Desenvolvimento de método para determinação multiclasse de resíduos de medicamentos veterinários em músculo suíno por UHPLC-MS/MS

Abstract

In swine farming, veterinary drugs are mainly applied to provide animal’s health. However, incorrect use can cause the presence of residues of these substances above the maximum limit allowed (MRL) in pork, and because of this, the development of analytical methods for the determination of these substances is extremely important. Thus, this study aimed to develop and validate a method for multiclass determination of veterinary drugs residues in pork by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). 36 compounds (33 antimicrobials and 3 β-agonists) were selected. During the development of the method, different chromatographic and sample preparation conditions were evaluated. In the extraction step, the solvent composition, the use of acidification and complexing agent, as well as the inclusion of a partition step with salts or at low temperature were tested. Then, the tests of the clean-up step were conducted with sorbents in dispersive or in cartridge mode, and the presence of co-extractives in the extract was verified by UHPLC-MS/MS and full scan analysis by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). The final sample preparation method consisted of extraction with an aqueous solution containing 0.1% (m/v) EDTA and 1% (v/v) formic acid, followed by the addition of acetonitrile, and clean-up of the extract by dispersive solid-phase extraction (d-SPE) using C18 sorbent. For determination of compounds with established MRLs (authorized), the extract was diluted in ultrapure water, while for the others it was necessary a step of evaporation of the extract and reconstitution in a solution of water:acetonitrile, prior to injection into the UHPLC-MS/MS system. Validation was performed following the parameters and criteria of acceptance of official documents, in which the MRL was considered for the permitted compounds, while the lowest calibration level (LCL) was adopted for prohibited and unauthorized compounds. Linearity showed r² > 0.99 for most compounds. The recoveries ranged from 83 to 113%, with a coefficient of variation (CV%) below 15%, except for erythromycin, which did not obtain a satisfactory result of CV% at one of the levels evaluated in the repeatability study. Decision limit (CCα) concentrations ranged from 5.3 to 447.8 μg kg-¹. The proposed method was applied to 25 pork samples and residues of enrofloxacin, oxytetracycline and ractopamine were detected. The results indicated that the method is suitable for its purpose and due to its simplicity, it can also be used in routine laboratories.