Detection of glucocorticoid bioactivity in bovine urine samples using a reporter gene assay.

The illegal use of anabolic substances in the meat producing industry is an ongoing problem due to the continual production of new synthetic compounds and/or the practice of low-level cocktail administration to avoid detection by the surveillance schemes of EU member states National Plan surveillance systems.

We present a highly sensitive reporter gene assay and sample extraction procedure based on a two step solid phase extraction and high performance liquid chromatography, developed for the detection of glucocorticoid abuse in bovine urine. The assay is capable of detecting compounds with glucocorticoid bioactivity and is extremely sensitive with an EC50 of 0.79 ng mL-1 for dexamethasone. New or unknown compounds with glucocorticoid bioactivity and low-level cocktail mixtures are detectable by this assay.

Cross-reactivity data for a range of 11ß-hydroxyglucocorticoids has been provided. This assay shows low interference from the 11-keto prohormones and other steroidal hormones. The assay may be suitable for application in other matrices such as hair. In conclusion this screening assay offers advantages over existing analytical techniques.

Rapid confirmatory method for the determination of sixteen synthetic growth promoters and bisphenol A in bovine milk using dispersive solid-phase extraction and liquid chromatography-tandem mass spectrometry

A rapid liquid chromatographic-tandem mass spectrometric (LC-MS/MS) multi-residue method for the simultaneous quantitation and identification of sixteen synthetic growth promoters and bisphenol A in bovine milk has been developed and validated. Sample preparation was straightforward, efficient and economically advantageous. Milk was extracted with acetonitrile followed by phase separation with NaCl. After centrifugation, the extract was purified by dispersive solid-phase extraction with C18 sorbent material. The compounds were analysed by reversed-phase LC-MS/MS using both positive and negative ionization and operated in multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for unambiguous confirmation. Total chromatographic run time was less than 10 min for each sample. The method was validated at a level of 1 mu g L-1. A wide variety of deuterated internal standards were used to improve method performance. The accuracy and precision of the method were satisfactory for all analytes. The confirmative quantitative liquid chromatographic tandem mass spectrometric (LC-MS/MS) method was validated according to Commission Decision 2002/657/EC. The decision limit (CC alpha) and the detection capability (CC beta) were found to be below the chosen validation level of 1 mu g L-1 for all compounds. (C) 2010 Elsevier B.V. All rights reserved.

Public Administration, Science and Risk Assessment: A Case Study of the U.K. Bovine Spongiform Encephalopathy Crisis

The bovine spongiform encephalopathy (BSE) outbreak in the United Kingdom is regarded as one of the worst public policy crises the British government has experienced during the postwar era. In material terms, it has led to the slaughter of 3.3 million cattle and estimated economic losses of £3.7 billion. In administrative terms, the crisis brought about the dissolution of the Ministry of Agriculture, Fisheries and Food. This article examines the istitutional context in which decisions about the scientific evidence on BSE were made. The authors argue that a centralized system in which government agencies control science for government is inherently vulnerable to alliances of experts and interest groups that undermine the credible assessment of public health and safety risks. Specific societal conditions may encourage risk-opportunistic behavior among policy makers that is conducive to delays and inaction until such time as the evidence of a health risk becomes overwhelming.

Bovine Serum Albumin Adsorption to Iron-Oxide Coated Sands Can Change Microsphere Deposition Mechanisms

Particulate colloids often occur together with proteins in sewage-impacted water. Using Bovine Serum Albumin (BSA) as a surrogate for protein in sewage, column experiments investigating the capacity of iron-oxide coated sands to remove latex microspheres from water revealed that microsphere attenuation mechanisms depended on antecedent BSA coverage. Dual pulse experiment (DPE) results suggested that where all BSA was adsorbed, subsequent multiple pore volume microsphere breakthrough curves reflected progressively reduced colloid deposition rates with increasing adsorbed BSA content. Modelling colloid responses suggested adsorption of 1 µg BSA generated the same response as blockage by between 7.1x108 and 2.3x109 deposited microspheres. By contrast, microsphere responses in DPEs where BSA coverage of the deposition sites approached/ reached saturation revealed the coated sand maintained a finite capacity to attenuate microspheres, even when incapable of further BSA adsorption. Subsequent microsphere breakthrough curves demonstrated the matrix’s colloid attenuation capacity progressively increased with continued microsphere deposition. Experimental findings suggested BSA adsorption on the sand surface approaching/ reaching saturation generated attractive deposition sites for colloids, which became progressively more attractive with further colloid deposition (filter ripening). Results demonstrate that adsorption of a single type of protein may either enhance or inhibit colloid mobility in saturated porous media.

