Study for quality control of meat products, meat speciation (halal test, meat adulteration) and veterinary drug residues using LC-MS/MS

Abstract

A sensitive, robust, and selective analytical method for determination of 17 Sulfonamides (SAs), 4 Tetracyclines (TCs) and Chloramphenicol (CAP) residues to be used in monitoring program for the analysis of studied veterinary drugs in chicken samples from different poultry farms of Egypt by cutting-edge LC-MS/MS QTRAP has been established and validated. Sample treatment based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) with several adjustments depending on the matrix and drugs group. Double extractions using acetified acetonitrile with 1% acetic acid are common practice in sample preparation of sulfonamides (SAs) and chloramphenicol (CAP) while extraction for tetracyclines (TCs) was by 70% methanol/DIW and pH adjusted by formic acid to be four. Extracts were cleaned-up using PSA for the dispersive solid phase extraction step for interference removal. Various conditions such as MS compatible solutions, LC column, mobile phase and gradient elution tested in order to save analysis time and for efficient chromatographic separation. Optimization of the various MS/MS experimental parameters (Declustering potential, collision cell exit potential, desolvation temperature, gas flow and collision energy) also carried out by full scan and selective reaction monitoring (SRM) with direct injection of single and mixed standard solutions. Matrix interferences monitored in extracts of honey, chicken and pork muscles for 22 antibiotics using recently developed and validated methods. Although a significant dispersion in the results observed for most of the compounds, ion suppression was the major issue. The optimized method validated, obtaining suitable results for all validation parameters in the evaluated matrices. Instrument linearity was established using multi-level calibration curve from 1-100µg/L for sulfonamides and tetracyclines and from 0.1-20µg/L for chloramphenicol; the correlation coefficient was ≥ 0.995 for all compounds. Methods linearity studied using different concentration levels, which lie in between the calibration points. The method proved to be linear for all compounds from Limit of quantitation (LOQ) to the highest level. Limit of quantitation was 10µg/L for Sulfonamides, Tetracyclines, and 0.2µg/L for chloramphenicol. Recovery values ranged from 70%-120%. Repeatability and reproducibility obtained at values lower than 20%. The limit of quantification (LOQ) established at 0.20, 10 and 15 µg/kg for CAP, SAs and TCs, respectively, which were lower than the maximum residue limit legally established by the European Union (EU). Method accuracy studied using various certified reference materials (CRMs) and proficiency tests (PTs) samples, results found to be valid within the acceptable limits. A full in-house validation of the method intended for routine analysis to support regulatory enforcement. Sixty fresh samples from different poultry farms in Egypt tested for the presence of studied compounds. Results showed that there were no positive samples detected except two samples contaminated with doxycycline but lower than the maximum residue limits (MRLs) established by European database (100µg/kg). Hence, more monitoring and risk assessment studies required to cover new generations of veterinary drugs.