Kristina Vanoosthuyze

Determination of Dexamethasone in Urine and Faeces of Treated Cattle with Negative Chemical Ionization - Mass Spectrometry.

For several years, the misuse of dexamethasone and its esters in livestock production has been clearly demonstrated. The first part of the present study deals with the elaboration of a sensitive and specific method for the determination of residues of dexamethasone in excreta at the ppb level. Sample preparation for urine and faeces, including high-performance liquid chromatography (HPLC) fractionation, was carried out. The detection was based on established methodology employing negative chemical ionization-mass spectrometry (NCI-MS) after oxidation of the dexamethasone. In comparison with previous literature, the yield of oxidized dexamethasone was substantially improved and the oxidation procedure was made more simple and robust. In the second part of the study, the relationship between the dose of dexamethasone administered and the levels of the drug in excreta was investigated using this method, as was the ratio between drug levels in urine and faeces. Treatment was carried out for 7 d with an oral dose of 50 mg d-1, the maximum levels found in urine and faeces were 980 and 744 ppb, respectively. While the elimination via faeces responded much slower at the start and the end of treatment, the final part of both excretion profiles were very similar and a level of 1 ppb was reached in both matrices 9 d after the end of treatment. Gas chromatography-mass spectrometry (GC-MS) results obtained for the urine samples were compared with those obtained with direct enzyme immunoassay.

Use of metal chelate affinity chromatography and membrane-based ion-exchange as clean-up procedure for trace residue analysis of tetracyclines in animal tissues and egg

A new and efficient procedure for the clean-up of tetracycline residues in animal tissues and egg prior to reversed-phase high-performance liquid chromatography is described. The principal steps involve homogenization of the tissues in sodium succinate buffer and methanol, followed by centrifugation and clean-up with metal chelate affinity chromatography (MCAC). After further concentration on an Empore extraction membrane with cation-exchange properties, the sample is analysed by HPLC with fluorescence detection. The method was tested on porcine kidney and muscle, bovine liver and whole chickens egg. The recoveries were determined from spiked tissues for oxytetracycline, tetracycline, chlortetracycline and doxycycline and ranged from 40 to 70%, with repeatabilities below 10% R.S.D.. The analytical responses were linear in the range up to at least 1000 ng/g. The detection limits of the method were estimated at 0.42 ng/g of oxytetracycline, 0.49 ng/g of tetracycline, 0.66 ng/g of chlortetracycline and 1.38 ng/g of doxycycline in porcine muscle, using signal-to-noise ratios of 4:1. Similar detection limits were estimated for kidney, liver and egg. The measured limits of quantification were 2 ng/g for oxytetracycline, 3 ng/g for tetracycline, 4 ng/g for chlortetracycline and 5 ng/g for doxycycline in porcine kidney. The advantage of this method over existing methods is its increased limit of detection.

SURVEY OF THE HORMONES USED IN CATTLE FATTENING BASED ON THE ANALYSIS OF BELGIAN INJECTION SITES.

Although the illegal use of orally administered compounds in cattle fattening has gained popularity, injection sites are still frequently found during control experiments on the carcasses in the slaughterhouses. The high concentrations of hormones in injection sites enable screening for the presence of 39 different hormones by a simple extraction followed by a fast and simple high-performance thin-layer chromatography analysis. Analysis of injection-site tissue is particularly successful for determining the hormones that are illegally injected. This data can not be obtained by analysis of other biological matrices like faeces, kidney fat or urine, owing to metabolization and selective excretion and/or deposition of these compounds. Since 1989, over 2000 injection sites have been analysed in our laboratory, which yielded a good survey of the hormones that were illegally injected. Over this period, the natural hormones estradiol and testosterone (mostly present as their esters) have obviously been used extensively. It is clear that since 1990 clostebol acetate has remained the most abused exogenous hormone. Additionally, some distinct trends were noticed, e.g., a tendency towards a highly decreased use of nandrolone, an increased use of progesterone and an increased occurrence of certain androgens like stanozolol and fluoxymesterone.

DEVELOPMENT OF A HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHIC METHOD FOR THE MULTI-SCREENING ANALYSIS OF CORTICOSTEROIDS.

Abstract In order to screen injection sites for the presence of corticosteroids, a high-performance thin-layer chromatographic (HPTLC) system was developed for the determination of 26 corticosteroid standards. Different mobile phases were tested and chromatography with chloroform-methanol was thoroughly evaluated. The development on preloaded Kieselgel 60 HPTLC plates with chloroform-methanol (92 + 8, v/v) yielded the best separation. After examining different detection methods, a resorcylaldehyde spray was selected, yielding the best colour differentiation and fluorescence at 366 nm for several components.

