Possible contamination with clenbuterol from treated veal calves to untreated pen mates.

To investigate whether clenbuterol-treated calves could contaminate untreated pen mates, three animal experiments were performed. (1) One calf of a pen of five was treated with clenbuterol by injection (Ventipulmin injection, REG NL 2532, 2.5 mL/100 kg) twice a day for 10 days. (2) In two pens, one animal was treated with clenbuterol via oral administration (Ventipulmin syrup, REG NL 2532, 4 mL/125 kg) for 4 weeks. (3) In two pens, one animal was treated with clenbuterol via the milk (Ventipulmin, REG NL 2532, 2.5 mL/100 kg body weight) twice a day for 10 days. Here, the animal was set apart during treatment, cleaned and put back into the group. Levels of clenbuterol were analysed in hair and urine with LC-MS/MS. Clenbuterol administered by injection could not be transferred from treated to untreated calves. In the second experiment, all pen mates were found positive for clenbuterol in the hair. This contamination was probably due to licking the mouth of the treated animal or saliva from the treated animal spoiling the floor. In the third experiment, no pen mates were found positive for clenbuterol in the hair. Clenbuterol was found in the urine and hair of only treated animals.

Illegal treatment of barrows with nandrolone ester: effect on growth, histology and residue levels in urine and hair.

The effect of 17ß-19-nortestosterone (17ßNT) treatment of barrows on residue levels and growth was evaluated. Five barrows were treated three times during the fattening period with 17ßNT phenylpropionate (Nandrosol, nandrolone phenylpropionate 50 mg/ml,1 mg/kg body weight). Another five barrows were untreated and five boars (untreated) were kept as positive control. Boars and treated barrows showed a 13 and 9% improvement in growth compared to untreated barrows, with mean final body weights of 121.6, 117.8 and 109.0 kg, respectively. The bulbourethral glands of the treated barrows were three times heavier than untreated barrows. The histology of the prostate and bulbourethral gland of the treated barrows was comparable to the boars, whereas the control barrows showed atrophic glands. Levels of 17ßNT ester in hair from treated barrows were high, whereas boars and untreated barrows did not show levels above LLQ. It is concluded that analysis of hair can detect illegal treatment with 17ßNT ester in barrows. The size of the bulbourethral gland can also be used for screening in the slaughterhouse.

Determination of beta-sympathomimetics in liver and urine by immunoaffinity chromatography and gas chromatography-mass-selective detection.

A specific and sensitive method for the determination of several beta-agonistic drugs in liver and urine is described. Following clean-up by immunoaffinity chromatography and two different derivatizations, gas chromatography-mass spectrometry with electron-impact ionization is performed. The immunoaffinity chromatography columns were packed with Sepharose-immobilized polyclonal antibodies raised against the beta-agonist clenbuterol. Owing to the high clean-up efficiency of the immunoaffinity column large sample volumes can be used (up to 100 ml urine or 25 gram liver). The immunoaffinity sample pretreatment is highly specific and no further sample pretreatment was necessary. Due to the combination of two different derivatizations only GC-MS with electron-impact ionization is necessary to fulfil legal requirements. The first confirmation step consists of a derivatization reaction between the hydroxyl group of the parent compound and trimethylsilane. The second confirmation method is a derivatization to a cyclic derivative with the hydroxyl group and the aliphatic nitrogen group. Limits of determination in liver as well in urine are at the 10 ng/kg or ng/l (ppt) level with acceptable signal-to-noise ratio. The method is suitable for identification and quantification of trace amounts of several similar beta-agonistic drugs either used separately or in combination and can be used also for quantification of clenbuterol in liver with regard to levels exceeding the maximum residue limit (MRL) of 1 microgram/kg (ppb).

Pour on application of growth promoters in veal calves: analytical and histological results.

To investigate the possibilities for screening and confirmation methods when the 'pour on' method of application is used for administration of growth promoters, an animal experiment was performed using a cocktail of a combination of growth promoters derived from (illegal) practice. Two cocktails were used, cocktail A consisting of stanozolol and estradiol benzoate and cocktail B consisting of stanozolol, estradiol benzoate and beclomethasone dipropionate. The intended dose per animal was 110 mg stanozolol, 25 mg estradiol and 10 mg beclomethasone. The experiment was performed on 20 male veal calves, 16 treated and 4 vehicle treated controls and 3 female veal calves, 2 treated and 1 vehicle treated control. Half of the animals were shaven prior to the application of the drugs. The cocktails were administered using two types of vehicles: vehicle A; Miglyol 840 with butylated hydroxytoluene and vehicle B; di(ethyleneglycol) monobutylether. During a 28 day treatment period, one group of animals was treated once a week, another group of animals was treated once every two weeks and slaughtered. Preliminary results showed that pour on application of anabolic steroids markedly increased growth performance of veal calves, the animals treated with cocktail A performed better than the animals treated with cocktail B. Macroscopically, the thymus was reduced in weight and size in the B animals. The bulbo-urethral glands were enlarged in all treated animals. Histologically all treated animals showed squamous metaplasia in the prostate, bulbo-urethral gland and Bartholins glands. Moreover, a changed secretion pattern was observed in both the prostate and the bulbo-urethral gland. Severe cortical atrophy was observed in the thymus and to a lesser extent the adrenals of the beclomethasone treated animals. The recently discovered 16 beta-hydroxy-metabolite of stanozolol was detected in urine, in relatively high concentrations. This is the first report of the excretion of this metabolite in urine after pour on administration showing the prospect for detection of dermal treatment. Estradiol levels were remarkably elevated (up to 200 micrograms l-1) exceeding the endogenous levels (< 1 microgram l-1).

Identification of an unknown beta-agonist in feed by liquid chromatography/bioassay/quadrupole time-of-flight tandem mass spectrometry with accurate mass measurement.

A new approach to the search for residues of unknown growth promoting agents such as anabolic steroids and beta-agonists in feed is presented. Following primary extraction and clean-up, samples are separated using gradient liquid chromatography (LC). The effluent is split towards two identical 96-well fraction collectors and an optional electrospray quadrupole time-of-flight mass spectrometry (QTOFMS) system for accurate mass measurement. One 96-well plate is used for a bioassay (enzyme-immuno assay, receptor assay) and will detect the bioactivity and position of the relevant peak in the chromatogram. The positive well in the second 96-well plate is used for identification by LC/QTOFMS/MS. The value of this LC/bioassay/QTOFMS/MS methodology is highlighted by the finding and structure elucidation of a new beta-agonist in a feed extract.

Multi-laboratory study of the analysis and kinetics of stanozolol and its metabolites in treated calves.

The European Union banned the use of anabolic steroids for cattle fattening in 1988. Analytical techniques able to detect trace amounts of the parent drugs and their metabolites are mandatory for the control of abuse. Stanozolol (Stan) is an anabolic steroid that is often found in injection sites and cocktails. However, it has never been detected in tissues (kidney fat, meat) or excreta (urine, faeces) taken during regulatory inspection. The difference between the structure of Stan and the other steroids (a pyrazole ring fused to the androstane ring system) is probably the cause of this phenomenon. In the multi-laboratory study described here, veal calves were treated with intramuscular doses of Stan. In the excreta of these calves the presence, absence and/or concentration of Stan and of its major metabolites 16 beta-hydroxystanozolol and 3'-hydroxystanozolol were determined. For the determination of these analytes the different laboratories used different extraction and clean-up procedures and also evaluated different analytical techniques such as GC-MS (negative chemical ionization) and LC-MS-MS. The aim of this investigation was to explore which analyte should be validated for veterinary inspection purposes.