Deliverables Confirmatory and identification method based on liquid chromatography coupled to mass spectrometry In this work analytical methods for the determination and identification of residues of 11 glucocorticosteroids have been developed: dexamethasone (DEX), betamethasone (BET), beclomethasone (BCL), prednisolone (PLN), prednisone, methylprednisolone (MPLN), flumethasone (FLU) and triamcinolone (TRI), triamcinolone acetonide, fludrocortisone, cortisone. Each step of these methods has been optimised in different biological matrixes, by means of comparison studies modifying the different critical parameters. Detection, confirmation and identification methods are based on liquid-chromatography with tandem-mass spectrometry (LC-MS/MS). Extraction and sample cleanup methods were developed for urine, liver, kidney and muscle samples. In order to achieve the highest concentrations of unconjugated compounds as possible, a 4 h (liver, kidney) or overnight incubation of the samples was necessary with Helix pomatia juice. Further extraction contained a liquid extraction followed by a solid phase extraction by either C18 SPE cartridges or OASIS columns. The extraction of hair samples is still under investigation. As for the chromatographic separation, it was important that epimers beta- and dexamethasone were separate adequately by using an appropriate HPLC column. More than 25 different combinations of columns and mobile phases have been studied in an HPLC-Diode array system. Best results for the separation of beta- and dexamethasone were obtained using a Hypercarb column, a column designed specifically for the separation of structurally similar compounds. The Synergi column also gave good results. As for the detection based on tandem mass spectrometry, electrospray (ESI) was used as ionisation mode. Postive ESI gave good gave good results for some glucocorticoids, but not for all. The negative ion mode however gave much more specific fragmentation, more specifically, the loss of formaldehyde, which good be found for all analysed glucocorticoids. The complete LC-MS/MS methods for all matrices(liver, kideny, muscle and urine) were validated according to European criteria. Detection capabilities (CCb)were below 0.86 µg/kg for all matrices or for compounds with an established MRL, the CCbs were not higher than 0.62 µg/kg above these MRLs. Repeatabilities and trueness were very satisfactory. A ringtest was organised amongst several European Laboratories and results were evaluated. From this ringtest it could be concluded that the developed LC-MS/MS methods were very reliable and very reproducible. The developed methods have been applied in the Spanish National Monitoring Plan for Veterinary Drug Residues and will also be applied in the routine analyses of the LAboratory of Food analysis of the Ghent University. A reporter gene-based bioassay to replace animal testing for measuring the pharmacological potency of glucocorticoids and their metabolites or derivatives Synthetic glucocorticoids belong to the most frequently administered drugs in livestock production. These synthetic hormones are employed for therapeutic purposes against inflammatory reactions, disorders of the musculoskeletal system, bovine ketosis and many other diseases of farm animals. A widespread illegal use of synthetic glucocorticoids to improve feed intake and weight gain has also been observed. To enforce the residue limits imposed on glucocorticoid drugs and preclude their illicit administration as growth promoters, it is necessary to establish high throughput analytical methods that can be applied to the screening of animal tissues. Here, we developed a luciferase reporter assay that detects a wide range of residues or contaminants with glucocorticoid activity. This new screening assay is performed by transfection of human cells with two reporter constructs followed by the measurement of two distinct luminescence signals, one of which serves as internal control to correct for unspecific matrix effects. The limit of detection in the assay medium ranges from 0.2nM for flumethasone to 3nM for methylprednisolone, while the maximal response reaches a 20- to 30-fold induction of luciferase activity. In combination with an appropriate sample clean-up method, this new assay has been successfully applied to the analysis of liver samples from calves treated with a single therapeutic injection of either dexamethasone or flumethasone. Thus, the dual reporter assay provides a sensitive, specific, highly responsive and robust screening tool to detect unwanted glucocorticoid activities in biological samples without knowledge of the precise chemical entity of the parent compounds or their metabolites. The methodology has been presented to representatives of the National Reference Laboratories at a practical workshop. The method is being presented at various International Congresses. Part of the methodology has already been published (van den Hauwe et al., J. Agric. Food Chem., Vol. 51, pp. 326-330, 2003). A second manuscript focussed on the reporter assay will be submitted to an International refereed Journal. All vectors and cell lines will be made available to interested laboratories. The glucocorticoid reporter assay