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Analytica Chimica Acta (v.586, #1-2)

Editorial Board (pp. co1).

Calendar of forthcoming meetings (pp. n1).

Editorial Board (pp. co1).

Calendar of forthcoming meetings (pp. n1).

Editorial Board (pp. co1).

Preface by C. Van Peteghem (pp. 1-1).

Why consumers behave as they do with respect to food safety and risk information by Wim Verbeke; Lynn J. Frewer; Joachim Scholderer; Hubert F. De Brabander (pp. 2-7).

In recent years, it seems that consumers are generally uncertain about the safety and quality of their food and their risk perception differs substantially from that of experts. Hormone and veterinary drug residues in meat persist to occupy a high position in European consumers’ food concern rankings. The aim of this contribution is to provide a better understanding to food risk analysts of why consumers behave as they do with respect to food safety and risk information. This paper presents some cases of seemingly irrational and inconsistent consumer behaviour with respect to food safety and risk information and provides explanations for these behaviours based on the nature of the risk and individual psychological processes. Potential solutions for rebuilding consumer confidence in food safety and bridging between lay and expert opinions towards food risks are reviewed. These include traceability and labelling, segmented communication approaches and public involvement in risk management decision-making.

Keywords: Consumer; Meat; Perception; Risk; Traceability

Why consumers behave as they do with respect to food safety and risk information by Wim Verbeke; Lynn J. Frewer; Joachim Scholderer; Hubert F. De Brabander (pp. 2-7).

Keywords: Consumer; Meat; Perception; Risk; Traceability

Why consumers behave as they do with respect to food safety and risk information by Wim Verbeke; Lynn J. Frewer; Joachim Scholderer; Hubert F. De Brabander (pp. 2-7).

Keywords: Consumer; Meat; Perception; Risk; Traceability

Calculation of the decision limit (CC α) and the detection capability (CC β) for banned substances: The imperfect marriage between the quantitative and the qualitative criteria by J. Van Loco; A. Jànosi; S. Impens; S. Fraselle; V. Cornet; J.M. Degroodt (pp. 8-12).

Initially in the Decision 2002/657/EC the criteria for the calculation of the decision limit (CC α) and the detection capability (CC β) have been estimated as purely quantitative ( α-error is 1% and β-error is 5%). In 2004, the European Commission has issued a document to provide guidance for the interpretation of the 2002/657/EC. In this document it is mentioned that also qualitative criteria should be fulfilled. Therefore, the calculated CC α and CC β must be verified by using fortified samples. The method should be able to detect/identify the target component in 50% of the cases at CC α and in 95% of the cases at CC β.Analytical methods for the analysis of nitroimidazoles, nitrofurans and corticosteroids with LC–MS/MS have been validated by fortifying blank samples below and above the MRPL. CC α and CC β were calculated using the ISO 11843 approach. In addition, the frequency of methodical compliance for the qualitative criteria was determined at each concentration level. It was observed that at the calculated CC α and CC β levels the qualitative criteria were not fulfilled. It was concluded that the detection capability of the analytical method should be calculated by using decreasing fortification levels at and below the MRPL.A protocol validating methods for banned substances by limiting the number of samples is presented and the qualitative criteria for the assessment of CC α and CC β were verified based on the same set of data without the need of performing additional validation experiments.

Keywords: Banned substances; Detection capability; ISO 11843; Method validation; Commission decision 2002/657/EC

Ultra-performance liquid chromatography coupled to time of flight mass spectrometry (UPLC–TOF): A novel tool for multiresidue screening of veterinary drugs in urine by Anton Kaufmann; Patrick Butcher; Kathryn Maden; Mirjam Widmer (pp. 13-21).

