Studies On Confirmation Analysis Of Piperazine In Chicken Muscle By LC-MS/MS

Piperazine is an effective type of anthelmintics which is widely used in animal husbandry. The inappropriate use of piperazine may result in drug residues in animal derived food and pose a threat to human health and ecological balance. At present, the methods for the detection of piperazine residues in animal derived food are rarely reported, particularly the application of LC-MS/MS technology. In this study, the HPLC-MS/MS method and UPLC-MS/MS method for the determination of piperazine in chicken muscle were developed and validated, using Jinghai yellow chicken as experimental material. The aim of this study was to establish standard methods of determination of piperazine in animal derived food. The main results were as follows:1. A HPLC-MS/MS method was developed for the extraction and detection of piperazine in chicken muscle. Piperazine was extracted from chicken muscle with acetonitrile and trichloroacetic acid solution. After the removal of fat using hexane, the supernatant was applied to the positive ion exchanging Strata-X-C cartridge for purification. Piperazine was washed with 2.0% formic acid aqueous solution, methanol, and water in turn, and finally eluted by 10.0% ammonium-acetonitrile. After evaporating the piperazine sample to dry using nitrogen, derived using 20.0% methanol aqueous solution, and 10.O?L supernatant was injected into HPLC. The mobile phase were 1.0% formic acid aqueous solution (A) and methanol (B) using the column of Waters SunFireTM C18(5?m,150.0mm×4.6mm i.d.), the flow rate was 0.4mL/min. The MS method was performed using Electrospray ionization positive ion mode (ESI+) and multiple reaction monitoring mode (MRM). Finally, piperazine was quantitatived by external standard method. At the level of 1.0?200.0?g/kg for piperazine in chicken muscle, the peak area of quantificational ions of the targets showed a linear correlation with the concentration (R2=0.9997). At spiked level 50.0,100.0 and 200.0?g/kg, the average recoveries were 86.43%±4.28%,88.63%±3.51% and 82.22%±1.28%, respectively, with the relative standard deviation (RSD) of 4.95%,3.97% and 1.56%, respectively. Intra-day RSD was within the range from 2.19% to 5.96%, and inter-day RSD was in the range from 3.21% to7.29%. Under the present detecting conditions of HPLC-MS/MS technology, the maximum limits of detection (LODs) of piperazine in chicken muscle was 0.3)?g/kg (S/N=3), and the maximum limits of quantitation (LOQs) was 1.0?g/kg(S/N=10). At the fortified levels of LOQs in specified sample, the recovery, intra-day RSD and inter-day RSD were 76.24%±4.73%,7.08% and 9.16%, respectively. The decision limit (CCa) and detection limit (CCP) were 103.0?g/kg and 106.0?g/kg, respectively.2. A UPLC-MS/MS method was developed for the extraction and detection of piperazine in chicken muscle. Piperazine was extracted from chicken muscle with acetonitrile and trichloroacetic acid solution. After removing fat using hexane, the supernatant obtained was applied to solid phase extraction for purification. The mobile phase were 0.1% formic acid aqueous solution (A) and methanol (B) using the column of Waters ACQUITY UPLC(?) HSS T3(1.8?m,100.0mm×2.1mm i.d.), and the flow rate was 0.2mL/min. MS method made use of ESI+and MRM. Finally, piperazine was quantitatived by external standard method. At the level of 1.0?200. O?g/kg for piperazine in chicken muscle, the peak area of qutantificational ions of the targets showed a linear correlation of the concentration (R2=0.9995). At spiked level of 50.0, 100.0 and 200.0?g/kg, the average recoveries were 102.93%±.70%,111.46%±5.88% and 105.68%±5.15%, respectively, with RSD of 4.57%,5.28% and 4.87%, respectively. Intra-day RSD was within the range from 2.43% to 4.32%, and inter-day RSD was in the range from 3.61% to 5.78%. Under the present detecting conditions of UPLC-MS/MS technology, the maximum limits of detection (LODs) of piperazine in chicken muscle was 0.4±g/kg (S/N=3), and the maximum limits of quantitation (LOQs) was 1.0?g/kg (S/N=10). At the fortified levels of LOQs in specified sample, The recovery, intra-day RSD, and inter-day RSD were 83.26%±8.92%,10.69%, and 12.83%, respectively?The Decision limit (CCa) and detection limit (CCP)were 106.0?g/kg and 113.0?g/kg, respectively.Comparing the two methods developed in this study, the method using UPLC-MS/MS had higher analysis speed and higher precision, better separating effect of the peaks and higher recovery, as well as economy of the mobile phase. Both of the two methods meet the requirements of the Chinese Ministry of Agriculture, the Food and Drug Administration of the United States, and the European Union. Both methods can be used in production practice for the determination of piperazine residue in chicken muscle.