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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 (%RSD20% for 86% of the compounds), reproducibility (%RSD40% 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.

Recently doubts have arisen on the usefulness of semicarbazide as marker residue for the illegal use of the antibiotic nitrofurazone (NFZ) in aquaculture and poultry production. Most notably azodicarbonamide (ADC) has been implicated as an alternative source of semicarbazide. ADC is used in some countries as a dough conditioner at concentrations up to 45 mg kg(-1). The use of ADC-treated flour or dough in coated or breaded food products may generate false non-compliant results in the analytical method for nitrofurazone metabolites, which is currently in use. During the dough preparation process ADC is largely reduced to biurea, which can be considered as an appropriate marker residue of ADC. Thus far no methods have been published for the determination of biurea in food commodities. Due to its polar nature it is very difficult to generate sufficient retention on conventional C18 HPLC columns. With a TSK amide HILIC type column good retention was obtained. A straightforward extraction-dilution protocol was developed. Using a mixture of dimethyl formamide and water biurea was nearly quantitatively extracted from a variety of fresh, coated and processed products. Mass spectrometric detection was performed with positive electrospray ionisation. The sensitivity and selectivity of the mass spectrometer for biurea was very good, allowing detection at concentrations as low as 10 microg kg(-1). However, in some extracts severe ion suppression effects was observed. To overcome the implications of ion suppression on the quantitative performance of the method an isotopically-labelled biurea internal standard was synthesized and incorporated in the method. The method developed can be used effectively in nitrofurazone analysis to eliminate the risk of false non-compliant results due to the presence of azodicarbonamide-treated components in the food product.

Toltrazuril (TZ), a triazinetrione derivative, and halofuginone (HFG), a quinazolinone derivative, have been licensed for the prevention and treatment of coccidiosis in broilers and turkeys, but have been excluded for use in laying hens. Little is known regarding the deposition of residues of toltrazuril and halofuginone in eggs and their rate of depletion. In this study, laying hens were treated with therapeutic doses of TZ (78 mg l −1 in the drinking water for 2 days, repeated after a 5-day interval for another 2 days) and HFG (3 mg kg −1 in the feed for 14 days). Eggs were collected before, during and after treatment. Residue concentrations of toltrazuril and its metabolite ponazuril (PZ) and halofuginone were determined in whole egg, as well as in the yolk and albumen. TZ and PZ residues were monitored daily in whole egg until 19 days post-treatment. PZ was found the predominant residue formed. Toltrazuril concentrations in whole egg reached a maximum during treatment of 1500 μg kg −1 , while ponazuril concentrations increased to 11,000 μg kg −1 . At the end of the post-treatment period levels for TZ in whole egg had dropped below the limit of detection (LoD) of 30 μg kg −1 but PZ was still present at 1600 μg kg −1 (LoD: 10 μg kg −1 ). Residues of TZ and PZ were mainly distributed in the egg yolk. HFG residues were monitored until 14 days post-treatment. Halofuginone was detected in egg up to a concentration of 450 μg kg −1 during the medication period and declined fairly rapidly after the end of administration. After 12 days withdrawal, residue levels reached the limit of detection of 2 μg kg −1 . Residue concentrations of HFG in yolk were approximately twice that in albumen.

Nifursol has recently been prohibited for use as a feed additive. Considering the similarity in structure between nifursol and the other nitrofurans, an analogous metabolism could be expected. To study the formation of tissue-bound residues in poultry, broilers were treated orally with nifursol during a period of 7 consecutive days, via medicated feed at a dosage of 50 mg/kg feed. Muscle, kidney, liver, bile and plasma samples were collected at the day of cessation of medication (day 0) and at days 3, 7, 14 and 21 after the end of medication. Samples were analysed for nifursol and the acid-hydrolysable side-chain of nifursol (DNSH; 3,5-dinitro salicylhydrazide). Samples were also analysed for the ratio between free (solvent-extractable) metabolites and tissue-bound (non-extractable) metabolites. The results obtained clearly indicate the formation of tissue-bound residues in poultry. Concentrations of non-extractable residue at zero withdrawal time averaged to 900 μg/kg in liver tissue, 2000 μg/kg in kidney tissue, 225 μg/kg in muscle tissue, 1000 μg/kg in bile and 1000 μg/kg in plasma. Taking into account an LoD of 1 μg/kg, non-extractable residues of DNSH can be detected for at least 3 weeks after administration in liver, kidney, bile and plasma and for up to 2 weeks in muscle tissue. The amounts of extractable residues were relatively low, in many instances less than 10% of the total amount of residue. In general terms the depletion data obtained show a similar behaviour of nifursol in broilers as previously found for furazolidone and furaltadone in broilers.

