| A method of a pre-column derivatization combined with liquid chromatography fluorescence detection(LC-FLD)methods were established for determination of piperazine residue in chicken tissues(muscle,kidney,liver),eggs(whole egg,albumen,yolk)and in pork in this study.The samples were gotten from Jinghai yellow chicken and Three-way cross hybrid pig(Duroc × Landrace × Yorkshire),after which the samples were extracted and purified by accelerated solvent extraction(ASE)and solid phase extraction(SPE).LC-FLD methods have been established.The main results of this study are shown as follows:1.The reaction conditions of the derivatization of piperazine and dansyl chloride(DNS-Cl)were optimized.It was confirmed that the single substitution reaction of piperazine was happened under optimized conditions,that is,100 ?L 1.0 ug/mL piperazine,600 ?L 1.0 mg/mL DNS-Cl and 100 ?L 0.12%triethylamine were put in a 10 mL centrifuge tube and reacted at 50°C for 20 min in the dark,then,monosubstituted dansyl piperazine(DNS-piperazine)was produced.2.A method was optimized for the extraction of piperazine in chicken tissues,eggs and pork by accelerated solvent extraction(ASE),that is,chicken tissues,eggs and pork were degrease with n-hexane under 80 °C and 1500 psi conditions,then,the piperazine residue from the chicken tissues,eggs and pork was extracted with 2%formic acid acetonitrile.This method is fast and the extraction efficiency is high.3.A high performance liquid chromatography-fluorescence detection(HPLC-FLD)method was established to determine piperazine residue in chicken tissues,eggs and pork.The chromatographic column was Athena C18(4.6×250 mm,5 ?m),the mobile phase consists of water and acetonitrile with gradient elution,the flow rate was 1.0 mL/min,and the column temperature was 25 °C.The excitation wavelength was 330 nm,and the emission wavelength was 531 nm.The results showed that when the piperazine concentration in chicken tissues,eggs and pork was within the range of LOQ-200.0 ?g/kg,the peak area of derivative product showed a good linear relationship with the additive concentration,and the coefficient of determination(R2)was greater than or equal to 0.9992.When the added concentration of piperazine was the limit of quantification(LOQ),0.5 MRL,1.0 MRL and 2.0 MRL,the recoveries was 78.49%~97.56%in the chicken tissues,the intra-day RSD were 2.52%?5.42%,the inter-day RSD were 3.32%?6.46%,and the limit of detection(LOD)and LOQ were 0.96?1.85 ?g/kg and 3.20?5.50 ?g/kg,respectively.The recoveries in eggs were 72.86%?89.26%,the intra-day RSD were 2.46%?6.07%,the inter-day RSD were 3.08%?6.48%,the LOD and LOQ were 1.92?2.50?g/kg and 6.50?7.50 ?g/kg respectively.The recoveries in porks were 86.92%?91.13%,the intra-day RSD were 3.71%?4.65%,the inter-day RSD were 4.75%?7.06%,and the LOD and LOQ were 1.15 ?g/kg and 3.52 ?g/kg,respectively.4.A method of ultra performance liquid chromatography-fluorescence detection(UPLC-FLD)for the determination of piperazine residue in chicken tissues,eggs and pork was established.In the method,ACQUITY UPLC HSS T3(2.1x100 mm,1.8 ?m)was used as chromatographic column,the mobile phase consists of water and acetonitrile(15:85,V:V),the flow rate was 0.2 mL/min,and the column temperature was 25 °C.The excitation wavelength was 330 nm,and the emission wavelength was 531 nm.The results showed that when the piperazine concentration in chicken tissues and pork was within the range of LOQ-200.0 ?g/kg and the piperazine concentration in eggs was within the range of LOQ-400.0 ?g/kg,the coefficient of determination(R2)was greater than or equal to 0.9991.When the added concentration of piperazine was LOQ,0.5 MRL,1.0 MRL and 2.0 MRL,the recoveries in the chicken tissues was 79.64%?99.77%,the intra-day RSD were 2.62%-6.71%,the inter-day RSD were 3.52%?7.77%,and LOD and LOQ were 0.5?1.20 ?g/kg and 1.80-3.50 ?g/kg,respectively.The recoveries in eggs were 77.07%?89.90%,the intra-day RSD were 2.44%?5.19%,the inter-day RSD were 3.10%?6.63%,the LOD and LOQ were 1.05?1.32 ?g/kg and 3.50?4.20?g/kg,respectively.The recoveries in porks were 87.10%?94.71%,the intra-day RSD were 2.50%?6.25%,the inter-day RSD were 2.58%?6.44%,and the LOD and LOQ were 0.62 ?g/kg and 1.85 ?g/kg,respectively.This method is efficient and sensitive,and provides a new method for the detection of piperazine residue.5.Comparing with HPLC-FLD,UPLC-FLD is more suitable for batch sample analysis since it has the advantages of high efficiency,the less consumption of mobile phase and high sensitivity.The two methods both meet the requirements of piperazine residue detection. |
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