Determination Of Piperazine Residuce In Chicken Muscle And Pig Tissues By High Performance Liquid Chromatography

Piperazine as effective anthelmintics is widely used in veterinary clinic.With piperazine unreasonable use or even abused, this kind of drug residuced in food will inevitably occur.Long-term consumption of animal food products containing piperazine will be likely to cause great harm to human health and even lead to poisoning.At present,the related research in piperazine residual,only have the method with Liquid Chromatography-Mass spectrometry and no standard methods for piperazine residuce released.Base on the above,this study intends to establish a method of piperazine residues in chickens and pigs tissue,not only to monitor the drug residue in the future,but also to improve the qulity of animal food,which is good to protect consumer’s health.1.Established a method of pre-column derivatization for piperazine:Accurately moved piperazine standard working solution100μL into a thimble tube(2mL),then added400μl of DNS-CL working solution and100μL of Na2CO3-NaHCO3buffer(pH10.0).The mixture was vortexed for lmin and then centrifuged for30s,reacted in a water bath at50℃for30min.After the mixture cold down, were used lmL of the ethyl acetate to extract for twice,combined the extraction to50ml of heart bottle and evaporated to dryness in a water bath at40℃.The residue was redissolved in lml acetonitrile under vortex and centrifuged at12000rmp for lOmin.An aliquot of20μl of the solution was injected into the HPLC after0.45μm of cylinder filter.After piperazine working solution derivated,the relative standard deviation of the retention time was0.37%, the standard deviationof the peak area was2.73%.It was show that the reliability of separation and quantitative in this experiment whose was been setting was high and also demonstrated that the derivative product was relatively stable in a certain period of time.2.Establish a method of residues detection for piperazine in the animal tissue:Animal tissues were extracted with the mixture of acetonitrile and perchloric acid,their extracts were defatted by hexane and then applied to positive ion exchanging MCX cartridge which was washed with2%phosphoric acid solution,methanol and ultra pure water in turn.The retained drugs were eluted by10%ammonium-acetonitrile. Elute was evaporated to dry by nitrogen gas,the dry residue was added the dansyl chloride and buffer to derive,then dissolved by acetonitrile and filtered. Derivatives were determined on C18column with ultraviolet absorption detection at278nm, using acetonitrile and water (78:22,V/V) as the mobile phase,quantified by external standard. The flow rate was1.0mL/min and the column temperature was35℃.3.Under these chromatography conditions established in the assay, the concentration of piperazine had a good linear coefficient relation(R2>0.9997) in the range of0.05～2mg/kg.On the level of drug (0.05、0.5、2mg/kg) which were added into blank, the average recovery of piperazine in chicken muscle were87.31%～91.83%,the intra-day coefficient of variation were3.51%～7.16%and inter-day coefficient of variation were3.91%～7.86%respectively; the average recovery of piperazine in pig muscle were87.77%-96.29%,the intra-day coefficient of variation were2.13%～7.04%and inter-day coefficient of variation were2.31%～8.85%respectively; the average recovery of piperazine in pig liver were78.80%～87.70%,the intra-day coefficient of variation were4.19%～6.45%and inter-day coefficient of variation were5.13%-7.06%respectively; the average recovery of piperazine in pig kidney were78.85%～84.84%,the intra-day coefficient of variation were3.06%～6.98%and inter-day coefficient of variation were3.11%～7.81%respectively. The limit of detection (LOD) and the limit of quantification (LOQ) of piperazine were0.03mg/kg and0.05mg/kg respectively.The extraction and purification and detection methods of piperazine residues in chicken and pig tissues established in this experiment apply to the determination of piperazine drug residues in chicken and pig tissues.