A multiresidue method for the screening of veterinary drugs in urine with ultra-performance liquid chromatography (UPLC) coupled to time of flight mass spectrometry (TOF) is proposed. The method covers currently more than 100 analytes belonging to different families of veterinary drugs. Urine samples are simply diluted and injected unfiltered into the UPLC–TOF. The short run time permit high-throughput of large series under a routine environment.The suggested approach includes the detection of some metabolites which are relevant to the urine matrix. The paper discusses the usefulness of metabolites as additional confirmation criteria for positive findings and suggests a revisiting of the identification point system in the light of additional resolution, as provided by the TOF and the UPLC technology. The proposed multiresidue approach was further broadened by monitoring drug group specific collision induced fragments (CID). Examples show the monitoring of generic CID fragments typical for sulfonamides and penicillines.

Keywords: Screening; Veterinary drugs; Ultra-performance liquid chromatography (UPLC); Time of flight mass spectrometry (TOF); Multiresidue

Multi-analyte approach for the determination of ngL⁻¹ levels of steroid hormones in unidentified aqueous samples by H. Noppe; K. Verheyden; W. Gillis; D. Courtheyn; P. Vanthemsche; H.F. De Brabander (pp. 22-29).

Since the 1970s, many analytical methods for the detection of illegal growth promoters, such as thyreostats, anabolics, β-agonists and corticosteroids have been developed for a wide range of matrices of animal origin, including meat, fat, organ tissue, urine and faeces.The aim of this study was to develop an analytical method for the determination of ngL⁻¹ levels of estrogens, gestagens, androgens (EGAs) and corticosteroids in aqueous preparations (i.e. drinking water, drinking water supplements), commercially available on the ‘black’ market. For this, extraction was performed with Bakerbond C₁₈ speedisk, a technique commonly used in environmental analysis. After fractionation, four fractions were collected using a methanol:water gradient program. Gas chromatography coupled to electron impact multiple mass spectrometry (GC–EI-MS²) screening for the EGAs was carried out on the derivatized extracts. For the detection of corticosteroids, gas chromatography coupled to negative chemical ionization mass spectrometry (GC–NCI-MS) was used after oxidation of the extracts. Confirmation was done by liquid chromatography coupled to electrospray ionization multiple mass spectrometry (LC–ESI-MS²). The combined use of GC and LC coupled to MS enabled the identification and quantification of anabolics and corticosteroids at the low ngL⁻¹ level. This study demonstrated the occurrence of both androgens and corticosteroids in different commercial aqueous samples.

Keywords: Gas chromatography; Liquid chromatography; Multiple mass spectrometry; Steroids; Corticosteroids; Anabolics; Residue analysis

The ultimate veal calf reference experiment: Hormone residue analysis data obtained by gas and liquid chromatography tandem mass spectrometry by Michel W.F. Nielen; Johan J.P. Lasaroms; Martien L. Essers; Marieke B. Sanders; Henri H. Heskamp; Toine F.H. Bovee; J. (Hans) van Rhijn; Maria J. Groot (pp. 30-34).

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 17β- and 17α-nortestosterone, on 17β- and 17α-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 17α-estradiol and 17α-testosterone. On one particular sampling day male veal calf urines showed very high levels of 17α-testosterone (up to 1000ngmL⁻¹), accompanied by lower levels of estrone and 17β-testosterone. Despite these extreme levels of natural testosterone, 17β-boldenone was never detected in the same urine samples; even 17α-boldenone and ADD were only occasionally beyond CCα (maximum levels 2.7ngmL⁻¹). The data from this unique experiment provide a set of reference values for steroid hormones in calf urine and demonstrate that 17β-boldenone is not a naturally occurring compound in urine samples.

Keywords: Steroid hormone; Reference data; Calf urine; Boldenone; Mass spectrometry; Liquid chromatography; Gas chromatography

Determination of anabolic steroids in dietary supplements by liquid chromatography–tandem mass spectrometry by C. Van Poucke; C. Detavernier; R. Van Cauwenberghe; C. Van Peteghem (pp. 35-42).