Depletion of the nitrofuran antibiotics furazolidone, furaltadone, nitrofurantoin and nitrofurazone and their tissue-bound metabolites AOZ, AMOZ, AHD and SEM from pig muscle, liver and kidney tissues is described. Groups of pigs were given feed medicated with one of the nitrofuran drugs at a therapeutic concentration (400?mg?kg(-1)) for ten days. Animals were slaughtered at intervals and tissue samples collected for analysis for six weeks following withdrawal of medicated feed. These samples were analysed both for parent nitrofurans (using LC-MS/MS and HPLC-UV), and for tissue-bound metabolites (using LC-MS/MS). The parent drugs were detectable only sporadically and only in pigs subjected to no withdrawal period whatsoever. This confirms the instability of the four major nitrofuran antibiotics in edible tissues. In contrast, the metabolites accumulated to high concentrations in tissues (ppm levels) and had depletion half lives of between 5.5 and 15.5 days. The metabolites of all four drugs were still readily detectable in tissues six weeks after cessation of treatment. This emphasizes the benefits of monitoring for the stable metabolites of the nitrofurans.

Use of nitrofuran drugs in food-producing animals has been prohibited within the EU because they may represent a public health risk. Monitoring compliance with the ban has focused on the detection of protein-bound nitrofuran metabolites which, in contrast to the parent compounds, are stable and persist in animal tissues. As part of the "FoodBRAND" project, an extensive survey of pork was undertaken across 15 European countries. Samples (n = 1500) purchased at retail outlets were analysed for the nitrofuran metabolites AOZ, AMOZ, AHD and SEM using LC-MS/MS determination of nitrobenzaldehyde derivatives. Limits of quantification for the method were 0.1 g/kg (AOZ, AMOZ), 0.2 g/kg (SEM) and 0.5 g/kg (AHD). Of the 1500 samples tested, measurable residues of nitrofuran metabolites were confirmed in 12 samples (0.8% incidence overall) of which 10 samples were purchased in Portugal (AOZ, 0.3 g/kg; AMOZ, 0.2?0.6 g/kg) and one sample each in Italy (AMOZ, 1.0 g/kg) and Greece (AOZ, 3.0 g/kg)

The origin, i.e. natural occurrence or illegal treatment, of findings of 17alpha-boldenone (alpha-Bol) and 17beta-boldenone (beta-Bol) in urine and faeces of cattle is under debate within the European Union. A liquid chromatographic positive ion electrospray tandem mass spectrometric method is presented for the confirmatory analysis of 17beta-boldenone, 17alpha-boldenone and an important metabolite/precursor androsta-1,4-diene-3,17-dione (ADD), using deuterium-labelled 17beta-boldenone (beta-Bol-d3) as internal standard. Detailed sample preparation procedures were developed for a variety of sample matrices such as bovine urine, faeces, feed and skin swab samples. The method was validated as a quantitative confirmatory method according to the latest EU guidelines and shows good precision, linearity and accuracy data, and CCalpha and CCbeta values of 0.1-0.3 and 0.4-1.0 ng/ml, respectively. Currently, the method has been successfully applied to suspect urine samples for more than a year, and occasionally to faeces, feed and swab samples as well. Results obtained from untreated and treated animals are given and their impact on the debate about the origin of residues of 17beta-boldenone is critically discussed. Finally, preliminary data about the degree of conjugation of boldenone residues are presented and a simple procedure for discrimination between residues from abuse versus natural origin is proposed.

