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.

Veterinary treatment of cows with isoxsuprine for a caesarian section may temporarily lead to residues in hair of both cow and calf.

Isoxsuprine is a beta-agonist that can be used for growth promotion in cattle, but it is also used as registered veterinary medicine. To investigate if veterinary treatment of cows could lead to residues of isoxsuprine in the hair of their newborn calves, an animal experiment was performed. Four cows, treated on veterinary indication with isoxsuprine lactate (Duphaspasmin) before a caesarian section, were included in the experiment. Hair samples from cows and from their calves were analyzed. The animals were shaved every week for 16 weeks and levels of isoxsuprine were measured in hair. In the cows, the levels of isoxsuprine were highest (>15 µg/kg) just after administration of the isoxsuprine lactate. After two weeks in two cows, a sort of plateau was reached and then the levels decreased. After approximately 10-15 weeks the levels were around the CCα level of the method used (0.5 µg/kg). In calves, for the first two weeks after birth, no isoxsuprine was found above CCα level in three of the four animals. At about 20-30 days old, a maximum concentration of 4 µg/kg was found. Then the levels dropped again under the CCα level, after 60 days no levels above CCα level were found. In one animal, the levels never reached CCα level. We conclude that veterinary treatment of cows with isoxsuprine may temporarily lead to low levels of isoxsuprine in the hair of their newborn calves which can be measured for a maximum of 60 days after birth.

Detectability of testosterone esters and estradiol benzoate in bovine hair and plasma following pour-on treatment.

The abuse of synthetic esters of natural steroids such as testosterone and estradiol in cattle fattening and sports is hard to detect via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. An interesting alternative can be provided by the analysis of the administered synthetic steroids themselves, i.e., the analysis of intact steroid esters in hair by liquid chromatography tandem mass spectrometry (LC/MS/MS). However, retrospective estimation of the application date following a non-compliant finding is hindered by the complexity of the kinetics of the incorporation of steroid esters in hair. In this study, the incorporation of intact steroid esters in hair following pour-on treatment has been studied and critically compared with results from intramuscular treatment. To this end animals were pour-on treated with a hormone cocktail containing testosterone cypionate, testosterone decanoate and estradiol benzoate in different carriers. The animals were either treated using injection and pour-on application once or three times having 1 week between treatments using injection and pour-on application. Animals were slaughtered from 10-12 weeks after the last treatment. Both hair and blood plasma samples were collected and analysed by LC/MS/MS. From the results, it is concluded that after single treatment the levels of steroid esters in hair drop to CCbeta levels (5-20 microg/kg) after 5-7 weeks. When treatment is repeated two times, the CCbeta levels are reached after 9-11 weeks. Furthermore, in plasma, no steroid esters were detected; not even at the low microgramme per litre level but--in contrast with the pour-on application--after i.m. injection, significant increase of 17beta-testosterone and 17beta-estradiol were observed. These observations suggest that transport of steroid esters after pour-on application is not only performed by blood but also by alternative fluids in the animal so probably the steroid esters are already hydrolysed and epimerized before entering the blood.

Effect of sample pre-treatment on the determination of steroid esters in hair of bovine calves.

The effect of three sample pre-treatment steps, washing, cutting and grinding on the determination of steroid esters in hair is studied. The study is performed by using hair samples obtained after pour-on application of steroid esters to bovine calves. After sample pre-treatment the hair is treated with a mild reducing agent [tris(2-carboxyethyl)phosphine hydrochloride] to extract the steroid esters. After a solid-phase extraction clean-up step the extracts are analysed by using liquid chromatography combined with triple-quadrupole mass spectrometric detection. For the washing step the use of non-organic washing solvents like (warm) water and a solution of 0.1% sodium dodecyl phosphate and organic solutions containing different percentages of methanol are tested. By using the non-organic solvents and the organic solvents with a percentage of methanol <20% the recovery results are as good as the results obtained without washing the hair. Cutting the hair samples increases the analyte recoveries of incurred steroid esters by 20% compared to the non-cut hair. The analyte recoveries of cut hair samples are about 60-80% that of ground hair samples. The obtained surface expansion of hair samples by grinding proves to be necessary in order to achieve the highest possible analyte yields. Finally the use of pressurised liquid extraction (PLE) for the extraction of steroid esters from plain (no washing, cutting or grinding) hair is investigated. The first results show lower (up to 40%) extraction recoveries in comparison with the classical solvent extraction procedures. If the limit of detection requirement is met, PLE may be an alternative for extracting large numbers of hair samples due to the short sample treatment procedure involved.