Nineteen different dietary supplements, ordered through the internet and intercepted by the Belgian pharmaceutical inspection at the post office, were analyzed by means of liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the presence of anabolic steroids. After a methanolic extraction the samples were screened for the presence of 49 compounds. This resulted in almost 60% of the samples being suspected of containing one of these 49 anabolic compounds and being subjected to a confirmatory product ion scan. In all of these suspected samples we were able to confirm at least one anabolic steroid with concentrations between 0.01 and 2.5mg unit⁻¹ (unit: one capsule or tablet or for liquids: the prescribed dose). The anabolic steroids that was mostly encountered was testosterone (50%) followed by β-boldenone (25%). These results once more confirm the dubious reputation of over-the-counter dietary supplements.

Keywords: Dietary supplements; Prohormones; Anabolic steroids; Doping; Liquid chromatography–tandem mass spectrometry (LC–MS/MS)

Determination of anabolic steroids in dietary supplements by liquid chromatography–tandem mass spectrometry by C. Van Poucke; C. Detavernier; R. Van Cauwenberghe; C. Van Peteghem (pp. 35-42).

Keywords: Dietary supplements; Prohormones; Anabolic steroids; Doping; Liquid chromatography–tandem mass spectrometry (LC–MS/MS)

Determination of anabolic steroids in dietary supplements by liquid chromatography–tandem mass spectrometry by C. Van Poucke; C. Detavernier; R. Van Cauwenberghe; C. Van Peteghem (pp. 35-42).

Keywords: Dietary supplements; Prohormones; Anabolic steroids; Doping; Liquid chromatography–tandem mass spectrometry (LC–MS/MS)

A downscaled multi-residue strategy for detection of anabolic steroids in bovine urine using gas chromatography tandem mass spectrometry (GC–MS³) by S. Impens; J. Van Loco; J.M. Degroodt; H. De Brabander (pp. 43-48).

Within the scope of the European Community member states’ residue monitoring plan, illicit administration of anabolic steroids is monitored at slaughterhouse level as well as on living animals. At farm level, urine is one of the target matrices to detect possible abuse of anabolic steroid growth promoters. Optimisation of the routinely applied analysis method resulted in a procedure for which high performance liquid chromatographic (HPLC) fractionation prior to GC–MSⁿ analysis was no longer required. Analytical results could be obtained within 1 day and only 5mL urine was needed tot carry out the screening procedure. Using the downscaled methodology, all validation criteria described in the European Commission document 2002/657/EC could be fulfilled, and the minimum required performance limits (MRPLs) established for anabolic steroids in urine, could be achieved.A higher GC–MS technique's specificity was achieved by detecting the steroids using GC–MS³. Nevertheless, it was decided to screen routinely sampled urine with GC–MS² whereas GC–MS³ was applied to confirm the presence of anabolic steroid residues in suspected sample extracts.

Keywords: Gas chromatography; Tandem mass spectrometry; Bovine urine; Sex steroid hormones; Anabolic steroid residues; Minimum required performance limits

Phytosterols and anabolic agents versus designer drugs by H.F. De Brabander; K. Verheyden; V. Mortier; B. Le Bizec; W. Verbeke; D. Courtheyn; H. Noppe (pp. 49-56).

Cholesterol is a well-known component in fats of animal origin and it also is the precursor of natural hormones. Phytosterols appear in plants and only differ slightly in structure from cholesterol. An important difference however is the low absorption in the gut of phytosterols and their saturated derivatives, the phytostanols. As a result, there is time for all kind of reactions in faecal material inside and outside of the gut. Determination of the abuse of natural hormones may be based on gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Abuse of natural hormones changes the¹³C/¹²C ratio of some metabolites during a relatively long time. The formation of (natural) hormones in the gut may interfere with this method. Designer drugs are mainly known from sports doping. In animal fattening, designer drugs may be used as well. Small changes in the structure of (natural) hormones may lead to a new group of substances asking for new strategies for their detection and the constatation of their abuse.

