Development of multi-residue analytical methods for quinolones, cephalosporins, and trimethoprim and application to the residue monitoring in livestock

Abstract

Three reliable analytical methods were developed based on a simple and rapid sample preparation followed by ultra performance liquid chromatography with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) for the determination of 13 quinolones, 9 cephalosporins, and trimethoprim in livestock and marine products (beef, pork, chicken, milk, egg, flatfish, jacopever, common eel, and shrimp). The proposed methods were validated according to the CODEX guidelines and applied in the survey of in a total of 1012 samples of livestock and marine products commercialized in Korea. In the first study, a simple and specific analytical method was developed for the simultaneous determination of 13 quinolones (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, norfloxacin, ofloxacin, orbifloxacin, oxolinic acid, pefloxacin, and sarafloxacin) using UPLC-ESI-MS/MS and all results fully complied with CODEX recommendation. Good linearities were achieved and the correlation coefficients were ranged between 0.9992 and 0.9999. The recoveries of 13 quinolones were higher than 80%, the limit of detection (LOD) and limit of quantitation (LOQ) were lower than 0.1 and 0.4 μg/kg, respectively. Besides, the matrix-matched calibration curve and internal standard (IS) played a significant role in compensating for the matrix effects. A survey for 13 quinolones residues was conducted on 310 livestock and marine products. Oxolinic acid, enrofloxacin, and flumequine were the most commonly detected antibiotics. The residues of quinolones were detected on 104 samples (33.5% incidence) but residue levels were below the MRLs. Detected concentrations were 0.1-100.0 μg/kg and the risk value (EDI/ADI, %) were safe levels between 0.0005% and 0.612%. However, pefloxacin was detected in one common eel sample above the legal residue limit and the detected concentration was 62.4 μg/kg. In the second study, a sensitive and reliable method was developed and validated for the simultaneous determination of 9 cephalosporins (cefacetrile, cefazolin, cephapirin, desacetyl cephapirin, cefalexin, cefalonium, cefoperazone, cefuroxime, and cefquinome) and all obtained results were satisfied with CODEX recommendation. The use of UPLC-ESI-MS/MS with polarity switching ionization mode improved the sensitivity and reduced analysis time, allowing the identification and quantification of 9 cephalosporins in 5 min. Because of the matrix effects, matrix matched calibration curves with IS were used for quantification to determine cephalosporin residues in samples. Good linearities were acquired and the LOD and LOQ were lower than 8 and 25 μg/kg, respectively. The survey for the 9 cephalosporins residues was conducted on 333 livestock and marine products. The residues were detected in only 12 livestock products (3.6% incidence) but residue levels were below the MRLs and the detected cephalosporins were cefalonium in beef and cefquinome in milk. Detected concentrations were from 1.10 to 9.77 μg/kg in beef and 7.50 μg/kg in milk. The risk value was very safe level of 0.02% in cephalonium and 0.01% in cefquinome. In the third study, a specific and simple analytical method was developed using UPLC-ESI-MS/MS for the determination of trimethoprim. The sample preparation through the ultrasonic-assisted extraction and SPE clean-up procedure was improved the recovery and reduced the matrix effect. The recoveries were higher than 70% and the LOD and LOQ were lower than 0.3 and 1.0 μg/kg, respectively. The survey for the trimethoprim residue was conducted on 369 livestock and marine products. The residues of trimethoprim were detected in 7 marine products (1.9% incidence) but residue levels were below the MRL. Detected concentrations were 1.17 to 16.43 μg/kg in jacopever, 40.0 μg/kg in flatfish, and 13.3 μg/kg in common eel. The risk value was safe level of 0.13%. In conclusion, the methods developed in this study were more reliable and accurate for screening, quantification, and identification of 13 quinolones, 9 cephalosporins, and trimethoprim residues in livestock and marine products and could be successfully applicable in field samples. The resulting residue level of 13 quinolones, 9 cephalosporins, and trimethoprim appeared to be relatively safe. However, the quinolones and 3rd - & 4th -generation cephalosporins were classified as critically important antimicrobials for human medicine (CIA) and trimethoprim and 1st - & 2nd -generation cephalosporins were classified as highly important (HIA) from World Health Organization (WHO). In addition, quinolones, cephalosporins, and trimethoprim were classified as veterinary critically important antimicrobials (VCIA) from the Office International des Epizooties (OIE). Thus, a strict guideline for the use of antibiotics and continuous survey on antibiotic residues is needed to ensure the safety of animal origin foods. The methods developed in this study will help to implement such guidelines of CODEX on the use of antimicrobials.