Screening method for the detection of residues of amphenicol antibiotics in bovine milk by optical biosensor.

An immunobiosensor assay was developed for multi-residue screening in bovine milk of the parent amphenicols, thiamphenicol and florfenicol, along with the metabolite florfenicol amine. A polyclonal antibody raised in a rabbit after immunisation with a florfenicol amine-protein conjugate was employed in the assay. Milk samples were subjected to acetonitrile extraction, reconstituted in buffer and diluted prior to biosensor analysis. Validation data obtained from the analysis of fortified samples has shown that the method has a detection capability of less than 0.25 µg kg-1 for florfenicol and less than 0.5 µg kg-1 for florfenicol amine and thiamphenicol. The cross-reactivity profile and validation data for the detection of these amphenicols is presented together with results obtained following the analysis of florfenicol incurred samples using the developed screening method along with a comparison of results obtained from the analysis of the same incurred samples using an MRM3 UPLC-MS/MS confirmatory method. Results are also presented obtained from the analysis of samples from both treated and non-treated animals which were co-housed and which show the potential for cross-contamination.

Screening method for the detection of residues of amphenicol antibiotics in bovine, ovine and porcine kidney by optical biosensor.

An immunobiosensor assay was developed for the multi-residue screening of the parent amphenicols, thiamphenicol and florfenicol, along with the metabolite florfenicol amine, in bovine, ovine and porcine kidney. A polyclonal antiserum raised in a rabbit after inoculation with a florfenicol amine-protein conjugate was employed in the assay. Sample homogenates were extracted directly into acetonitrile, reconstituted in buffer and diluted prior to biosensor analysis. Validation data obtained from the analysis of fortified samples has shown that the method has a detection capability (CCß) of less than 25µgkg-1 (1/2 MRL) for thiamphenicol in the kidney of all three species, less than 150µgkg-1(1/2 MRL) for florfenicol and florfenicol amine and less than 250µgkg-1 (1/2 MRL) for florfenicol and florfenicol amine in bovine/ovine and porcine kidney respectively. Intra-assay variation (n=10) was calculated at 4.5% and 2.6% at concentrations of 10µgkg-1 and 150µgkg-1respectively for bovine kidney while inter-assay variation (n=3) was determined to be 5.0% and 16.5% respectively at the same concentrations. The cross-reactivity profile and validation data for the detection of these amphenicols is presented together with the results obtained following the analysis of florfenicol incurred samples using the developed method.

Screening method for the detection of a range of nitrofurans in avian eyes by optical biosensor.

An immunobiosensor assay was developed for the multi-residue screening of a range of nitrofuran compounds in avian eyes. A polyclonal antibody which binds at least 5 of the major parent nitrofurans was raised in a rabbit after inoculation with a nitrofuran mimic-protein conjugate. Sample homogenates were extracted into 0.1M hydrochloric acid and subjected to clean-up by solid phase extraction and micro-centrifugation prior to biosensor analysis. Validation data obtained from the analysis of 21 fortified samples has shown that the method has a detection capability (CCß) of less than 1 ng eye(-1) for nitrofurazone (NFZ). In addition, cross-reactivity data and the analysis of a smaller number of fortified samples have shown that the method will also detect a range of other major parent nitrofurans including furazolidone (FZD), furaltadone (FTD), nitrofurantoin (NFA) and nifursol (NFS). Intra-assay variation (n=10) was calculated at 12.9% and 10.1% at concentrations of 1 ng eye(-1) and 2 ng eye(-1) NFZ respectively. Inter-assay variation (n=3) was determined to be 10.8% and 4.7% at the same NFZ concentrations respectively. The cross-reactivity profile and validation data for the detection of these nitrofurans are presented together with the results obtained following the analysis of a small number of incurred samples using the developed method.

Development of an optical biosensor based immunoassay to screen infant formula milk samples for adulteration with melamine.

The illegal adulteration of milk with melamine in 2008 in China led to adverse kidney and urinary tract effects in hundreds of thousands of children and the reported deaths of six. The milk had been deliberately adulterated to elevate the apparent protein content, and subsequently melamine was detected in many milk-related products which had been exported. This led to the banning of imports of milk and milk products from China intended for the nutritional use of children and to the implementation of analytical methods to test products containing milk products. An optical biosensor inhibition immunoassay has been developed as a rapid and robust method for the analysis of infant formula and infant liquid milk samples. A compound with a chemical structure similar to that of melamine was employed as a hapten to raise a polyclonal antibody and as the immobilized antigen on the surface of a biosensor chip. The sensitivity of the assay, given as an IC(50), was calculated to be 67.9 ng mL(-1) in buffer. The antibody did not cross-react with any of the byproducts of melamine manufacture; however, significant cross-reactivity was observed with the insecticide cyromazine of which melamine is a metabolite. When sample matrix was applied to the assay, a limit of detection of <0.5 µg mL(-1) was determined in both infant formula and infant liquid milk. The development of the immunoassay and validation data for the detection of melamine is presented together with the results obtained following the analysis of melamine-contaminated milk powder.

