents are described as follows.(1) Determination of malachite green, crystal violet and their leuco-metabolites in fishby HPLC-VIS detection after immunoaffinity column clean-upA high performance liquid chromatography method with visible detection(HPLC–VIS) for the determination of malachite green (MG)，crystal violet (CV),leucomalachite green (LMG)， and leucocrystal violet (LCV) in fish has beendeveloped after clean-up through an IAC. The analytes were simultaneously extractedfrom fish muscle with acetonitrile and ammonium acetate buffer. The leuco-forms，LMG and LCV，were oxidized quantitatively to the chromic CV and MG by reactionwith2,3-dichloro-5,6-dicyano-1,4-benzoquinone. The extracts were then purified onan IAC which prepared by immobilizing the anti-MG-CV antibodies by the sol–gelmethod. Finally, the eluents were analyzed by HPLC-VIS. The limits of detectionwere0.15,0.1,0.18and0.14ng/g for MG, CV, LMG and LCV, respectively. Theaverage recoveries in samples fortified with MG, CV, LMG and LCV over the range 0.5–10ng/g were from71.6%to96.8%with RSDs of5.1–12.3%(n=6). This novelmethod was confirmed by liquid chromatography tandem mass spectrometry withelectrospray interface in positive mode using multiple reaction monitoring.(2) Determination of sulfonamides in milk and powder milk by HPLC-UV afterimmunoaffinity column clean-upA high performance liquid chromatography-UV method was established for thedetermination of16sulfonamides residues in milk and powder milk after clean up byIAC. The milk samples were extracted with ethyl acetate, then the clear part of theextractant was loaded on the IAC column for purification. The mobile phase ismethanol-1.1%acetic acid-0.01mol PBS with gradient elution, the chromatographicanalysis was performed by HPLC. The recoveries of the method were in the range of80–110%，the RSD was below10%，the limit of quantification for milk and powdermilk was0.5μg/L and5.0μg/kg，respectively.