Search

A method is described for the quantitative determination of quinoxaline-2-carboxylic acid (QCA), the marker residue for the veterinary drug carbadox, in swine liver. Tissue is subjected to alkaline hydrolysis followed by liquid–liquid extraction. QCA residues are cleaned up using automated solid phase extraction (SPE), before a final liquid–liquid extraction step. Analysis is based on LC coupled to positive ion electrospray MS-MS, monitoring product ions at m/z 129, 102 and 75 for QCA and at m/z 106 for the internal standard (d₄-QCA). The method has been validated according to draft revised EU criteria for analysis of veterinary drug residues, and is suitable for monitoring tissues taken under national surveillance schemes. The method has been validated at 3, 10, 30, 100 and 300 μg kg−1. The method performance characteristics, CCα (decision limit) and CCβ (detection limit) were determined to be 0.16 and 0.27 μg kg−1, respectively. The described method, which is relatively rapid and applicable to large sample numbers, correlates well (r2 = 0.9799) with a widely used GC-MS assay for QCA.

European Union Member States are now required to monitor poultry meat for the presence of coccidiostat residues. Among other factors contributing to the production of residue-free food is the ability of animal feed manufacturers to produce medication-free feedstuffs, ensuring the proper observance of withdrawal periods prior to slaughter. Carry-over of medication was investigated in a local poultry feed mill that was using monensin as its principal coccidiostat for broilers. Monensin, at levels in excess of 5% of the therapeutic dose (approximately 110 mg kg–1), was present in 22.5% of 40 withdrawal feeds. Subsequent studies in the mill indicated that most of the contamination occurred during the processing of feeds after the mixing stage. The mill altered its manufacturing process as a result of this study. The consequence of this was that the incidence of monensin withdrawal feeds, at levels greater than 5% of the therapeutic dose, fell from 22.5 to 2.5%. This collaborative study has helped the feed compounder to produce more effective withdrawal feeds, thereby reducing the potential exposure of consumers to unwanted residues of monensin in poultry meat.

The administration of chloramphenicol (CAP) is banned in food animals in the European Union (EU). It is, therefore, important to have adequate screening methods to determine if residues of CAP and its major metabolite, chloramphenicol-glucuronide (CAP-Gluc), are present in samples taken for monitoring purposes. Six castrated male cattle were treated with a single intramuscular injection of 10 mg kg–1 CAP. Animals were sampled once daily for urine and were slaughtered at 3 and 6 d post-injection. Samples of bile, kidney, liver and diaphragmatic muscle were removed at slaughter. All matrices were analysed using the four plate test (FPT) bioassay, the Charm II radioimmunoassay and a Ridascreen CAP-Glucuronid competitive enzyme immunoassay (EIA). The FPT detected CAP residues in urine samples taken up to 2 d post-treatment. The Charm assay detected CAP in the urine for up to 4 d post-treatment. The EIA detected CAP throughout the 6 d sampling period. Samples of bile were positive by both the EIA and the Charm assay at day 3 and day 6. No zones of inhibition were obtained using the FPT in bile or diaphragm either with or without sample pre-treatment with β-glucuronidase. However, the kidney and the liver from one animal killed at day 6 gave larger zones of inhibition after treatment with β-glucuronidase, indicating the presence of CAP. The kidneys of all treated animals slaughtered at day 3 were positive by both the EIA and the Charm assay but none of the kidneys at day 6 tested positive by either method. Owing to technical difficulties, the Charm assay was not suitable for the analysis of liver. The EIA failed to detect CAP in the liver of any treated animal. It is concluded that urine appears to be the best matrix for screening purposes. The sensitivity of the FPT is inadequate for the determination of CAP residues where minimal withdrawal periods have been observed. The Charm assay and the EIA were suitable for the detection of both CAP and CAP-Gluc in tissues and body fluids for longer periods post-administration. The EIA was more sensitive for the determination of low concentrations of CAP and its metabolite.