Comprehensive screening and quantification of veterinary drugs in milk using UPLC-ToF-MS.

Ultra-performance liquid chromatography combined with time-of-flight mass spectrometry (UPLC-ToF-MS) has been used for screening and quantification of more than 100 veterinary drugs in milk. The veterinary drugs represent different classes including benzimidazoles, macrolides, penicillins, quinolones, sulphonamides, pyrimidines, tetracylines, nitroimidazoles, tranquillizers, ionophores, amphenicols and non-steroidal anti-inflammatory agents (NSAIDs). After protein precipitation, centrifugation and solid-phase extraction (SPE), the extracts were analysed by UPLC-ToF-MS. From the acquired full scan data the drug-specific ions were extracted for construction of the chromatograms and evaluation of the results. The analytical method was validated according to the EU guidelines (2002/657/EC) for a quantitative screening method. At the concentration level of interest (MRL level) the results for repeatability (%RSD < 20% for 86% of the compounds), reproducibility (%RSD < 40% for 96% of the compounds) and the accuracy (80-120% for 88% of the compounds) were satisfactory. Evaluation of the CCbeta values and the linearity results demonstrates that the developed method shows adequate sensitivity and linearity to provide quantitative results. Furthermore, the method is accurate enough to differentiate between suspected and negative samples or drug concentrations below or above the MRL. A set of 100 samples of raw milk were screened for residues. No suspected (positive) results were obtained except for the included blind reference sample containing sulphamethazine (88 microg/l) that tested positive for this compound. UPLC-ToF-MS combines high resolution for both LC and MS with high mass accuracy which is very powerful for the multi-compound analysis of veterinary drugs. The technique seems to be powerful enough for the analysis of not only veterinary drugs but also organic contaminants like pesticides, mycotoxins and plant toxins in one single method.

Multiresidue analysis of beta-agonists in bovine and porcine urine, feed and hair using liquid chromatography electrospray ionisation tandem mass spectrometry.

The use of beta-agonists as growth promoters in cattle breeding is forbidden in many countries for reasons of fair trade and consumer protection. In recent years the use of liquid chromatography (LC) tandem mass spectrometry (MS/MS) has been shown to be the method of choice for the control of beta-agonists. In this study an LC-MS/MS multiresidue analysis method is presented for trace analysis of 22 beta-agonists. A truly generic concept has been designed based on mixed-mode solid-phase extraction and positive electrospray ionisation LC-MS/MS operated in the multiple reaction monitoring mode. This method allows application to a wide variety of sample matrices such as urine, feed and hair, following minor modifications to the analysis procedure only. The method features fit-for-purpose sensitivity in urine as shown by CCalpha and CCbeta values of less than 0.2 and less than 0.5 microg/l respectively, for all beta-agonists studied (terbutaline and reproterol, less than 0.3 and less than 1.0 respectively). Similar but semiquantitative application to feed and hair showed CCbeta values of less than 10.0 and less than 5.0 microg/kg, respectively. A further simplification and improvement is demonstrated using Ultra Performance LC (UPLC) and fast-switching MS/MS. The successful validation of this method following the latest EU requirements and its application to real samples demonstrate that a new versatile tool has been achieved for veterinary control of beta-agonists.

Screening for estrogen residues in calf urine: comparison of a validated yeast estrogen bioassay and gas chromatography-tandem mass spectrometry.

Within the European Union, the control for residues of illegal hormones in food-producing animals is based on urine analysis for a few target analytes using gas chromatography/mass spectrometry and/or liquid chromatography-tandem mass spectrometry. Recently, we developed a robust yeast bioassay screening tool for estrogens, which was validated as a qualitative screening method in accordance with EC decision 2002/657/EC. In this study, we present long-term performance data and a comparison of urine data obtained with this bioassay, and data from an established gas chromatography-tandem mass spectrometry (GC/MS/MS) confirmatory analysis method. More than 120 calf urine samples from a controlled reference experiment were analysed using both protocols. According to the GC/MS/MS method, only the natural estrogens 17alpha-estradiol and estrone were present in the non-compliant samples. The bioassay was less sensitive than GC/MS/MS for the relatively weak estrogenic compound 17alpha-estradiol, in accordance with expectations. Assuming that application of the mass spectrometric method is considered beyond reasonable doubt, the bioassay performed very well: only 5.6% of the calf urine samples found compliant in GC/MS/MS were screened false suspect in the bioassay screening method. The bioassay results of non-compliant urine samples under routine conditions were as predicted, taking into account the relative estrogenicity of the natural estrogens 17alpha-estradiol and estrone vs. 17beta-estradiol. Only one sample was screened false negative for 17alpha-estradiol and estrone. Application of this fast and simple estrogen bioassay in routine surveillance and control can significantly reduce GC/MS/MS sample workload and allow higher percentages of animals to be screened for potential hormone abuse.

