Validation of ELISA test kits for detection of beta-agonist residues in meat (original) (raw)
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Validation methods for the determination of β agonists residues in feed
Veterinary drugs are widely used in modern animal husbandry. Their usage is not only for therapeutic and prophylactic purposes but also for better breeding efficiency. A part of veterinary drugs are used as growth promoters in form of specified compounds or mixtures of compounds. Therefore, among other compounds, steroids and other substances that have similar pharmacological activity are used to improve efficiency of protein conversion. As a result of enhanced protein conversion the growth of animals is faster which lead to earlier slaughter. The aim of this study was to detect levels of β – agonist residues since they are used as additives in the feed. By using ELISA method level of β – agonist was determinate. Total of 49 feed samples were screened for the presence of β–agonist as a part of national monitoring residue plan. The feed samples were collected and delivered by the authorised veterinary inspectors within period of 1 year. The validation process was carried out accordin...
2018
The use of β-agonists in livestock production is prohibited in many countries because the residues of β-agonists in food pose a potential risk to human health. The present work describes the development and validation of the LC-MS/MS method for detection of ten β-agonists in bovine urine according to Commission Decision 2002/657/EC requirements. Linearity of the method resulted with coefficient of correlation >0.990. The decision limits (CCα) ranged from 0.127 ng/mL to 0.646 ng/mL, and the detection capability (CCβ) resulted in the range 0.140 ng/mL to 0.739 ng/mL. The observed recoveries in the fortified bovine urine samples were satisfactory at every fortification level with values from 73.67% to 118.80%. The coefficient of variation (CV, %) at three fortification levels for each β-agonist was in complete agreement with the requirements from Commission Decision 2002/657/EC. The CV for intraday precision varied from 1.619% to 15.472% and the CV for interday precision varied from...
Food Analytical Methods, 2018
A robust and sensitive methodology, utilizing GC-MS, for the identification and quantitation of seven β-agonist residues (mabuterol, clenbuterol, brombuterol, mapenterol, clenpenterol, clenproperol, and clencyclohexerol), in liver and meat samples, is described. Different extraction procedures, followed by GC/MS measurement, were tested and compared in order to achieve optimum extraction conditions and eliminate matrix effects. The optimized method consisted of consecutive extractions with hydrochloric tris(hydroxymethyl)aminomethane (TRIS-HCl) and tert-butyl-methyl ether (TBME), defatting with hexane and solid-phase extraction (SPE) with C18 cartridges. Ν,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) was used as a derivatization agent and the GC-EI-MS determination was performed using a MDN-5S capillary column in single-ion monitoring (SIM) acquisition mode. The method was validated according to the Commission Decision 2002/657/EC, fulfilling all the EU criteria. Quantification was performed via internal standard calibration using deuterated analogs of the compounds. Recoveries ranged from 83 to 118% (mabuterol at concentration levels of 2.0 and 1.5 μg kg −1 , respectively) and precision, expressed as %relative standard deviation (% RSD), was in every case lower than the % RSDs obtained from Horwitz equation. The obtained decision limit (CCα) and detection capability (CCβ) values varied from 0.21 ng g −1 (clenbuterol) to 0.49 ng g −1 (clenproperol, clencyclohexerol) and from 0.60 (mapenterol) to 0.69 ng g −1 (clenpenterol), respectively.
Rapid Methods for detection of Veterinary Drug residues in Meat
Veterinary World, 2010
The use of substances having hormonal or thyreostatic action as well as b-agonists is banned in many countries. However, sometimes forbidden drugs may be added to feeds for illegal administration to farm animals for promoting increased muscle development or increased water retention and thus obtain an economical benefit. The result is a fraudulent overweight of meat but, what is worse, residues of these substances may remain in meat and may pose a real threat to the consumer either through exposure to the residues, transfer of antibiotic resistance or allergy risk. This has exerted a great concern among the meat consumers. The control of the absence of these forbidden substances in animal foods and feeds is regulated in the European Union by Directive 96/23/EC on measures to monitor certain substances and residues in live animals and animal products. Analytical methodology, including criteria for identification and confirmation, for the monitoring of compliance was also given in Decisions 93/256/EEC and 93/257/EEC. More recently, Decision 2002/657/EC provided rules for the analytical methods to be used in testing of official samples. New substances with anabolic properties are being detected year by year increasing the list of forbidden compounds to be tested. Furthermore, the extended practice consisting in the use of "cocktails" (mixtures of low amounts of several substances that exert a synergistic effect) to have a similar growth promotion, reduces the margin for an effective analytical detection. Thus, the evolution of the "black market" is making really difficult to have an effective analytical control of the residues of these substances in foods of animal origin. Control laboratories must face an increasing demand of analysis like the growing number of residues to be analysed in different types of samples, the strict guidelines for analytical methodologies according to the latest Directives, the increased costs of such new methodologies, the variety of residues to search per sample and the need to invest on powerful new instruments for identification and confirmatory purposes. Rapid and versatile screening methodologies make its control easier and reduce the number of non-compliant samples to be confirmed through tedious and costly confirmatory analytical methodologies. For instance, the multiresidue analysis can be performed better by using fast LC methods. Thus, the availability of new screening methodologies and the improvement of the existing ones will contribute to a better safety assurance of meat and other foods of animal origin.
