DEVELOPMENT AND IMPLEMENTATION OF UPLC-MS/MS METHOD FOR THE DETERMINATION OF PIPERAZINE RESIDUES IN PIG TISSUES

The manuscript presents the results of the development and validation of UPLC-MS/MS method for the determination of piperazine residues in muscle and parenchymal tissues of pigs. Piperazine and its derivatives are well-known anthelmintic drugs used in human and veterinary medicine. Piperazine is a heterocyclic compound without any chromophore groups with a low molecular weight (86.1 g/mol), which makes its detection in biological samples by LC-MS/MS difficult. We have developed the confirmatory technique for the direct UPLC-MS/MS assay of piperazine residues (without prior derivatization) in animal tissues after sample purification using solid-phase extraction. The proposed sample preparation includes homogenization, liquid-liquid extraction using acetonitrile, concentration and purification of samples from matrix components using SPE cartridges Oasis HLB (Waters) and with additional concentration of the eluate by drying. The analysis of the prepared samples was carried out by the method of ultra high performance liquid chromatography with tandem mass spectrometric detection using the precursor ion of 87.0 m/z and product ions of 43.9 and 70.0 m/z (ES+ ionization, multiple reactions monitoring mode). In order to experimentally confirm the fitting-for-purpose of the developed confirmatory method for clinical studies of veterinary drugs based on API piperazine and to assess the safety of food products of animal origin for piperazine residues, the validation of the developed UPLC-MS/MS method for the determination of piperazine in muscle tissues and liver of pigs was carried out using approaches described in EMEA/CVMP/VICH/463202/2009 and in accordance with Regulation (EU) 2021/808. In the process of UPLC-MS/MS technique validation, various key operating parameters were established for both investigated matrices: limit of detection, limit of quantification, linearity, specificity, accuracy, trueness. In particular, it was established that the developed method provides determination of piperazine residues in muscle tissues and liver of pigs with a sensitivity of 50 and 400 μg/kg, respectively.The developed and validated confirmatory UPLC-MS/MS method for the determination of residual amounts of piperazine is highly sensitive, highly selective and rapid, appropriate for the control and monitoring of the safety of animal meat and liver samples, as well as to provide analytical support for clinical studies of veterinary drugs based on API piperazine and its derivatives.