provides an alternative screening tool for residue analysis and, therefore, the initial objectives were met. Production of antibodies and incurred radiolabelled and unlabelled samples A number of highly sensitive and in many cases highly specific antibodies have been successfully produced to 5 of the glucocorticoids. Although no single antibody can be regarded as "generic" it was considered unlikely at the commencement of the project that a single antibody with significant cross reactivity for all compounds could be raised. Cross reactivity profiles of the antibodies raised show that 2 antibodies could be used during final assay development to allow detection of all glucocorticoids included in the project. Within the course of the project several calves were treated with synthetic glucocorticoids and slaughtered. Their tissues and fluids were collected and used for analysis. In a first study, nine calves were divided randomly into 3 equal groups. Prior to treatment blood and urine samples were collected for nine days. Group A calves received dexamethasone, group B triamcinolone and group C methyl prednisolone. The three calves within each group received a single injection of glucocorticoid. Prior to slaughter blood and urine samples were taken daily. At slaughter plasma, serum, urine, bile, injection site, fat, kidney, liver, muscle and skin were collected. All samples were immediately frozen and stored at 20°C prior to transport to the project partners. In a second study, four calves received four separate glucocorticoid treatments. Each calf received an intramuscular injection containing the relevant drug at a concentration of 10mg/50kg bodyweight. Calf 1 received two injections at 10mg/kg; one for each of the two drugs. Blood and urine samples were taken for three consecutive days prior to treatment and for two days after treatment. At slaughter plasma, serum, urine, bile, injection site, fat, kidney, liver, muscle and skin were collected. All samples were immediately frozen and stored at 20°C prior to transport to the project partners. Incurred samples were prepared for use in both the screening and confirmatory ring tests. Six bovines were administered glucocorticoids then allowed a period of drug withdrawal prior to slaughter. Urine was collected from the experimental animals for 10 days prior to administration of the corticosteriods and throughout the withdrawal period. At slaughter liver, kidney and urine samples were removed and stored frozen until required. At the end of the project a workshop was organised to disseminate the results achieved during the project to European national reference laboratories. At the workshop the results achieved were presented and practical demonstrations of the reporter gene assay and proteomics were given. Website for internal communication between the project partners and external website for dissemination of results A public Internet site was used as a major means to disseminate project information to interested parties outside the project. A specific domain name (http://www.glucocorticoid.org) designated for the project site was registered. The site was administered by a webmaster from one of the project partners. In order to facilitate the accessibility to the web site, relevant key words were submitted and the site added to the following search engines: - Alta Vista at http://www.altavista.com - Google at http://www.google.com - Yahoo at http://www.yahoo.com Use of the site was also promoted through direct contacts with potential users, e.g. via distribution of a printed information leaflet. During a 13-day period the project site had about 300-400 visits from people interested in the content of the project, which makes an average of about 25 visits per day. The Webmaster of the site has also obtained requests about papers listed in the publication list at the website, and chemicals produced in the project, which shows that the site has attracted the attention of interested parties and worked as an intermediate link to the scientists working in the project. A printed leaflet about the project was produced and distributed to National Reference Laboratories and other key persons at the Fourth International Symposium of Hormone and Veterinary Drug Residue Analysis in Antwerp, Belgium, June 4-7 2002. One of the aims is to direct readers toward the public website of the project. Dissemination and coordination within the project was facilitated by the use of a restricted access conference web site named The GlucBoard. Proteome screening for identification of markers of glucocorticoid exposure The goal of workpackage 4 was to determine proteins in blood or tissue that are specifically related, either induced or suppressed, to glucocorticoid administration to cattle. For this identified biomarker or set of biomarkers, one or more specific immunoassays were to be developed, validated on samples of treated animals and ringtested by several laboratories. An innovative approach was developed in which the analysis was not directly targeted towards the minute amounts of glucocorticoids in body fluids, excreta or tissues, but towards the biological effects of glucocorticoids in the body. This enables detection even without knowing which