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.

Multi-detection of corticosteroids in sports doping and veterinary control using high-resolution liquid chromatography/time-of-flight mass spectrometry.

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), 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 beta2-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 microg L(-1) (human urine, sports doping) and 2 microg 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 microg L(-1), respectively. The method can be easily extended with other banned substances of interest, as demonstrated by the addition of 21 beta2-agonists to the original analyte mixture in urine, without causing any interferences.

The determination of biurea: a novel method to discriminate between nitrofurazone and azodicarbonamide use in food products.

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.

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.

Residue analysis of tetracyclines in poultry muscle: shortcomings revealed by a proficiency test.

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.

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.

Transfer of nitrofuran residues from parent broiler breeder chickens to broiler progeny.

The use of nitrofuran antibiotics in food-producing animals is prohibited within the EU. Countries in the EU, as well those intending to export food to the EU, must ensure that their products are free from nitrofuran residues. As a result of recent global problems where chicken meat from a wide range of countries has been contaminated with nitrofuran metabolites, an investigation was performed to discover whether or not residues of the nitrofurans might be transferred from parent breeder chickens to their offspring broilers. Four groups of broiler breeders were each treated with one of the nitrofurans: furazolidone, nitrofurazone, nitrofurantoin or furaltadone. Residues of their side-chain metabolites, AOZ, SEM, AHD and AMOZ, were detected in the fertilised eggs at concentrations up to 1567 microg/kg. However, in the chicks that subsequently hatched from these eggs, residue concentrations of SEM, for example, were only found up to 26.6 and 32.5 microg/kg in liver and muscle, respectively, for 1-d-old chicks. Residue concentrations in tissues had fallen below the detection limit of the analytical method for 40-d-old broiler chicks, for all compounds except for semicarbazide (SEM, the nitrofurazone metabolite). Relatively high concentrations of nitrofurans are available to the newly hatched chick through the egg yolk. However, most of these residues are neither utilised nor deposited in the liver or muscle.

Depletion of four nitrofuran antibiotics and their tissue-bound metabolites in porcine tissues and determination using LC-MS/MS and HPLC-UV.

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.

The occurrence of nitrofuran metabolites in the tissues of chickens exposed to very low dietary concentrations of the nitrofurans.

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.

Determination of nifursol metabolites in poultry muscle and liver tissue. Development and validation of a confirmatory method.

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 microg kg(-1) in muscle and liver tissue. A decision limit (CC(alpha)) was obtained of 0.04 and 0.025 microg kg(-1) in muscle and liver, respectively. Similarly a detection capability (CC(beta)) was obtained of 0.10 and 0.05 microg 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.

Confirmatory analysis of 17beta-boldenone, 17alpha-boldenone and androsta-1,4-diene-3,17-dione in bovine urine, faeces, feed and skin swab samples by liquid chromatography-electrospray ionisation tandem mass spectrometry.

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.

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.

Optimizing the balance between false positive and false negative error probabilities of confirmatory methods for the detection of veterinary drug residues.

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.

Rapid analysis of beta-agonists in urine by thermospray tandem mass spectrometry.

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.

Possibilities for confirmatory analysis of some beta-agonists using two different derivatives simultaneously.

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.

The Dutch PCB/Dioxin Study. Development of a method for the determination of dioxins, planar and other PCBs in human milk.

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.

Confirmatory analysis of clenbuterol using two different derivatives simultaneously.

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.

Simultaneous determination of planar chlorobiphenyls and polychlorinated dibenzo-p-dioxins and -furans in Dutch milk using isotope dilution and gas chromatography-high-resolution mass spectrometry.

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 chlorobiphenyls 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.

Automated clean-up procedure for the gas chromatographic-high-resolution mass spectrometric determination of polychlorinated dibenzo-p-dioxins and dibenzofurans in milk.

A highly automated extraction and clean-up method for polychlorinated dibenzodioxins and polychlorinated dibenzofurans is described. The method includes the use of gel permeation chromatography, alumina clean-up and porous graphitized carbon chromatography, followed by analysis by gas chromatography-high-resolution mass spectrometry. The procedure allows for the analysis of six milk samples per day in addition to two quality control samples and a blank. Detection limits on a fat basis for the individual congeners in milk samples are in the sub-ppt range. Long-term performance was investigated and data are given for reproducibility, precision and accuracy.