Surveillance study of a number of synthetic and natural growth promoters in bovine muscle samples using liquid chromatography-tandem mass spectrometry

A simple, new method permitting the simultaneous determination and confirmation of trace residues of 24 different growth promoters and metabolites using liquid chromatography-mass spectrometry was developed and validated. The compounds were extracted from bovine tissue using acetonitrile; sodium sulphate was also added at this stage to aid with purification. The resulting mixture was then evaporated to approximately 1 ml and subsequently centrifuged at high speed and an aliquot injected onto the LC-MS/MS system. The calculated CC values ranged between 0.11 and 0.46 mu g kg-1; calculated CC were in the range 0.19-0.79 mu g kg-1. Accuracy, measurement of uncertainty, repeatability and linearity were also determined for each analyte. The analytical method was applied to a number of bovine tissue samples imported into Ireland from third countries. Levels of progesterone were found in a number of samples at concentrations ranging between 0.28 and 30.30 mu g kg-1. Levels of alpha- and beta-testosterone were also found in a number of samples at concentrations ranging between 0.22 and 8.63 mu g kg-1 and between 0.16 and 2.08 mu g kg-1 respectively.

Screening and Quantitative Confirmatory Method for the Analysis of Glucocorticoids in Bovine Milk Using Liquid Chromatography-Tandem Mass Spectrometry

An LC/MS/MS method was developed and validated for the simultaneous identification, confirmation, and quantification of 12 glucocorticoids in bovine milk. The method was validated in accordance with the criteria defined in Commission Decision 2002/657/EC. The developed method can detect and confirm the presence of dexamethasone, betamethasone, prednisolone, flumethasone, 6 alpha-methylprednisolone, fluorometholone, triamcinolone acetonide, prednisone, cortisone, hydrocortisone, clobetasol propionate, and clobetasol butyrate in bovine milk. Milk samples are extracted with acetonitrile; sodium chloride is subsequently added to aid partition of the milk and acetonitrile mixture. The acetonitrile extract is then subjected to liquid-liquid purification by the addition of hexane. The purified extract is evaporated to dryness and reconstituted in a water acetonitrile mixture, and determination is carried out by LC/MS/MS. The method permits analysis of up to 30 samples in 1 day.

Development of a rapid, multi-class method for the confirmatory analysis of anti-inflammatory drugs in bovine milk using liquid chromatography tandem mass spectrometry

A rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous identification, confirmation and quantitation of seven licensed anti-inflammatory drugs (AIDS) in bovine milk. The method was validated in accordance with the criteria defined in Commission Decision 2002/657/EC. Two classes of AIDS were investigated, corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs). The developed method is capable of detecting and confirming dexamethasone (DXM), betamethasone (BTM), prednisolone (FRED), tolfenamic acid (TV), 5-hydroxy flunixin (5-OH-FLU). meloxicam (MLX) and 4-methyl amino antipyrine (4-MAA) at their associated maximum residue limits (MRLs). These compounds represent all the corticosteroids and NSAIDs licensed for use in bovine animals producing milk for human consumption. These compounds have never been analysed before in the same method and also 4-methyl amino antipyrine has never been analysed with the other licensed NSAIDs. The method can be considered rapid as permits the analysis of up to 30 samples in one day. Milk samples are extracted with acetonitrile; sodium chloride is added to aid partition of the milk and acetonitrile mixture. The acetonitrile extract is then subjected to liquid-liquid purification by the addition of hexane. The purified extract is finally evaporated to dryness and reconstituted in a water/acetonitrile mixture and determination is carried out by LC-MS/MS. Decision limit (CC alpha) values and detection capability (CC beta) values have been established for each compound. (C) 2009 Elsevier B.V. All rights reserved.

Development and validation of dry reagent time-resolved fluoroimmuno assays for zeranol and alpha-zearalenol to assist in distinguishing zeranol abuse from Fusarium spp. toxin contamination in bovine urine

Zeranol, an oestrogenic growth promoter in food animals, is banned within the European Union (EU). However, commercially available immunoassay kits for zeranol cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the validation of a specificity enhanced, rapid dry reagent time-resolved fluoroimmunoassay (TR-FIA) for zeranol (recovery 99%, limit of detection 1.3 ng ml(-1)) demonstrating that up to 150 ng ml(-1) of Fusarium spp. toxins in urine do not lead to false-positive results. This assay will assist EU Member States to implement Council Directive 961 23\EC, which requires states to monitor for potential abuses of zeranol. A similar TR-FIA for the Fusarium spp. toxin a-zearalenol, using the same sample extract, is also described (recovery 68%, limit of detection 5.6 ng ml(-1)). Only the addition of diluted sample extract is required to perform these dry-reagent TR-FIAs, the results being available within 1 h of extract application. The EU-funded project 'Natural Zeranol' (FAIR5-CT97-3443) will use these fluoroimmunoassays to screen bovine urine in four Member States to gather data on the seasonality of Fusarium spp. toxin contamination of urine and the incidence of zeranol screening test positives.