| Piperazine, as an effective insect repellent, has been widely applied in veterinary clinic. However, the improper use of piperazine causes a severe issue of drug residue in animal tissues, which will be a threat to human health after long-term consumption of animal-origin product containing piperazine. At present, few research in this field was found at home and abroad. Therefore, this study integrated new techniques or methods, for example, pre-column derivatization, accelerated solvent extraction, and solid phase extraction, The present study established a GC-MS/MS detection method including pre-treatment procedures as well as validation process, with Jinghai Yellow chickens, Yingtaogu duck, Yangzhou geese, and Duroc-landrace-yorkshire swine as test materials, to determine piperazine residue levels in animal tissues of meat, liver, kidney as well as skin and fat, so as to provide scientific reference for the establishment of national standard on detection method of piperazine related residue as well as technological means of resudue monitoring in animal-origin product. Main experimental results are shown as follows:1. The experimental conditions of piperazine derivation with acetic anhydride was established and optimized as follows:20 μg piperazine (dissolved in methylene dichloride solvent) reacted with 50 μL acetic anhydride and 100 uL triethylamine (both were overdosed) in closed glassware for 30 min at 50℃ and this derivatization was successfully applied to gas chromatography analysis, which showed improved chromatography feature of sharp target peak and guaranteed a precise quantification.2. Extraction method of piperazine residue from animal tissues using technique of accelerated solvent extraction (ASE) was established and optimized, samples of animal tissues were mixed with anhydrous Na2SO4 and diatomite in turn and were loaded into the extract pond. The extraction process underwent at a device condition of 80 ℃ and 1500 psi with n-hexane as de-fat solvent and acetonitrile as extraction solvent of piperazine. At spiked level of LOQ,50.0, 100.0,500.0,1000.0 and 2000.0 μg/kg, the average recoveries were 80.98%～96.26%, with the relative standard deviation (RSD) of 1.58～4.53%. This method of extraction is safe, automatic and also have advantages of less consunption of extraction solvent and matrix effect as well as higher extraction efficiency.3. A GC-MS/MS method was established, optimized, and validated, for the first time, to determine piperazine residue levels in tissues of mussels, livers, kidneys, as well as skin and fat from chickens, ducks, geese, and pigs. Electron ionization was adopted, with SCAN and SIM mode, and Auto SRM was used for qualitative or quantitative purpose. Within the dosing level of 4.5～2500.0 μg/kg for piperazine in animal tissues, the peak area of quantificational ion, m/z170.1 >68.1, showed a good linear correlation with concentration (R2=0.9995). Under current experimental method and instrumental conditions, intra-day RSD and inter-day RSD were within the range of 1.36%-7.76% and 1.86%-8.10%, respectively. The limit of detection (LOD) was within the range of 1.4～1.6 μg/kg (S/N≥3) and the limit of quantification (LOQ) was within the range of 4.8～5.2 μg/kg (S/N≥10). This method is of simplicity, high precision, and high sensitivity and the validation parameters of this method meet the drug residue requirements of the Ministry of Agriculture, the European Union and the United States. |
PDF Full Text Request