Keywords: Cholesterol; Phytosterols; Phytostanols; Anabolic agents; Designer drugs

Phytosterols and anabolic agents versus designer drugs by H.F. De Brabander; K. Verheyden; V. Mortier; B. Le Bizec; W. Verbeke; D. Courtheyn; H. Noppe (pp. 49-56).

Keywords: Cholesterol; Phytosterols; Phytostanols; Anabolic agents; Designer drugs

Phytosterols and anabolic agents versus designer drugs by H.F. De Brabander; K. Verheyden; V. Mortier; B. Le Bizec; W. Verbeke; D. Courtheyn; H. Noppe (pp. 49-56).

Keywords: Cholesterol; Phytosterols; Phytostanols; Anabolic agents; Designer drugs

Mass spectrometric detection of and similarities between 1-androgens by K. Verheyden; B. Le Bizec; D. Courtheyn; V. Mortier; M. Vandewiele; W. Gillis; P. Vanthemsche; H.F. De Brabander; H. Noppe (pp. 57-72).

Regularly new anabolic steroids appear on the black market. In most cases these substances are marketed on websites or are confiscated during inspections. 1,(5α)-Androstene-17β-ol-3-one, also known as 1-testosterone, is one of these substances presented to body-builders as a nutritional supplement or a pro-hormone. 1-Testosterone closely resembles the natural hormone testosterone except for a 1,2-double bound instead of a 4,5-double bound. 1-Androstene-3β,17β-diol is transformed into 1-testosterone after oral administration.1-Testosterone, 1-androstene-3β,17β-diol and some other related ‘new’ anabolic steroids were studied with gas chromatography coupled to mass spectrometry (GC–MS) and Liquid chromatography coupled to tandem mass spectrometry (LC–MS²) methods. Similarities in spectra to known analytes, which may lead to pitfalls in the interpretation of the derivatised analytes, are discussed.

Keywords: 1-Testosterone; 1-Androstenediol; Multiple mass spectrometry

Modification of mRNA expression after treatment with anabolic agents and the usefulness for gene expression-biomarkers by Martina Reiter; Vanessa M. Walf; Arne Christians; Michael W. Pfaffl; Heinrich H.D. Meyer (pp. 73-81).

With this feasibility study a first step towards a new monitoring system for hormonal treatments was done. Screening of regulation and function of anabolic sex steroids via modified gene expression of mRNA in various tissues could be a new approach to trace treatments with unknown drugs or newly combined cocktails.In the study, uterus, liver and muscle tissue from 24 cycling heifers were taken after the animals were treated either with Melengestrol Acetate (MGA), Finaplix-H^® (200mg Trenbolone Acetate) or Ralgro^® (36mg Zeranol) for 56 days. In every treatment group always two heifers were given 1-fold, 3-fold and 10-fold doses of the standard preparation, the control group without any treatment consisted of two animals. The different tissue gene expression profiles were investigated via the candidate gene approach. Totally 57 candidate genes were selected according to their functionality by screening the actual literature and composed to functional groups: angiogenesis, apoptosis, cell cycle, endocrine factors, energy metabolism, inflammatory factors, muscle function, oncogenes, protein metabolism and transcription factors. Gene expression was measured using quantitative real-time RT-PCR (qRT-PCR) technology.From 24 tested candidate genes in the liver, 17 showed a significant regulation. Eight genes were influenced by MGA, 9 by Finaplix-H^®, and 4 by Ralgro^®. For the muscle tissue 19 genes were tested with the result that in the neck muscle 11 genes were regulated and in the hind limb muscle 8 genes. In the neck 5 genes were affected by MGA, 6 by Finaplix-H^® and 3 by Ralgro^®. Only 2 genes were influenced by MGA in the hind limb muscle. Finaplix-H^® affected 6 and Ralgro^® 4 genes. In the uterus 29 target genes were tested and 13 were significantly influenced by the anabolic sex steroids. Under Finaplix-H^® treatment eight target genes were regulated and Ralgro^® and MGA showed a significant regulation in four target genes.The highest gene expression changes under anabolic treatment were observed in the uterus. The analyzed genes showed significant regulations but further studies, testing different animal husbandry conditions will be needed to identify meaningful expression patterns for the different tissues.With the investigation of the regulation and possible function of anabolic sex steroids via gene expression, a preparatory work for the development of an expression pattern for drug screening was made.