Use of nitrofuran drugs in food-producing animals has been prohibited within the EU because they may represent a public health risk. Monitoring compliance with the ban has focused on the detection of protein-bound nitrofuran metabolites which, in contrast to the parent compounds, are stable and persist in animal tissues. This paper describes a solid phase extraction clean-up procedure for the protein-bound metabolite of furazolidone, 3-amino-2-oxazolidone (AOZ), allowing detection of the derivatised metabolite, NPAOZ, at residue level. Validation studies for the method on porcine liver samples fortified at 5 and 25 ng g −1 show mean recovery of 84±19.2 and 90±16.3%, respectively. Comparison between liquid chromatography with UV or tandem mass spectrometric detection shows agreement within 10% for incurred porcine liver samples.

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.

This paper reviews current approaches to validation of analytical chemical methods. It identifies some shortcomings of existing validation schemes such as insufficient coverage of variability in space or time, and mismatches between validation criteria and intended use of the method, e.g. the use in regulatory control. A general statistical modelling approach for combining different aspects of validation is recommended, and illustrated with an example. This type of modelling, based on components of variation, is advocated as the basis for the development of new statistically underpinned validation schemes which integrate current validation and quality assurance activities.

GC-MS data on veterinary drug residues in bovine urine are used for controlling the illegal practice of fattening cattle. According to current detection criteria, peak patterns of preferably four ions should agree within 10 or 20% from a corresponding standard pattern. These criteria are rigid, rather arbitrary and do not match daily practice. A new model, based on multivariate modeling of log peak abundance ratios, provides a theoretical basis for the identification of analytes and optimizes the balance between the avoidance of false positives and false negatives. The performance of the model is demonstrated on data provided by five laboratories, each supplying GC-MS measurements on the detection of clenbuterol, dienestrol and 19 beta-nortestosterone in urine. The proposed model shows a better performance than confirmation by using the current criteria and provides a statistical basis for inspection criteria in terms of error probabilities.

The chemometric analysis of low-level analytical data is hampered by the common presence of interfering compounds, by the frequent absence of measurement signals and by a non-constant measurement variability which is related to concentration level in a non-linear way. A model is presented to handle this type of data in the context of the practical problem of multivariate detection from gas chromatography/mass spectrometry (GC-MS) data. The model, based on log ratio modelling, is compared with previous approaches to parts of the problem. The basic idea behind the model is to define for the multivariate detection problem a null hypothesis for the values of log ratio measurements and to estimate variability as a function of total measured intensity. In practice it is often impossible to anticipate all kinds of interference which may occur. Therefore we propose to use expert assessments of the probability that certain expected peak ratios are generated by the analyte rather than by interferences. These expert assessments can then be used to define a proper null hypothesis for the multivariate detection test. The application of the model is illustrated for the detection of the illegal growth promoter clenbuterol in urine by selected ion-monitoring GC-MS.

Monolayer cultures of pig hepatocytes were used to investigate the role of the liver in the biotransformation of the veterinary drug dimetridazole (1,2-dimethyl-5-nitroimidazole). 14C-Labeled dimetridazole (DMZ) was primarily hydroxylated to 1-methyl-2-hydroxymethyl-5-nitroimidazole (up to 90%) and to a minor extent N-demethylated to 2-methyl-(4,5)-nitroimidazole (6-10%). Prolonged incubation with the parent drug but also the 2-hydroxymethyl metabolite resulted in the formation of two other minor metabolites. The major one of these was identified as the glucuronide of the 2-hydroxymethyl metabolite, based on its molecular mass and the succesful hydrolysis with β-glucuronidase. The second showed a molecular mass of 144 and is hypothesized to be 2-hydroxymethyl-5-nitroimidazole. No evidence was obtained for the formation of a cysteine or glutathione conjugate involved in the detoxification of reactive intermediates. In addition to free metabolites, there was a time-related formation of protein-bound metabolites up to a maximum of 30 pmol/mg of protein after exposure to 50 μM DMZ for 48 h. In general, these metabolites accounted for 0.06-0.15% of the metabolized DMZ. Unextractable metabolites were also observed after incubation of cells with the 2-hydroxymethyl and 1-desmethyl metabolites. It is concluded that the 1-desmethyl and, in particular, the 2-hydroxymethyl metabolite are the major metabolites formed by pig hepatocytes. These compounds are thus far the only metabolites identified in vivo, but together with the parent compound, they accounted for only a small part (

