Determination Of Levamisole,Mebendazole And Its Two Metabolite Residues In Poultry Meat And Eggs By HPLC-MS/MS

This study used Haiyang Yellow chicken,Jinghai Yellow chicken,Gaoyou duck and Yangzhou goose as experimental material,and liquid-liquid extraction(LLE)combined with solid phase extraction(SPE)technology was used to extract the target compound.HPLC-MS/MS method for the simultaneous determination of levamisole,mebendazole and its two metabolites(aminomebendazole and hydroxymebendazole)residues in poultry meat and eggs was established and optimized.The main results of this research are shown as follows:1.LLE-SPE method was developed and optimized for the simultaneous extraction of levamisole,mebendazole,aminomebendazole and hydroxymebendazole residues from poultry meat,using alkaline ethyl acetate as the extractant.The sample was added sequentially 0.5 mL of 50%potassium hydroxide(KOH),8 mL of ethyl acetate,and 8 mL of 0.1 mol/L hydrochloric acid to extract the target compound and then purified by an Oasis(?)MCX solid phase extraction cartridge.After sample pretreatment,the extract was blown dry by nitrogen,and then re-dissolved by initial mobile phase,and finally detected by the instrument.The results showed that the recovery of the four analytes in poultry meat were all above 87.58%.2.LLE-SPE method was developed and optimized to extract levamisole,mebendazole,aminomebendazole and hydroxymebendazole residues in poultry eggs.The sample was added sequentially 0.5 mL of 50%KOH and 8 mL of acetonitrile:water(90:10,V/V),and then vortexed and ultrasonically extracted.The supernatant was collected,and the above extraction step was repeated once.Afier sample extraction,an Oasis(?)MCX solid phase extraction cartridge was used to purify,and then blown dry by nitrogen and redissolved by initial mobile phase.After HPLC-MS/MS detection and analysis,the recoverie of the four analytes in poultry eggs were all above 86.00%.3.HPLC-MS/MS method was developed and optimized for the simultaneous determination of levamisole(LMS),mebendazole(MEB),aminomebendazole(AMEB)and hydroxymebendazole(HMEB)in poultry meat and eggs.The chromatographic conditions were performed on an XbrdgeTM C18 column(4.6×150 mm,5 μm)at 35℃,and 0.1%formic acid in water and acetonitrile were used as mobile phases.The elution program adopted gradient elution,and the flow rate was 0.6 mL/min.The detection instrument was equipped with a triple quadrupole mass spectrometer and an electrospray ion source(ESI).The detection of target compounds used a positive ion scanning and multiple reaction monitoring(MRM)mode.Levamisole,mebendazole and their two metabolites were added to blank poultry meat and eggs at these concentrations within the limit of quantification(LOQ)of-25 μg/kg and LOQ-150 μg/kg,which showed that the peak area of the target compound has a good linear relationship with the concentration,and the coefficient of determination(R2)were higher than 0.9990.When the added concentrations of LMS,MEB,HMEB and AMEB were the LOQs amount,0.5,1.0 and 2.0 Maximum Residue Limit(MRL),the average recoveries in blank poultry meat were 87.58%-95.95%,86.77%-95.21%,88.36%-95.63%and 89.00%-96.94%,respectively.The intra-day relative standard deviations(RSDs)in blank poultry meat were 2.0%-5.13%,2.07%-4.67%,2.39%-4.99%and 1.75%-3.92%,respectively,and the inter-day RSDs were 2.99%-5.37%,2.62%-4.75%,2.96%-5.52%and 2.54%-4.96%,respectively.The limits of detection(LODs)in blank poultry meat were 0.05-0.07,0.04-0.06,0.15-0.18 and 0.23-0.30 μg/kg,respectively,and the LOQs were 0.16-0.22,0.12-0.16,0.50-0.60 and 0.62-0.80 μg/kg.At four concentration levels,the average recoveries in blank poultry eggs were 86.04%-96.64%,85.98%-96.55%,88.44%-96.48%and 87.89%-97.38%,respectively,the intra-day RSDs were 1.67%-5,15%,1.41%-5.85%,1.40%-5.00%and 1.53%-5.09%,respectively,and the inter-day RSD were 2.49%-5.44%,2.60%-6.32%,2.34%-5.35%and 2.73%-5.34%,respectively.The LODs in blank poultry eggs were 0.03-0.07,0.03-0.05,0.15-0.23 and 0.25-0.33 μg/kg,respectively,and the LOQs were 0.10-0.22,0.08-0.20,0.50-0.80,0.80-1.0 μg/kg.The developed method is rapid,efficient and sensitive,providing a technical support for the simultaneous determination of levamisole,mebendazole,aminomebendazole and hydroxymebendazole residues in animal-derived foods.