Application of high-performance thin-layer chromatography and gas chromatography-mass spectrometry to the detection of new anabolic steroids used as growth promoters in cattle fattening

Abstract The misuse of natural and synthetic hormones as growth promotors in cattle fattening, although forbidden within the European Community, is well known. Frequently these hormones are injected into the animal as highly concentrated mixtures (the so-called “hormone cocktails”), which usually stay locally at the site of injection from where they are distributed by a slow diffusion process. The analysis of these injection sites by an HPTLC method following a simple and unselective extraction yields a good picture of the compounds which are misused. Although almost 40 hormone reference standards are run with the sample, unknown spots regularly appear on the HPTLC plates, demonstrating that attempts are continuously made to bypass the laboratory controls by introducing new products on the black market. By continuously gathering HPTLC data for a broad range of hormones which were not yet known to be used in cattle fattening, it was possible to elucidate quickly the identities of two new spots that appeared on the chromatogram in the period between the end of 1992 and early 1993. These new compounds that were found in injection sites were the gestagens delmadinone acetate and algestone acetophenide. Their identities were confirmed by GC-MS analysis.

Screening of calf urine for 19-nortestosterone : matrix effect in some immunoassays

The performances of three enzyme linked immunosorbent assays (ELISAs) and one radioimmunoassay (RIA) in 17 alpha-19-nortestosterone (17 alpha-19-NT) analysis of bovine urine have been evaluated. Sample preparation was performed by enzymic deconjugation and solid-phase extraction. The main object of the study was the evaluation of the matrix effect, which is generally great in 19-NT immunoassays. Fifty-seven bovine urine samples have been analysed by immunoassay and by gas chromatography-mass spectrometry (GC-MS). The detection limit of GC-MS analysis was 1 microgram l-1. The mean sample blank values in urine analysis were close to 1.5 micrograms l-1 for one ELISA, and 0.4-0.8 microgram l-1 for the other assays (n = 55, samples coming from several breeds in different geographic areas). The detection limits, calculated in compliance to the EEC criteria, were 2.67, 1.97, 2.05 and 5.0 micrograms l-1 for RIA, ELISAs 1, 2 and 3, respectively. The percentage of false positive results adopting this criteria were: 1.8, 3.6, 1.8 and 3.6%. The probability of the occurrence of false negative results was evaluated by adding 2 micrograms l-1 of 19-NT to a negative sample, was high in each assay. Two samples from experimentally treated animals gave positive results both in GC-MS and in immunoassays. The results obtained confirm that by employing immunoassay and solid-phase extraction as sample clean-up, it is not possible to apply the action level (2 ppb) suggested by the European Union (EU).

Development of a competitive enzyme immunoassay for 17α-19-nortestosterone

Because 17 beta-19-nortestosterone and its esters are frequently used anabolic steroids in cattle in Europe, there is a need for an assay that can also detect certain metabolites. The enzyme immunoassay described here involves the detection and quantitation of the major metabolite 17 alpha-19-nortestosterone in urine. The assay is based on the coating of an antibody raised in a rabbit against 17 alpha-19-nortestosterone-3-carboxy-methyloxime-bovine serum albumin (17 alpha-19-NT-3-CMO-BSA), the competitive incubation of 17 alpha-19-NT and the 17 alpha-19-nortestosterone-3-CMO-horseradish peroxidase label, followed by the detection of the blue colour developed by the action of the enzyme on tetramethylbenzidine. The 3-CMO conjugate of 17 alpha-19-nortestosterone was used to produce an antibody with selective affinity for the 17 alpha-epimer. For the optimization of the assay, different coatings and incubation conditions were tested. The standard curve ranged between 0.98 and 4000 pg per well, with a B/B0 50% of +/- 65 pg per well. Colour was measured with a microtitre plate reader. The method was validated by means of certified blank and spiked cattle urine samples.

Use of an immunomagnetic separation‐ELISA technique for the fast detection of growth promoters in cattle urine

The detection of illegal growth promoters in cattle urine by conventional immunoassays, such as radioimmunoassay (RIA) and enzyme‐linked immunosorbent assay (ELISA) has been extensively described. However, improved speed and simplicity, with the development of a fast and simple on‐site test as the ultimate goal, remains an interesting immunochem‐ical challenge. This paper describes two approaches to this problem. To avoid time‐consuming sample preparations, attempts were made to omit the extraction and hydrolysis step. Secondly, instead of the classical microtiter plate as the solid phase, magnetizable beads, which provided a solid phase that was dispersed throughout the sample, were used. In this design, the diffusion limitations of the free immunoreagents to the solid‐phase‐bound immunoreagent (which slow down the kinetics of the antibody‐antigen reaction) were circumvented. This principle was applied to the detection of the β‐agonist clenbuterol and the anabolic steroid nortestosterone in cattle urine....