A method is described for the analysis of beta-agonists in urine of cattle. The method uses solid-phase extraction (SPE), followed by analysis of the resulting extract by flow injection thermospray tandem mass spectrometry (TSP-MS-MS). Sample preparation is performed using a mixed-bed SPE procedure using a sorbent having both hydrophobic and ionic properties. MS-MS analysis following thermospray ionization, is performed in single-reaction monitoring parent mode. In that way isotope dilution can be used for quantitation of clenbuterol. Data are presented on precision and accuracy for clenbuterol and related compounds. Furthermore, data acquisition was performed in full-scan neutral loss mode to indicate the suitability of flow injection analysis (FIA)-TSP-MS-MS for exploratory analysis. Detection of beta-agonists in this mode is based on the presence of the N-tert.-butyl-beta-ethanolamino moiety and, in that respect, detection of known as well as unknown compounds having this moiety will take place. This feature is exemplified by the analysis of samples containing several compounds.

1. The major metabolite resulting from the biotransformation of furaltadone (5-morpholinomethyl-3-[5-nitrofurfurylidene-amino]-2-oxazoli dinone) by pig hepatocytes was shown to result from the N-oxidation of the tertiary nitrogen in the morpholino-ring, leaving the nitrofuran ring unchanged. 2. No evidence could be obtained for the formation of an open-chain cyano-metabolite, a minor metabolite in the case of the related nitrofuran drug furazolidone (N-(5-nitro-2-furfurylidene)-3-amino-2-oxazolidinone). This metabolite was the major metabolite, following incubation of furaltadone and furazolidone with Salmonella typhimurium bacteria. 3. The N-oxide was not further metabolized by pig hepatocytes or bacteria, and gave negative test results in the Ames-test (TA 100, no S9-mix) at the highest tested dose of 1 microgram/plate. Furaltadone gave a positive result at 10 ng/plate. 4. The biotransformation of both drugs by pig hepatocytes and bacteria resulted in the formation of protein-bound metabolites, with no clear quantitative differences between the two drugs. The intact 3-amino-2-oxazolidinone (AOZ) and 5-morpholinomethyl-3-amino-2-oxazolidinone (AMOZ) side-chains of furazolidone and furaltadone, respectively, could be released from these metabolites by mild acid treatment. 5. Hepatocytes incubated with the AMOZ side-chain of furaltadone showed a decreased monoamine oxidase activity at high dose levels (IC50 3.7 mM), whereas exposure to the AOZ side-chain of furazolidone resulted in a clear inhibition at 10,000-fold lower concentrations (IC50 0.5 microM). In the presence of 1% dimethylsulphoxide (DMSO), the MAO-inhibition by AMOZ and especially AOZ was remarkably reduced. 6. It is concluded that protein-bound metabolites containing an intact and releasable side-chain might be present in tissues of animals treated with furaltadone. However, these residues might be of less toxicological concern than those of furazolidone.

A method is described for the simultaneous determination of planar chlorobiphenyls and dioxins in milk using isotope dilution and gas chromatography—high-resolution mass spectrometry (GC-MS). The method is based on gel permeation chromatography, alumina clean-up and carbon chromatography and is highly automated, making a high sample throughput possible. Data on recovery, accuracy and reproducibility of results obtained with quality control samples are presented. Data for both dioxins and planar chlorbiphenyls from the analysis of samples of Dutch milk from several areas in the Netherlands are also presented. Possible interference of the chlorobiphenyls in the determination of the dioxins in the GC-MS method is discussed.

