Development and Validation of a Multi-detection Confirmatory Method for Antibiotics Determination in Piglet Kidneys by UHPLC-TOF-MS According Commission Decision 2002/657/EC

Abstract

The combination of ultra-high-performance liquid chromatography (UHPLC) with time-of-flight mass spectrometry (ToF-MS) is one of the most powerful analytical tools in terms of selectivity, sensitivity and speed. However, due to the gap on regulation and guidelines concerning the validation criteria for the use of ToF-MS methodology not only for screening but also for confirmatory and quantification purposes, this study presents, for the first time, an approach on how to use the referred methodology for confirmatory and quantification purposes. Thus, an UHPLC-ToF-MS method was developed and validated for the simultaneous determination of 43 antibiotic residues from 7 different families (tetracyclines, sulfonamides, quinolones, penicillins, macrolides, cephalosporins and trimethoprim) in piglet kidneys as a confirmatory method. A simple sample preparation consisting of extraction with a mixture of acetonitrile and ethylenediaminetetraacetic acid (EDTA) followed by solid-phase extraction (SPE) with a hydrophilic-lipophilic balanced (HLB) cartridge and finalized with a defatting step with n-hexane was used. The methodology was validated according the Commission Decision 2002/657/EC, by defining suitable criteria for antibiotic identification, as well as by evaluating required parameters like selectivity, specificity and ruggedness that fulfil the required criteria. The calculated values of decision limit (CCα) and of detection capacity (CCβ) for antibiotics with established maximum residue limits (MRLs) fully complied with required criteria. For antibiotics for which no MRL was set, values ranging from 0.2 to 0.5 μg kg−1 and from 0.5 to 0.8 μg kg−1 were achieved for CCα and for CCβ, respectively. Linearity (all R2 > 0.95), recovery (81.4–110.0%) (lowest values of 4.6% and 7.1% were obtained for repeatability and for reproducibility, respectively), LODs (0.04–13.0 μg kg−1), LOQs (0.09–32.0 μg kg−1) and matrix effects (35.5–118.1 %) were also evaluated. After validation, the method was applied to analyse piglet kidney samples. Antibiotic residues were determined in 10 of the 15 analysed samples. However, all the antibiotics concentrations were below the MRLs.