Improved screening method for the detection of a range of nitroimidazoles in various matrices by optical biosensor.

An immunobiosensor assay was developed for the multi-residue screening of a range of nitroimidazole compounds in various species and sample types including porcine, bovine and ovine kidney, avian liver, serum and eggs and bovine milk. A polyclonal antibody which binds at least seven of the major nitroimidazoles and their metabolites was raised in a sheep after inoculation with a metronidazole protein conjugate. Sample homogenates were extracted into acetonitrile and subjected to micro-centrifugation prior to biosensor analysis. Validation data obtained from the analysis of 20 fortified samples has shown that the method has a detection capability (CCbeta) of less than 1 microgkg(-1) (or microgL(-1)) for dimetridazole (DMZ) in all species and matrices investigated. In addition, cross-reactivity data and the analysis of a small number of fortified samples have shown that the method will also detect a range of other major parent nitroimidazoles and their metabolites including ronidazole (RNZ), ipronidazole (IPZ), metronidazole (MNZ), hydroxymetronidazole (MNZOH), hydroxydimetridazole (DMZOH) and hydroxyipronidazole (IPZOH). The cross-reactivity profile and validation data for the detection of these nitroimidazoles are presented together with the results obtained following the analysis of a small number of incurred samples using the developed method.

Effective monitoring for ractopamine residues in samples of animal origin by SPR biosensor and mass spectrometry.

Ractopamine (RCT) is a member of the beta-2-agonist (beta-agonist) family. It is licensed for use as an animal growth promoter in more than 20 countries worldwide, including the United States and Canada, but is either not licensed or prohibited by over 150 others, including those within the European Union. The issue of the use of RCT in livestock bound for human consumption has risen to prominence recently following the decision by The People's Republic of China to ban the import of pork from a number of processing plants after finding traces of RCT in shipments from the U.S.A. In order to monitor for the illegal use of such compounds within Europe, there is a requirement to have a robust and reliable testing scheme capable of the detection of low concentrations of RCT. In the present study an optical biosensor screening assay was developed. The developed assay was compared with a liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) confirmatory procedure. These methods were used to study the ability to detect RCT in pigs following treatment. Both testing procedures were capable of detecting low microgkg(-1) concentrations of the drug in urine and liver. Liver was found to be a less suitable sample matrix, with RCT residue levels being undetectable after 5 days withdrawal of the drug. Urine samples however still contained detectable RCT residues several weeks after withdrawal. The correlation (as measured by r(2)) between the biosensor and LC-MS/MS methods was 0.99 and 0.97 for urine and liver samples, respectively. It is concluded that testing regimes based on RCT analysis in liver are less likely to detect illegal administration of the drug than those based on urine analysis. Urine samples provide an excellent matrix for the detection of RCT residues for an extended period post withdrawal.

The development of a multi-nitroimidazole residue analysis assay by optical biosensor via a proof of concept project to develop and assess a prototype test kit.

An assay based on optical biosensor technology has been developed to detect a broad range of nitroimidazole drug residues and their metabolites (dimetridazole (DMZ), metronidazole (MNZ), ronidazole (RNZ), hydroxymetronidazole (HO-MNZ) and hydroxydimetridazole (HO-DMZ)) in chicken muscle. The detection limit for the procedure was determined as 0.5 ppb for DMZ and detection capabilities (CCbetas) ranged from <1 ppb for DMZ, MNZ and RNZ to <2 ppb for HO-MNZ and HO-DMZ. Intra-assay variation (n=6) was calculated as 11.6% at a concentration of 1 ppb DMZ and 4.7% at a concentration of 2 ppb DMZ. Inter-assay variation (n=3) was determined to be 14.2% at a concentration of 1 ppb DMZ and 3.5% at a concentration of 2 ppb DMZ. A prototype kit based on this assay was produced and a multinational study was undertaken to independently evaluate its performance. The resulting data showed that the kit can be implemented with little difficulty in laboratories of varying expertise and is sensitive enough to meet the standards required by international law. Feedback from this study led to the incorporation of some minor improvements to the kit. The commercial partner in the project, XenoSense Ltd., was consulted with regards to producing a commercial test kit based on the prototype assay. As feedback from the collaborative study had been positive with respect to speed, ease of use and performance of the kit, the decision to commercialise the kit was taken. In conclusion, the prototype nitroimidazole kit was shown to offer numerous advantages over existing analytical techniques.