Keywords: Abbreviations; AM; arithmetic mean; bp; base pairs; CP; crossing point; DNA; desoxyribonucleic acid; for; forward primer; RIN; RNA integrity number; qRT-PCR; quantitative reverse-transcription polymerase chain reaction; rev; reverse primer; RG; reference gene; mRNA; messenger ribonucleic acid; CG; candidate geneAnabolic agents; Expression profiling; Candidate gene approach; Melengestrol acetat; Trenbolone acetat; Zeranol; Real-time quantitative reverse transcriptase polymerase chain reaction

Multi-residue screening of a minimum package of anabolic steroids in urine with GC–MS by P.R. Kootstra; P.W. Zoontjes; E.F. van Tricht; S.S. Sterk (pp. 82-92).

The method comprises the screening of two groups of anabolic compounds, the stilbenes and several steroids. All compounds, inclusive their metabolites when possible, for which gas chromatography–mass spectrometry (GC–MS) currently is the preferred analytical technique, are included. Two different derivatives are prepared. One group, including the stilbenes, is detected as HFB derivative (Method 1), the second group is detected as TMS derivative (Method 2). The method is used to perform a qualitative and semi-quantitative analysis of a minimum package of anabolic steroids to be included in National Residue Control Plans based on Council Directive 96/23 and complies with the current Minimum Required Performance Limits. The method has been validated according to Commission Decision 2002/657/EC. The CCα and CCβ values are based on the detection of the most abundant ion. Results of validation experiments are presented.The method is flexible and due to the non-specific sample clean-up more and new anabolic compounds can be easily added in order to new monitoring requirements.

Keywords: Anabolic steroids; Gas chromatography–mass spectrometry (GC–MS); Residue control plan

Multi-residue screening of a minimum package of anabolic steroids in urine with GC–MS by P.R. Kootstra; P.W. Zoontjes; E.F. van Tricht; S.S. Sterk (pp. 82-92).

Keywords: Anabolic steroids; Gas chromatography–mass spectrometry (GC–MS); Residue control plan

Multi-residue screening of a minimum package of anabolic steroids in urine with GC–MS by P.R. Kootstra; P.W. Zoontjes; E.F. van Tricht; S.S. Sterk (pp. 82-92).

Keywords: Anabolic steroids; Gas chromatography–mass spectrometry (GC–MS); Residue control plan

Development and validation of a multi-residue method for the detection of a wide range of hormonal anabolic compounds in hair using gas chromatography–tandem mass spectrometry by Lauriane Rambaud; Fabrice Monteau; Yoann Deceuninck; Emmanuelle Bichon; François André; Bruno Le Bizec (pp. 93-104).

The monitoring of anabolic steroid residues in hair is undoubtedly one of the most efficient strategies to demonstrate the long-term administration of these molecules in meat production animals. A multi-residue sample preparation procedure was developed and validated for 28 steroids. A 100mg hair sample was grinded into powder and extracted at 50°C with methanol. After acidic hydrolysis and extraction with ethyl acetate, phenolsteroids, such as estrogens, resorcyclic acid lactones and stilbens in one hand, are separated from androgens and progestagens in the other hand. Solid phase extractions were performed before applying a specific derivatisation for each compound sub-group. Detection and identification were achieved using gas chromatography–tandem mass spectrometry with acquisition in the selected reaction monitoring mode after electron ionisation. The method was validated according to the 2002/657/EC guideline. Decision limits (CCα) for main steroids were in the 0.1–10μgkg⁻¹ range.

