Steven R. H. Crooks

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.

Benzimidazole carbamate residues in milk: Detection by Surface Plasmon Resonance-biosensor, using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method for extraction

A surface plasmon resonance (SPR) biosensor screening assay was developed and validated to detect 11 benzimidazole carbamate (BZT) veterinary drug residues in milk. The polyclonal antibody used was raised in sheep against a methyl 5(6)-[(carboxypentyl)-thio]-2-benzimidazole carbamate protein conjugate. A sample preparation procedure was developed using a modified QuEChERS method. BZT residues were extracted from milk using liquid extraction/partition with a dispersive solid phase extraction clean-up step. The assay was validated in accordance with the performance criteria described in 2002/657/EC. The limit of detection of the assay was calculated from the analysis of 20 known negative milk samples to be 2.7mugkg(-1). The detection capability (CCbeta) of the assay was determined to be 5mugkg(-1) for 11 benzimidazole residues and the mean recovery of analytes was in the range 81-116%. A comparison was made between the SPR-biosensor and UPLC-MS/MS analyses of milk samples (n=26) taken from cows treated different benzimidazole products, demonstrating the SPR-biosensor assay to be fit for purpose.

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 Peoples 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 rapid immunobiosensor screening method for the detection of residues of sulphadiazine

A rapid immunoassay using an optical biosensor (BIAcoreTM) for determining the presence of sulphadiazine (SDZ) residues in pig bile was developed. SDZ was immobilised onto the surface of a dextran-coated silicon chip and a solution containing SDZ antibody, sample and buffer was injected over the chip surface. The level of antibody binding to the chip was determined after 20 s and the surface of the chip was then regenerated over a 1-min period prior to another sample injection taking place. Standard curves were constructed to allow quantification of SDZ presence in sample. Concentrations ranging from 0 to 10.64 mu g ml-1 SDZ were detected in bile samples taken from experimentally fed pigs and randomly selected pigs taken from a local slaughterhouse. These results were compared to the concentrations of SDZ detected by high-performance liquid chromatography in associated tissues. When concentrations in bile exceeded 0.6 mu g ml-1 SDZ, the corresponding edible tissue was above the maximum residue level (MRL)...

On-line detection of sulfamethazine and sulfadiazine in porcine bile using a multi-channel high-throughput SPR biosensor.

Abstract The performance of a prototype multi-channel optical biosensor in both the laboratory and on site at an abattoir was evaluated. The high-throughput surface plasmon resonance (SPR) instrument allows either simultaneous analysis of eight samples for a single analyte or multi-analyte analysis. In conjunction with an automated sample pipetting station, direct analysis of up to 650 bile samples for sulfamethazine (SMZ) and sulfadiazine (SDZ) per day was possible. Instrument performance was assessed in a laboratory based trial by comparing results of the prototype assay method with the routine Biacore 1000 procedure used in the laboratory. During the assay of 1751 samples, false positive rates were calculated as 0.86% for SMZ and 1.48% for SDZ using the prototype biosensor as compared to 0.63% (SMZ) and 0.69% (SDZ) when using the Biacore 1000 instrument. During a 2-month on-site study at an abattoir, a total of 6069 bile samples were analysed. No false negative results were recorded while extremely low false positive rates (0.13% for SMZ and 0.74% for SDZ) were found. The present study clearly demonstrates the potential of high-speed SPR biosensor technology for high-throughput veterinary drug detection.

Immunobiosensor—an alternative to enzyme immunoassay screening for residues of two sulfonamides in pigs†

A rapid immunoassay using an optical biosensor (BIAcore) for determining the presence of sulfamethazine (SMT) residues in pig bile was developed. The assay was used in a routine screening laboratory alongside a previously described biosensor method for sulfadiazine (SDZ). Sulfonamide bile concentrations, determined by enzyme immunoassay (EIA), have already been shown suitable for use in predicting the extent of sulfonamide accumulation in kidney. The ability of immunobiosensor based bile screening to predict violative tissue residues (greater than the maximum residue limit; MRL) was compared with results achieved using two conventional EIAs for two of these drug residues (SMT and SDZ). Analysis of 2081 samples for both sulphonamide residues, over an 8 month period, showed the false positive prediction rate of biosensor analysis to be 0.14% and 0.34% for SMT and SDZ, respectively, compared with false positive rates of 1.54% and 1.44% by EIA. Biosensor analysis showed no false negative predictions for either SMT or SDZ while EIA showed a false negative prediction rate of 0.14% for SMT and 0.24% for SDZ. The present study has clearly demonstrated that immunobiosensor assays can be developed for veterinary drug residue screening programmes. These methods have the potential for generating faster and more reliable results than conventional immunoassay methods.

Determination of Sulphadimidine (Sulfamethazine) Residues in Milk, Plasma, Urine and Edible Tissues by Sensitive ELISA

A highly sensitive immunoassay for sulphadimidine (SDM) is described, demonstrating the benefit of using spacer arms with different chemical compositions in the hapten immunogen and peroxidase tracer. The antiserum used in the optimized SDM assay exhibited 50% binding inhibition in buffer at approximately 0.15 microg l -1 , while the developed enzyme-linked immunosorbent assay (ELISA) exhibited approximately 100% cross-reactivity between SDM and its major metabolite N 4 -acetyl-SDM, relatively slight reaction with sulphamerazine (5.2%) and negligible reaction with 25 other related antimicrobials. The assay was evaluated using control and SDM-spiked milk, plasma, urine and edible tissue samples. The recovery of added SDM at the concentration of regulatory interest varied around 100%. The precision of the ELISA was below 5 and 15% for inter-and intra-assay variability, respectively. The highly sensitive method has been demonstrated to be an extremely effective way of overcoming problems caused by matrix int...

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.

Determination of sulphadimidine in animal feedstuffs by an enzyme‐linked immunoassay

An enzyme immunoassay technique for the determination of sulphadimidine in animal feedstuffs has been developed. The antibody showed limited cross-reactivity with other drugs, including sulphonamides, used as feed additives. Using spiked samples recoveries of 80-88% were obtained. The limit of detection of the assay was 70 ng/g.

A review of coccidiostats and the analysis of their residues in meat and other food

Coccidiostats are used in the control of protozoan infections in different food producing animals. They are most widely used as feed additives in intensively reared species such as pigs and poultry to maintain animal health and in some cases enhance feed conversion. However, a number of these drugs are used in the control of infections in beef and lamb production. Coccidiostat residues have been frequently reported in meat and eggs in a number of countries since the late 1990s. This has prompted increased research and surveillance of coccidiostat residues in food. This paper reviews the various coccidiostat agents used in animal production, including their chemical properties, mode of action and activity. Legislation concerning coccidiostats, limits for residues in food, monitoring and occurrence of residues in food is discussed. Methods for residue determination in food, including screening and physicochemical methods are discussed in depth. The paper concludes with a synopsis of the current state of coccidiostat residue analysis and future perspectives.