Liguria e Valle d Aosta
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1 GR CALUX in illegal treatment detection: from targeted to untargeted screening strategy Danilo Pitardi Amsterdam, the Netherlands is under the Ministery of Health Veterinary Public Health Our activities are focused on 1. Control.. foodstuffs, feedingstuffs and live animals 2. Detection.. contaminats drug residues agents of zoonoses animal diseases. Research.. 1
2 They are widely used as therapeutic agents in veterinary practice; metabolic diseases, shock, stress and inflammatory disorders In some European Countries they are also utilized illegally as growth promoters, either alone or in association with anabolic steroids Our activity in live animals Synthetic glucocorticoids 1. Correct use as therapeutic agents; respect of withdrawal time 2. Illicit treatment of producing animals Synthetic glucocorticoids Testing activity in live animals Urine samples (above 2 ppb) Screening analysis (ELISA) (-) Confirmatory analysis (LC-MS/MS) What s the matter? Both screening and confirmatory analyses are limited to a short list of known molecules. 2
3 What s the matter? ELISA Specificity ELISA Limits of Detection Compound % Cross-Reactivity Urine Urine (IAC) Dexamethasone 100 Dexamethasone 2.5 ng/ml 0.1 ng/ml Dexamethasone 21 Acetate 162 Flumethasone 5.0 ng/ml 0.2 ng/ml Dexamethasone 21 Phospate.6 Betamethasone 5.8 ng/ml 0.25 ng/ml Flumethasone 50 Betamethasone 4 Betamethasone 21 Acetate 7 Betamethasone 21 Phosphate 2.0 Triamcionlone 1.0 Prednisolone 1.7 Cortisol <0.5 Cortisone <0.5 LC-MS/MS analysis: simultaneous detection of six glucocorticoids (dexamethasone, flumethasone, betamethasone, prednisolone, prednisone, methylprednisolone). GR-CALUX Detection based on the glucocorticoid activity of each molecule, not on the signature of each molecule Potentially it could detect every molecule with glucocorticoid activity at a very low level Unimportant effect of natural glucocorticoids 1. Blank samples (CALUX, ELISA, LC-MS/MS) Study design 2. Pure compouds (EC50, REP). Spiked samples (CALUX, ELISA, LC-MS/MS) Partners: TEST VERITAS,, BDS
4 1. Blank samples I 20 urine samples (calves) animals bred under control conditions samples collected at farm : samples and LC- MS/MS natural GCs VERITAS: ELISA BDS: CALUX Screening positive 2 ppb 1. Blank samples II urine samples (bulls) animals bred under control conditions samples collected at slaughterhouse : samples and LC- MS/MS natural GCs VERITAS: ELISA BDS: CALUX Screening positive 2 ppb VS 4
5 2. Pure compounds BDS evaluated the EC50 and REP of the most used synthetic GCs VERITAS provided the standards Mandatory detection capability 2 ppb Difference between dex BDS and dex VERITAS Probably due to different formulation (acetate, phosfate)..under investigation... Spiked samples samples VERITAS spiking BDS CALUX analysis 5
6 CONCLUSIONS 1. The physiological levels of endogenous GCs don t seem to influence CALUX measurements 2. At the slaughterhouse the increase in GCs levels could invalidate the analysis. GR CALUX showed, using pure compounds, a sensitivity lower than the mandatory detection capability (CCβ), comparable with a confirmatory test 4. Prohormones can t be detected by GR CALUX (prednisone) 5. The technique looks a promising screening tool for the detection of illicit treatments in animals but more focused experiments are needed NEXT STEP.. GR CALUX validation as screening test for GCs detection in bovine urine sample Thank you Acknowledgments Test Veritas Srl (Barbara Cini) c/o Area Science Park, Bldg. R, Loc. Padriciano Trieste (TS) Italy phone fax info@testveritas.com BioDetection Systems b.v. Science Park XH Amsterdam The Netherlands tel: fax: info@bds.nl 6
Liguria e Valle d Aosta
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1 Heparan Sulfate ELISA Kit (Cat: No. 280564-1) Technical Information: Contents General Description 2 Basic Performance 3 Reactivity: 4 1: Specificity of Heparan Sulfate: Intersection Reactivity with Various
Validation Report for the Neogen Fentanyl Kit for ELISA Screening of Whole Blood and Urine Specimens
Validation Report for the Neogen Fentanyl Kit for ELISA Screening of Whole Blood and Urine Specimens This document describes the validation of a Neogen Fentanyl kit for the semi-quantitative analysis of