Keywords: Anabolic; Steroid; Validation; Hair; Gas chromatography–tandem mass spectrometry (GC–MS/MS)

Exposure assessment of prepubertal children to steroid endocrine disrupters by Frédérique Courant; Jean-Philippe Antignac; Daniel Maume; Fabrice Monteau; Anna-Maria Andersson; Niels Skakkebaek; François Andre; Bruno Le Bizec (pp. 105-114).

Global concern has been raised in recent years over adverse effects that may result from exposure to chemicals that may interfere with the endocrine system. A specific question is related to low-dose effects and long-term exposure consequences, especially for critical populations (foetus, new born, prepubertal children). In this context, we decided to focus our attention on steroid hormones as they are the most potent endocrine disrupters. Our general goal is to investigate whether the steroid intake through food may represent a risk for prepubertal children, from an endocrine disruption point of view, especially with regard to the corresponding endogenous production level in this target population. As a starting point, it was estimated that a (re)-evaluation of the endogenous production of natural estrogens for this population was necessary, on the basis of a very sensitive and specific confirmatory measurement technique (gas chromatography–tandem mass spectrometry or gas chromatography–high resolution mass spectrometry). Thus, a new ultra-sensitive approach for steroid trace measurement in biological samples was developed, which was mainly based on a specific derivatisation (pentafluorobenzyl derivative) and negative chemical ionisation (NCI). Preliminary results obtained by applying this method on plasma samples from healthy prepubertal children demonstrated that estradiol endogenous level in prepubertal children is unsurprisingly very low. Estrone was determined in almost all samples at concentration in the 2–70ngL⁻¹ range while 17α and 17β estradiol were quantified in only few samples at concentrations ranging from 2 to 6ngL⁻¹. Exogenous contributions of estrogens will therefore constitute a relatively higher proportion of sex hormone activity in the immature child.

Keywords: Serum; Children; Estrogens; Methodology; Mass spectrometry

Gas chromatography–mass spectrometry/mass spectrometry analysis to determine natural and post-administration levels of oestrogens in bovine serum and urine by S. Biddle; P. Teale; A. Robinson; J. Bowman; E. Houghton (pp. 115-121).

A novel analytical approach has been developed and shown to be capable of detecting the isomers of oestradiol in the low ppt (pgmL⁻¹) range in bovine serum and urine. Following extractive derivatisation the analytes were detected as their 3-pentafluorobenzoyl 17-trimethylsilyl ether derivatives by gas chromatography–mass spectrometry/mass spectrometry (GC–MS/MS), using electron capture negative ion chemical ionisation. The isomers of oestradiol were quantified in both blank and post-administration urine and serum samples, with a view to setting action/threshold levels for these compounds, to allow discrimination between normal samples and samples from animals treated with growth promoting ear implants. A non-parametric statistical assessment of the data resulted in proposed action levels (with a false positive probability of 1 in 1000) of 1.6 and 2.7ngmL⁻¹ for 17α-oestradiol, in male and female urine, respectively, and 40 and 44pgmL⁻¹ for 17β-oestradiol, in male and female urine, respectively. An action level of 20pgmL⁻¹ was proposed for 17α- and 17β-oestradiol in male serum. In female serum the proposed action levels were 40 and 20pgmL⁻¹ for 17α- and 17β-oestradiol, respectively.

Keywords: Growth promoters; Cattle; Oestrogen; Endogenous; Gas chromatography/mass spectrometry; Electron capture

Screening and confirmation criteria for hormone residue analysis using liquid chromatography accurate mass time-of-flight, Fourier transform ion cyclotron resonance and orbitrap mass spectrometry techniques by M.W.F. Nielen; M.C. van Engelen; R. Zuiderent; R. Ramaker (pp. 122-129).