A toxicokinetic experiment with four cows fed polychlorinated dibenzo-p-dioxins and -furans in the dry period for about 100 days is described. The excretion of the dioxins in milk after calving was characterized by a distribution and an elimination phase. The distribution phase lated for about 7 days. The duration of the elimination phase depended on the kind of congener. The longest elimination half-life was found for hexachlorinated congeners

A method has been developed for the determination of aflatoxin B1 in cattle feed which is an automated version of the method which was ring-tested by the European Community. The method uses a two-column clean-up with intermediate solvent evaporation and is coupled on-line to a high-performance liquid chromatographic system. The automated and manual methods are compared with regard to repeatability, accuracy and recovery of the analyte. Both methods have comparable repeatability at a concentration of 5 μg/kg of aflatoxin B1; however, due to the lower recovery of the automated method, it differs significantly from the manual method with respect to accuracy. Nevertheless, the method may be used for screening purposes due to its good reproducibility.

A liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) method was developed using the latest high-resolution LC column technology, the ultra performance liquid chromatography (UPLC (TM)), and electrospray ionization (ESI) in the positive ion mode. Gradient UPLC separation conditions were optimized for a group of 22 analytes comprising 17 glucocorticosteroids, specific designer steroids such as tetrahydrogestrinone (THG) and specific beta(2)-agonists such as formoterol. The UPLC/TOFMS separation obtained required 5.5 min only for all the substances tested. Even the critical pair of dexamethasone and betamethasone isomers was almost completely resolved. Thanks to the over 10,000 full-width at half maximum (FWHM) mass resolution and high mass accuracy features of TOFMS 50 mDa window accurate mass chromatograms could be reconstructed for the individual analytes. Sensitive screening in human and calf urine samples fortified at the glucocorticosteroids minimum required performance limit (MRPL) of 30 mu g L-1 (human urine, sports doping) and 2 mu g L-1 (calf urine, veterinary control) could be obtained. The potential of UPLC/TOFMS for confirmatory analysis was shown by determining the accurate mass of all compounds and fragment ions upon in-source collision-induced dissociation (CID) at different energies. The exact mass measurement errors for all glucocorticosteroids were found to be within 6 ppm. Considering veterinary control, limits of detection (LOD) and limits of quantification (LOQ) were determined for most of the analytes in calf urine and found to range from 0.1 to 3.3 and from 0.4 to 4.4 mu g L-1, respectively. The method can be easily extended with other banned substances of interest, as demonstrated by the addition of 21 beta(2)-agonists to the original analyte mixture in urine, without causing any interferences. (c) 2006 Elsevier B.V. All rights reserved.

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.

A lifetime controlled reference experiment has been performed using 42 veal calves, 21 males and 21 females which were fed and housed according to European regulations and common veterinary practice. During the experiment feed, water, urine and hair were sampled and feed intake and growth were monitored. Thus for the first time residue analysis data were obtained from guaranteed lifetime-untreated animals. The analysis was focused on the natural hormones estradiol and testosterone and their metabolites, on 17 beta- and 17 alpha-nortestosterone, on 17 beta- and 17 alpha-boldenone and androsta-1,4-diene-3,17-dione (ADD), and carried out by gas chromatography tandem mass spectrometry (GC/MS/MS), an estrogen bioassay and liquid chromatography (LC) MS/MS. Feed, water and hair samples were negative for the residues tested. Female calf urines showed occasionally low levels of 17 alpha-estradiol and 17 alpha-testosterone. On one particular sampling day male veal calf urines showed very high levels of 17 alpha-testosterone (up to 1000 ng mL(-1)), accompanied by lower levels of estrone and 17 beta-testosterone. Despite these extreme levels of natural testosterone, 17 beta-boldenone was never detected in the same urine samples; even 17 alpha-boldenone and ADD were only occasionally beyond CC alpha (maximum levels 2.7 ng ng mL(-1)). The data from this unique experiment provide a set of reference values for steroid hormones in calf urine and demonstrate that 17 beta-boldenone is not a naturally occurring compound in urine samples. (c) 2006 Elsevier B.V. All rights reserved.