glucocorticoid is administered. The analysis of the biological effect at the level of the gene expression was initially planned by measurements of proteins (proteomics or proteome analysis). In a later phase the expression of genes by measurements of messenger RNA (mRNA) was also added to the workpackage. The intended measurements of liver and blood samples with proteomics were not possible. In stead, a U937 macrophage cell system and a L6 myotubes system were used to detect proteins and genes that could be regarded as specific biomarkers. Several proteins and genes were identified as potentially interesting, some of which were confirmed by mass spectrometry. Within the timeframe available it was not possible to further investigate the selectivity of the biomarkers and the presence in body fluids or tissues. As a consequence, it was not possible to develop one or more immunoassays for the direct detection of the biomarkers in for example blood. Hence, the validation and the ringtest could not be organised. Nevertheless, the technologies developed have greatly improved the possibilities for the testing the biological effects of pharmacologically active substances in well defined cell systems, the analysis of proteins with proteomics and mass spectrometric techniques, the analysis of genes (mRNA) with DNA arrays (transcriptomics) and multivariate statistics for advanced data evaluation. This generally applicable panel of methods can and will be used for new research projects. Screening by chemiluminescence immunoassay Synthetic corticosteroids, are widely used in live-stock breeding. After application of such corticosteroids a delay time of 48 hours for milk and 72 hours for meat is prescribed. Corticosteroids are found in cattle feed and are suspected to be used as growth-promotors. In all European Community countries however, the use of growth promoters has been banned since 1986. During this project we have developed an immunoassay for a number of corticosteroids. This CORTICOSTEROID-EIA is a competitive enzyme immunoassay for the screening of urine samples on the presence of corticosteroids like dexamethasone, betamethasone, flumethasone and triamcinolone. This EIA system uses antibodies raised against protein conjugated dexamethasone. With this EIA-kit 96 analyses can be performed. Samples and standards are measured in duplicate, which means that in total 40 samples can be analysed. The EIA kit contains all the reagents, including standards to perform the test. Chemicals for the SPE and preparation of the urine samples are not included. This test makes no distinction between the different corticosteroids and can therefore only be used as a screening method. For the confirmation of the presence and the identification of the specific steroid, more specific analytical methods (HPLC or GC-MS) are recommended. PRINCIPLE OF THE DEXAMETHASONE EIA: The microtiter plate based EIA kit consists of 12 strips, each 8 wells, precoated with sheep antibodies to rabbit IgG. In one incubation step, specific antibodies (rabbit anti-dexamethasone), enzyme labelled dexamethasone (enzyme conjugate) and dexamethasone standards or samples are added to the precoated wells. The specific antibodies are bound by the immobilised anti-rabbit antibodies and at the same time dexamethasone (in the standard solution or in the sample) and enzyme labelled dexamethasone compete for the specific antibody binding sites (competitive enzyme immunoassay). After an incubation time of one hour at 37C, the non-bound (enzyme labelled) reagents are removed in a washing step. The amount of bound enzyme conjugate is visualized by the addition of substrate chromogen (tetramethylbenzidine, TMB). Bound enzyme transforms in 30 minutes the chromogen substrate into a coloured product. The substrate reaction is stopped by the addition of sulphuric acid. The colour intensity is measured photometrical at 450 nm and is inversely proportional to the steroid concentration in the standard solution or the sample. Total time to perform the assay for 25 samples, including sample preparation is one day. SPECIFICITY AND SENSITIVITY: The dexamethasone EIA utilizes antibodies raised in rabbits against protein conjugated dexamethasone. The calibration curve is linear between 0.125 to 2 ng/ml of dexamethasone. Cross-reactions: - Flumethasone 179%; - Dexamethasone 100%; - Betamethasone 55%; - Triamcinolone 49%; - Methylprednisolone 8%; Cross-reactivities were determined in the competitive EIA and calculated at 50% inhibition. A sample preparation method with glucuronidase treatment and solid phase extraction (SPE)to minimise the matrix effect of urine samples is included. A sample preparation method was also developed for the analysis of corticosteroids in meat using an organic extracion and SPE. This has been validated in LCMS-MS but is also applicable in the EIA. The EIA can be implemented in a slaughtering house environment using urine samples or pre-urines obtained from the slaughtered animals. A simple laboratory instrumentation is necessary to perform the sample preparations and the EIA which contains easy and ready to use reagents. Some laboratory skills are however requested. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