Screening for the presence of anabolic growth promoters in urine samples from cattle grown for meat production can be performed by (semi)quantitative methods such as immuno-, receptor- or cell-based assays or by quantitative methods with mass spectrometric detection which can also include confirmation of compounds. In this study conventional immunoassays used at two different institutes [Veterinary Sciences Division (VSD) in Northern Ireland and TNO Nutrition and Food Research Institute (TNO) in The Netherlands] were compared with the oestrogen radioreceptor assay (ORRA), with GC-MS as the reference method. Urine samples were generated by treating calves (n = 2 per group) intramuscularly with ethynyloestradiol (EE2), diethylstilbestrol (DES) or α-zearalanol (zeranol, ZER). Urine samples were collected up to 21 d after administration of the oestrogenic compounds. Samples were screened by enzyme immunoassay or radioimmunoassay and by the ORRA and also by GC-MS. Values found by VSD were lower by a factor of 1–20 than those measured by TNO. These differences could be explained by differences in sample clean-up (immunoaffinity chromatography versus solid-phase extraction) and by differences in cross-reactivities between the antisera used. The ORRA and GC-MS showed similar results for EE2 and DES, but produced lower results (by a factor of ca. 3) for ZER owing to the relatively low affinity of ZER for the oestrogen receptor. The most important finding was that the withdrawal period for calves treated with EE2, DES or ZER was similar for each of the screening methods used. Therefore, it is concluded that the choice of screening method does not affect the probability of finding a positive sample.

Chlortetracycline (CTC) is one of the few antibiotics that can be used without any withdrawal period in chickens laying eggs intended for human consumption. 6-Iso-CTC and 4-epi-6-iso-CTC have recently been identified as the principal metabolites of CTC in eggs. Although not covered by the European Union (EU) maximum residue limit (MRL) for CTC, these compounds, taken together, accumulate in the eggs of birds treated therapeutically with CTC to a mean concentration equivalent to more than twice the EU MRL (200 µg kg–1) in eggs. Plateau concentrations in eggs were achieved after approximately 3 d of medication. Following withdrawal of medication, mean egg concentrations of these compounds were maintained for 48 h, before falling below a level equivalent to the MRL after 5 d. Feeds containing typical sub-therapeutic contamination concentrations of CTC did not produce mean concentrations of 6-iso-CTC plus 4-epi-6-iso-CTC, combined, greater than 200 µg kg–1. It is not known whether these compounds are formed as a result of metabolism or of chemical degradation. However, analysis of ovules pre-lay showed that all of the CTC present in this matrix was in the form of 6-iso-CTC and 4-epi-6-iso-CTC, and not as the parent drug. Although microbiologically inactive, the toxicological properties of 6-iso-CTC and 4-epi-6-iso-CTC are not known.

The drug salbutamol (SBL) is a β-agonist that may be used illegally as an animal growth promoter. SBL is also a good example of a drug which is excreted in the form of glucuronides and sulfates. Such metabolites cause complexities in analysing for the presence of drug residues. In the majority of cases a process of deconjugation and sample clean-up is required prior to analysis. This is both time consuming and causes some loss of accuracy. In this study, the urine of calves treated with SBL orally for 3 d was collected during and after medication. Samples were assayed before and after hydrolysis by two different methods, radioimmunoassay (RIA) and a newly developed biosensor immunoassay (BIA). Some samples were also analysed by GC-MS. The results clearly showed that both screening assays (RIA and BIA) found high concentrations of SBL residues throughout the study. This was especially true in the BIA method. It was also demonstrated that urine sample analysis without the need for deconjugation or clean-up could be achieved. Results obtained by GC-MS tended to be an order of magnitude lower than the corresponding screening test results. This work showed that biosensor based veterinary drug residue testing procedures can be developed which can generate results in real time without the need for time consuming sample preparation.