The abuse of esters of natural androgenic steroids in cattle fattening and sports is hard to control via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. In veterinary control strange findings of 17beta-testosterone and 17alpha-testosterone in urine are often ignored because of the lack of statistically sound reference data of naturally occurring levels. An interesting alternative for inconclusive urine analyses in veterinary control can be provided by the analysis of the administered steroids themselves, i.e. the analysis of intact steroid esters in hair. Unfortunately, the analysis of intact steroid esters is complicated not only by the vulnerability of the esters which precludes alkaline hydrolysis of the hair, but also by the wide polarity range of short and long-chain esters yielding very poor recoveries for either the one or the other. In this study, a multi-steroid esters LC/MS/MS screening method is presented for trace analysis of the synthetic intact esters of 17beta-testosterone and the undecylenate ester of 17beta-boldenone in bovine hair. The method, requiring only 200 mg of pulverised hair, features a mild digestion procedure using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and the use of four deuterium-labelled steroid esters as internal standards covering the wide polarity range of the analytes. In spiked hair samples for most of the analytes the limit of detection and the accuracy using isotope dilution were 2-5 ng/g and 97-105%, respectively. The applicability was demonstrated using hair samples from a controlled experiment in which six bovines were injected intramuscularly with two different doses of two commercial mixtures of testosterone esters, and with two different doses of boldenone undecylenate. Depending on the dose all administered testosterone- and boldenone esters were found to be incorporated in bovine hair following a single intramuscular injection, except testosterone propionate which dose might have been too low.

A proficiency test for tetracycline drug residues in poultry muscle was organized according to the guidelines of International Laboratory Accreditation Cooperation (ILAC) ILAC-G13:2000 (2000). For the proficiency test, three test materials were prepared. The homogeneity and stability of the materials during the study were demonstrated. Sixteen laboratories accepted the invitation to participate in the proficiency test; 11 laboratories reported results within the time frame of the study. Most notably, only four of the participating laboratories complied with the definition of the maximum residue limit (MRL) concerning the inclusion of 4-epimers as stated in European Commission Regulation 281/96 (1996). Most participants reported values for the decision limit (CCalpha) and detection capability (CCbeta) and hence were already in compliance with European Commission 2002/657/EC (2002) for this aspect of method validation. However, some CCalpha and CCbeta values were not in agreement with the actual within-laboratory reproducibility calculated from the results reported in this proficiency test. Although most laboratories obtained satisfactory results, it is clear that an effort is needed to include 4-epiOTC, 4-epiTC and 4-epiCTC in the analytical methods. Moreover, reconsideration of values determined for CCalpha and CCbeta with respect to their accuracy may be necessary in some cases.

New anabolic steroids show up occasionally in sports doping and in veterinary control. The discovery of these designer steroids is facilitated by findings of illicit preparations, thus allowing bioactivity testing, structure elucidation using NMR and mass spectrometry, and final incorporation in urine testing. However, as long as these preparations remain undiscovered, new designer steroids are not screened for in routine sports doping or veterinary control urine tests since the established GC/MS and LC/MS/MS methods are set up for the monitoring of a few selected ions or MS/MS transitions of known substances only. In this study, the feasibility of androgen bioactivity testing and mass spectrometric identification is being investigated for trace analysis of designer steroids in urine. Following enzymatic deconjugation and a generic solid-phase extraction, the samples are analyzed by gradient LC with effluent splitting toward two identical 96-well fraction collectors. One well plate is used for androgen bioactivity detection using a novel robust yeast reporter gene bioassay yielding a biogram featuring a 20-s time resolution. The bioactive wells direct the identification efforts to the corresponding well numbers in the duplicate plate. These are subjected to high-resolution LC using a short column packed with 1.7-microm C18 material and coupled with electrospray quadrupole time-of-flight mass spectrometry (LC/QTOFMS) with accurate mass measurement. Element compositions are calculated and used to interrogate electronic substance databases. The feasibility of this approach for doping control is demonstrated via the screening of human urine samples spiked with the designer anabolic steroid tetrahydrogestrinone. Application of the proposed methodology, complementary to the established targeted urine screening for known anabolics, will increase the chance of finding unknown emerging designer steroids, rather then being solely dependent on findings of the illicit preparations themselves.

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.

