Multiresidue Screening of Veterinary Drugs in Meat, Milk, Egg, and Fish Using Liquid Chromatography Coupled with Ion Trap Time-of-Flight Mass Spectrometry.

New approaches to veterinary drug screening based on liquid chromatography-mass spectrometry (LC-MS/MS) and time-of-flight mass spectrometry (ToF/MS) are rapid and have high selectivity and sensitivity. In this study, we developed a multiresidue method for screening over 100 veterinary drug residues using ion trap (IT)-ToF/MS. The screened compounds comprised major drug classes used in veterinary practice, representing the following: amphenicols, anthelmintics, benzimidazoles, ß-lactams, coccidiostats, ionophores, macrolides, non-steroidal anti-inflammatory drugs, quinolones, sulfonamides, tetracyclines, and tranquilizers. The method was developed based on chromatographic retention time, specific accurate mass, isotope distribution, and fragment data. Each compound was validated at three levels, and the mass accuracy, accuracy, and repeatability were calculated. All parameters showed acceptable values and conformed to the Commission Decision 2002/657/EC criteria. This screening method can simultaneously analyze over 100 veterinary drugs in meat, milk, eggs, and fish in a single analytical run.

Magnetic nanoparticle based purification and enzyme-linked immunosorbent assay using monoclonal antibody against enrofloxacin.

Monoclonal anti-enrofloxacin antibody was prepared for a direct competitive enzyme-linked immunosorbent assay (ELISA) and purification system using monoclonal antibody (mAb) coupled magnetic nanoparticles (MNPs). The IC50 values of the developed mAb for enrofloxacin (ENR), ciprofloxacin, difloxacin, sarafloxacin, pefloxacin, and norfloxacin were 5.0, 8.3, 9.7, 21.7, 36.0, and 63.7 ng/mL, respectively. The lowest detectable level of ENR was 0.7 ng/mL in the prepared ELISA system. To validate the developed ELISA in the food matrix, known amounts of ENR were spiked in meat and egg samples at 10, 20 and 30 ng/mL. Recoveries for ENR ranged from 72.9 to 113.16% with a coefficient of variation (CV) of 2.42 to 10.11%. The applicability of the mAb-MNP system was verified by testing the recoveries for ENR residue in three different matrices. Recoveries for ENR ranged from 75.16 to 86.36%, while the CV ranged from 5.08 to 11.53%. Overall, ENR-specific monoclonal antibody was prepared and developed for use in competitive to ELISAs for the detection of ENR in animal meat samples. Furthermore, we suggest that a purification system for ENR using mAb-coupled MNPs could be useful for determination of ENR residue in food.

Veterinary drug residues in domestic and imported foods of animal origin in the Republic of Korea.

The Korean National Residue Programme comprises three different approaches for evaluating domestic and imported foods of animal origin: monitoring, surveillance/enforcement and an exploratory test programme. Monitoring and surveillance/enforcement testing programmes are routinely implemented by 17 Provincial Veterinary Services for domestic products and regional offices of the Animal and Plant Quarantine Agency (QIA) for imported products. The exploratory project conducted at QIA headquarters is designed to test substances that are not included in monitoring and enforcement testing programmes. Here, we carried out exploratory testing for determining the presence of 42 veterinary drugs that have no established Korean maximum residue limits and analysed their levels simultaneously, in a total of 3108 samples of domestic and imported animal-origin foods. Of the tested drugs, acetylsalicylic, paracetamol, clopidol, diclazuril, amprolium, toltrazuril and its metabolites (toltrazuril sulphone and toltrazuril sulphoxide) and phenylbutazone and its metabolites (oxyphenylbutazone) were detected.

Chemical residues and contaminants in foods of animal origin in Korea during the past decade.

Residues of veterinary drugs, pesticides, and environmental contaminants in domestic and imported foods of animal origin were monitored by the National Residue Program and inspection service in Korea in the past decade. In all, 134 substances were analyzed in the monitoring plan; 35 substances were examined in the surveillance and enforcement testing program, and 27 substances were investigated in exploratory projects. The overall trend of violation rates gradually decreased over the past decade. Pesticides were not found in any domestic samples of animal origin. The violation rates of chlortetracycline and oxytetracycline decreased, but quinolone and penicillin detections increased in Korea. Several kinds of residue violations of veterinary drugs, endosulfan, or dioxins were found in the imported products each year. In an example event in 2008, the Korea monitoring plan contributed globally to investigate the dioxin contamination from Chilean pork. Continuous monitoring based on internationally harmonized standards and methods provides the essential scientific basis to manage and ensure food safety.

Overexpression of salicylic acid carboxyl methyltransferase reduces salicylic acid-mediated pathogen resistance in Arabidopsis thaliana.

We cloned a salicylic acid/benzoic acid carboxyl methyltransferase gene, OsBSMT1, from Oryza sativa. A recombinant OsBSMT1 protein obtained by expressing the gene in Escherichia coli exhibited carboxyl methyltransferase activity in reactions with salicylic acid (SA), benzoic acid (BA), and de-S-methyl benzo(1,2,3)thiadiazole-7-carbothioic acid (dSM-BTH), producing methyl salicylate (MeSA), methyl benzoate (MeBA), and methyl dSM-BTH (MeBTH), respectively. Compared to wild-type plants, transgenic Arabidopsis overexpressing OsBSMT1 accumulated considerably higher levels of MeSA and MeBA, some of which were vaporized into the environment. Upon infection with the bacterial pathogen Pseudomonas syringae or the fungal pathogen Golovinomyces orontii, transgenic plants failed to accumulate SA and its glucoside (SAG), becoming more susceptible to disease than wild-type plants. OsBSMT1-overexpressing Arabidopsis showed little induction of PR-1 when treated with SA or G. orontii. Notably, incubation with the transgenic plant was sufficient to trigger PR-1 induction in neighboring wild-type plants. Together, our results indicate that in the absence of SA, MeSA alone cannot induce a defense response, yet it serves as an airborne signal for plant-to-plant communication. We also found that jasmonic acid (JA) induced AtBSMT1, which may contribute to an antagonistic effect on SA signaling pathways by depleting the SA pool in plants.

Microarray-based screening of jasmonate-responsive genes in Arabidopsis thaliana.

Jasmonates comprise a family of plant hormones that regulate gene expression to modulate diverse developmental and defensive processes. To screen a set of jasmonate-responsive Arabidopsis genes, we performed a microarray analysis using an Affymetrix GeneChip containing about 8,300 gene probes synthesized in situ. External treatment with 100 microM methyl jasmonate resulted in significant changes (more than twofold increases or decreases) in the expression levels of 137 genes in the rosette leaves of 5-week-old Arabidopsis plants. Of these, 74 genes were up-regulated, including those involved in jasmonate biosynthesis, defense responses, oxidative stress responses, senescence, and cell wall modification. In contrast, the expression of genes involved in chlorophyll constitution and photosynthesis was down-regulated. Most importantly, the jasmonate treatment significantly reduced transcripts of abscisic acid-responsive cold/drought-stress genes, which suggests that an antagonistic interaction occurs between the jasmonate and abscisic acid signaling pathways in abiotic stress responses. Northern blot analysis of some selected genes revealed that the jasmonate-responsive genes exhibited unique time-course expression patterns after the external jasmonate treatment. Based on the basic clustering of the genes, we established a likely regulation scenario: the genes induced early after treatment are involved in signaling mechanisms that activate or repress other genes, whereas intermediate- and late-accumulating genes are activated by the signaling mechanisms and are subsequently involved in the ultimate jasmonate-modulated cellular responses.