Can high-performance liquid chromatography coupled with fluorescence detection under all conditions be regarded as a sufficiently conclusive confirmatory method for B-group substances?

Commission Decision 2002/657/EC requires confirmatory analysis of B-group compounds when detected at levels above the permitted limit. In contrast to banned substances, for B-group substances, the use of mass spectrometric techniques is not obligatory and several techniques including liquid chromatography (LC)-ultraviolet light (UV) on two different LC columns and (single-column) high-performance liquid chromatography (HPLC)-fluorescence (Flu) are considered to deliver sufficient evidence for the identification of the detected substance. The analysis of sodium salicylate in animal drinking water collected at poultry farms is presented here as an example to show that even in a simple matrix such as animal drinking water, fluorescence detection in some cases may provide inadequate specificity. Of 50 samples analysed by LC-Flu, 18 tested positive for sodium salicylate. However, only in one sample was the presence of the analyte confirmed with mass spectrometric detection; the others were blank. Consequently, the LC-Flu results obtained were false-non-compliant for sodium salicylate. A second case concerning the analysis of avermectins in milk by HLPC-Flu is briefly described. For a number of samples analysed in the framework of a proficiency test, false non-compliant results for emamectin were reported due to a background interference sometimes present that practically co-eluted with the analyte. The observed retention time difference (1%) was well below the criterion (2.5%) specified in Commission Decision 2002/657/EC. Considering the impact of positive findings on individual farmers as well as on trade, product image and food safety perception by the consumer, it is concluded that also for B-group substances false-non-compliant results should be avoided whenever possible. This is especially important when the results are treated as and are expected to have the same repercussions as in the case of banned A-group substances. In these circumstances, only results obtained by mass spectrometry should be considered for confirmatory purposes.

Liquid chromatographic-tandem mass spectrometric determination of selected sulphonamides in milk.

Liquid chromatography-tandem mass spectrometry is used for the quantitative analysis of selected sulphonamides in milk. Ultrafiltration is the only sample pre-treatment technique which is required. Consequently, sample throughput is much higher than with conventional procedures, and analyte recoveries are high. As for quantification, both external standard and isotope dilution calibration yield satisfactory results. The method is fully validated for five sulphonamides with a maximum residue limit of 100 microg/kg, and which are included in the Dutch control programme on residues. Furthermore, results are presented on the applicability of the method to detect compounds at a much lower concentration level exemplified by a banned sulphonamide, dapsone, which has a provisional action limit of 5 microg/kg. The main conclusion is that the present, novel approach to the trace-level determination of veterinary drugs is simple and straightforward and has a wide-ranging application potential which is briefly exemplified by the analysis of selected benzimidazoles in milk by essentially the same procedure.

Development and validation of a method for the determination of sub-additive levels of virginiamycin in compound animal feeds by liquid chromatography.

A method for the detection of virginiamycin M1 as a marker compound of virginiamycin at sub-additive level in pig, calf, piglet, sow, poultry, cattle and laying hen feeds was developed and validated. Both UV detection at 230 nm and MS detection were applied. Virginiamycin M1 was extracted from animal feeds with ethyl acetate after wetting of the feed with water followed by clean-up on Sep-Pak silica gel and OASIS HLB cartridges. Analysis of extracts was carried out on an Inertsil ODS-2 column with acetonitrile-water-formic acid as the mobile phase and UV detection at 230 nm. The limit of quantification (LOQ) of the method was 2.7 mg kg(-1). The proposed method was validated at a target species dependent minimum required performance limit (MRPL), at 2MRPL and at 5MRPL levels in pig, calf, piglet, sow, poultry, cattle and laying hen feeds. Recoveries at target species dependent MRPL levels ranged from 38 to 67%, within-day repeatabilities from 7 to 19% and within-laboratory reproducibilities from 13 to 27%. The proposed UV method is primarily suitable for screening purposes at subadditive levels, but semi-quantitative data can also be produced. Three MS detection modes (ion-source CID, full MS and MS2) were tested as an alternative and/or extension to UV detection. The selectivity and sensitivity of both LC-MS2 and LC-MS were much better than those of UV detection at 230 nm.