A method is described for the identification and quantitative determination of 3,5-dinitrosalicylic acid hydrazide (DSH), the marker residue of nifursol metabolites in poultry (turkey, broiler) muscle and liver tissue. The method is based on the acid-catalysed hydrolysis of tissue-bound metabolites to free DSH and in situ derivatisation with 2-nitrobenzaldehyde to the corresponding nitrophenyl derivative NPDSH. A structural analogue of DSH, 4-hydroxy-3,5- dinitrobenzoic acid hydrazide (HBH) was synthesised to serve as an internal standard. The analytes were isolated from the matrix by liquid-liquid extraction with ethyl acetate. Determination was performed by LC-MS/MS with negative electrospray ionisation. The [M - H]+ ions of NPDSH and NPHBH at m/z 374 were fragmented by collision induced dissociation (CID) producing transition ions at m/z 182, 183 and 226. The transition ions at m/z 182 and 226 were selected for monitoring of NPDSH while the transition ion at m/z 183 was selected for NPHBH. The method has been validated according to the EU criteria of Commission Decision 2002/657/EC at 0.5, 1.0 and 1.5 μg kg-1 in muscle and liver tissue. A decision limit (CCα) was obtained of 0.04 and 0.025 μg kg-1 in muscle and liver, respectively. Similarly a detection capability (CCβ) was obtained of 0.10 and 0.05 μg kg-1 in muscle and liver, respectively. The introduction of HBH as an internal standard did not lead to a significant improvement of the quantitative performance of the method. In fact for liver better performance characteristics were obtained when the IS was not taken into account. Nevertheless, as a qualitative marker for recovery, HBH could still be very useful in the analysis of unknown samples. © The Royal Society of Chemistry 2005.

A liquid chromatography (LC)-based method with mass spectrometric (MS/MS) detection was developed for the determination of estradiol benzoate residues in hair of cattle. First hair samples were pulverized with a ball mill followed by treatment with the reducing agent Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). After liquid/liquid extraction samples were further purified by solid-phase extraction. Finally samples were analyzed with LC–MS/MS using deuterated estradiol benzoate as internal standard. The method was validated following the latest EU guidelines for screening methods using blank hair samples spiked at 5 ng g−1. The detection capability (CCβ) was less than 5 ng g−1 and the decision limit (CCα) was 1.6 ng g−1. The recovery was 27% at the 5 ng g−1 level and the accuracy was 95%. Confirmation, with two MS/MS transitions, was possible at a concentration of 5 ng g−1 or higher. Incurred samples obtained from different animal experiments were analyzed and the presence of estradiol benzoate was confirmed. Finally hair samples from different slaughterhouses were analyzed.

The global problem of food products contaminated by residues of the banned carcinogenic nitrofuran drugs has prompted research into how such residues accumulate in tissues. In the study described here, two aspects have been investigated where the nitrofurans accumulate in tissues from chickens exposed to either a dietary or an environmental source of contamination. Twenty groups of broilers were fed a diet containing one of the nitrofurans: furazolidone, nitrofurazone, nitrofurantoin or furaltadone at concentrations of 30, 100, 300, 1000 and 3000 microg kg(-1). At the lowest concentration of furazolidone contamination (0.01% of the therapeutic dose) tissue bound AOZ metabolite residues were detected in liver (1.1 +/- 0.2 microg kg(-1)) and in muscle (0.33 +/- 0.03 microg kg(-1)). Similar results were obtained for AMOZ (0.6 +/- 0.2 microg kg(-1) in liver), the tissue bound metabolite of furaltadone. There was no appreciable accumulation of nitrofurantoin in chicken muscle. The AHD metabolite was not detected in muscle from birds fed nitrofurantoin at either 30 or 100 microg kg(-1). For nitrofurazone the concentrations of the SEM metabolite were higher in muscle than in liver for all dietary concentrations. The potential for a contaminated environment to cause nitrofuran residues in chickens was investigated. Six chickens were placed in a pen that was previously occupied by birds fed a diet containing 3000 microg kg(-1) of furazolidone. After 24 hours' exposure of the chickens to the litter in the pen, AOZ residues of 0.13 +/- 0.04 and 0.10 +/- 0.03 microg kg(-1) were detected in liver and muscle, respectively. The results of both experiments have implications for the poultry industry in trying to eliminate nitrofurans from their production systems, and for regulatory analysts faced with the detection of low concentrations of the drugs, both in tissues and in feedingstuffs.