Food Additives & Contaminants: Part A, 2008
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UPLC-ESI-MS-MS Determination of Three β2-Agonists in Pork
Chromatographia, 2010
Matrix-induced ion suppression and different polarity usually interfere with extraction, purification and drug analysis, therefore a method was developed for determination of three b 2-agonists namely, salbutamol, ractopamine and clenbuterol in pork. After enzymatic hydrolysis, a 2.0 g homogenized pork sample was extracted, with perchloric acid-methanol (9:1, v/v) to precipitate protein, and then cleaned by mixed-mode Bond Elut Plexa PCX solid phase extraction (SPE) cartridges. Finally, the evaporated extract was reconstituted to 2 mL with initial mobile phase. The analytes were determinated by ultra performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS-MS). Results indicate that the method is effective to reduce ion suppression phenomena. Recoveries of the three b 2-agonists in most cases were better than 85% with relative standard deviations between 2 and 11%. The limits of detection (LOD) were 0.07-0.12 lg kg-1. The limits of quantitation (LOQ) ranged from 0.22-0.38 lg kg-1 .
Analytica Chimica Acta, 1993
TWO rehable gas chromatograpluc-mass spectrometnc @C-MS) procedures for the unequwocal determmahon of &agomsts m urme of meat-producmg ammals are d-bed The B-agomsw drugs are extracted usmg disposable nuxed sohd-phase extration columns The unne sample (10 ml) 1s buffered (PH 6 0) and apphed to a Clean Screen DAU carmdge The analytes are eluted wth ethyl acetate contammg 3% (v/v) of concentrated (32%) ammonia solution The extracted analytes are derwa&ed to either their tnmethylsrlyl (TMS) or cychc 2_(dlmethyl&morphohne (DMS) derrvatlves and analysed by GC-MS under electron impact (EI) and positwe-ion chermcal lomzatlon (PCI) conQtrons Cychc DMS denvatwes proved to be useful for screenmg for or confimung the presence of clenbuterol analogues m the EI mode If necessary, they could also be confirmed m the PC1 mode using ammonia as reagent gas With regard to TMS denvatwes, theu use prowded a rapid and efticrent method for screening for (EI) and confirmmg (PCI) the presence of at least thirteen /3-agonists at the low ng ml-' level
Analytica Chimica Acta, 2009
A bioassay was developed for the detection of a broad range of beta-agonist compounds in animal feeds. A solubilised beta2-adrenoceptor was utilised as the binding protein in the assay. This protein was found to be highly stable when stored at 80 degrees C. The assay was developed and initially validated to determine the sensitivity and relative selectivity against a panel of commonly used beta-agonist compounds. It was also shown that when beta-agonists were present as cocktails in samples a pronounced synergistic effect could be measured. The method was further validated according to EC Decision 2002/657 and proved capable of detecting 250ng clenbuterol equivalents per gram of sample. This is well below the quantities normally associated with beta-agonist medicated feeds. The beta2-adrenoceptor used in the study only failed to bind the compound zilpaterol, raising doubts as to whether this compound is a true beta2-adrenergic drug.
2000
Data are reported here on the assessment of the potential use of an enzyme-linked immunosorbent assay (ELISA) to monitor the presence of the b-agonist, clenbuterol in 142 samples of sheep urine and 53 samples of eye tissues from the same sheep. Data obtained showed that the clenbuterol levels ranged in the urine and eye tissues from not detected to 0.272 ppb and not detected to 1.54 ppb tissue, respectively. For con®rmation, all samples that showed to be ELISA positive for clenbuterol residues were analyzed by GC±MS and were all found to be negative.