A new approach to the search for residues of known and unknown estrogens in calf urine is presented. Following enzymatic deconjugation and solid-phase extraction, a minor part of the samples is screened for estrogen activity using a recently developed rapid reporter gene bioassay. The remainder of the bioactive extracts is analyzed by gradient liquid chromatography (LC) with, in parallel, bioactivity and mass spectrometric detection via effluent splitting toward a 96-well fraction collector and an electrospray quadrupole time-of-flight mass spectrometer (QTOFMS). The LC fractions in the 96-well plate are used for the detection of estrogen activity using the bioassay. The biogram obtained features a 20-s time resolution, and the suspect well numbers can be easily correlated with the LC/QTOFMS retention time. The mass spectral data from the thus assigned relevant parts of the chromatograms are background subtracted, followed by accurate mass measurement, element composition calculation, and identification. The method allows estrogen activity detection and identification of (un)known estrogens in urine at the 1-2 ng/L level, in compliance with current residue analysis performance for hormone abuse in cattle. The applicability of this LC/bioassay/QTOFMS approach for the identification of estrogens in real-life samples is demonstrated by the analysis of several calf urine samples, and preliminary data from a pig feed sample.

Microcolumn liquid chromatography (μLC) combined with electrospray tandem mass spectrometry is used for the determination of intact glycosylated cytokinins and the corresponding aglycons at picomole and sub-picomole levels in plant tissue. Routine analysis was done on C8-bonded silica using a methanol–water gradient. Data acquisition was performed by multiple reaction monitoring. Quantification was carried out by using isotopically labelled analogues and applying linear regression to the response factor versus concentration data. For routine analysis a calibration range from 0.5 to 10 pmole injected on-column was used. The limits of detection ranged from 50 to 100 fmole injected on-column. The μLC procedure was used to analyse plant tissue extracts from transgenic homozygote and hemizygote as well as wild-type Nicotiana tabacum species, and cauliflower samples. The data were compared with results obtained by conventional immunoassay and a satisfactory correlation was found. Validation data are presented.

A previously described method for the confirmatory analysis of clenbuterol with chemical ionization GC-MS was extended to fit more beta-agonists. The method has been used routinely for about one year and a large number of samples have been analysed according to the described procedure. This paper summarizes the results obtained with this method with regard to clenbuterol and furthermore discussion is focused on applicability to beta-agonists other than clenbuterol with respect to the European Community requirements for confirmatory analysis.

A method for the determination of dioxins, planar PCBs and other PCBs in human milk is described. The work is part of a big Dutch project, (seven institutes participates) in which 400 human milk samples from two areas (Rotterdam and Groningen) taken at two different times (10 and 42 days) after delivery had to be analysed for planar PCBs, dioxins and other PCBs (1st sampling) and only other PCBs (2nd sampling). Planar compounds are determined with GC/HRMS, other PCBs with GC/ECD. As type of analytes for 1st and 2nd sampling are different the method was focussed on these analytes. After fat extraction, clean up was carried out on GPC and alumina columns. Only in case of analysis of planar compounds and other PCBs, chromatography on porous graphitised carbon was carried out after which GC-HRMS (planar compounds) and GC/ECD (other PCBs) was performed; otherwise GC/ECD after alumina clean up was performed. The method is validated through recovery experiments with standards (other PCBs, labelled planar compounds), comparison of results for other PCBs obtained with the two different clean-up procedures, reproducibility of "quality control samples" for planar compounds blanc chemicals.

To meet the requirements of the European Community for confirmatory analysis of clenbuterol using low-resolution mass spectrometry, usually two different techniques (i.e. electron impact and chemical ionization) have to be applied to confirm unambiguously its presence in extracts of urine. This paper describes the application of two different derivatives and the simultaneous analysis of these two different derivatives in one gas chromatographic—mass spectrometric analysis. With the proposed combination of techniques, Community requirements can more easily be met in only one analytical run. Examples of the analysis of some urine samples are presented, as well as data on linearity, repeatability and equivalence of the combined technique to separate determinations.