An emerging trend is recognised in hormone and veterinary drug residue analysis from liquid chromatography tandem mass spectrometry (LC/MS/MS) based screening and confirmation towards accurate mass alternatives such as LC coupled with time-of-flight (TOF), Fourier transform ion cyclotron resonance (FTICR) or Fourier transform orbitrap (FT Orbitrap) MS. In this study, mass resolution and accuracy are discussed for LC/MS screening and confirmation of targeted analytes and for the identification of unknowns using the anabolic steroid stanozolol and the designer β-agonist “Clenbuterol-R” as model substances. It is shown theoretically and experimentally that mass accuracy criteria without proper mass resolution criteria yield false compliant (false negative) results, both in MS screening and MS/MS confirmation of stanozolol. On the other hand, previous medium resolution accurate mass TOFMS/MS data of the designer β-agonist were fully confirmed by high resolution FT Orbitrap MSⁿ experiments. A discussion is initiated through a proposal for additional criteria for the use of accurate mass LC/MS technologies, to be implemented in Commission Decision 2002/657/EC.

Keywords: Liquid chromatography mass spectrometry; Accurate mass; Criteria; Steroid; Residue analysis; Fourier transform mass spectrometry; Orbitrap

A confirmatory method for detection of a banned substance: The validation experience of a routine EU laboratory by R. Galarini; A. Piersanti; S. Falasca; S. Salamida; L. Fioroni (pp. 130-136).

The Commission Decision 2002/657/EC is a fundamental reference document for the UE laboratories involved in residue analysis although its implementation has caused some difficulties in the requirements interpretation. In this work a pragmatic validation approach of a quantitative confirmatory method for the detection of 17-alpha-(α-NT) and 17-beta-19-nortestosterone (β-NT) in bovine urine by gas chromatography mass spectrometry is proposed. The 19-nortestosterone is a banned anabolic steroid for which no minimum required performance limit (MRPL) has been laid down, therefore the limit reported in Italian Residue Monitoring Plan (2μgL⁻¹) has been considered the reference level to evaluate the method performances. The decision limit (CCα) and the detection capability (CCβ) were obtained by the calibration curve procedure. The minimum required performance level ( mrpl), which represents the starting concentration of the calibration curves, was preliminary fixed estimating the results dispersion of blank urine samples fortified at 2μgL⁻¹ for each isomer. The found CCα and CCβ were 1.5 and 1.9μgL⁻¹ for α-NT and 1.2 and 1.4μgL⁻¹ for β-NT. The precision (repeatability and within-laboratory reproducibility) and recoveries were suitable for the investigated concentration range (1–3μgL⁻¹). Finally, the method ruggedness (minor and major changes) has been also demonstrated.

Keywords: Decision 2002/657/EC; Validation; Confirmatory method; 19-Nortestosterone

A confirmatory method for detection of a banned substance: The validation experience of a routine EU laboratory by R. Galarini; A. Piersanti; S. Falasca; S. Salamida; L. Fioroni (pp. 130-136).

Keywords: Decision 2002/657/EC; Validation; Confirmatory method; 19-Nortestosterone

A confirmatory method for detection of a banned substance: The validation experience of a routine EU laboratory by R. Galarini; A. Piersanti; S. Falasca; S. Salamida; L. Fioroni (pp. 130-136).

Keywords: Decision 2002/657/EC; Validation; Confirmatory method; 19-Nortestosterone

Multi-detection of corticosteroids in sports doping and veterinary control using high-resolution liquid chromatography/time-of-flight mass spectrometry by M.E. Touber; M.C. van Engelen; C. Georgakopoulus; J.A. van Rhijn; M.W.F. Nielen (pp. 137-146).

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 β₂-agonists such as formoterol. The UPLC/TOFMS separation obtained required 5.5min 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 50mDa 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 30μgL⁻¹ (human urine, sports doping) and 2μgL⁻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 6ppm. 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 4.4μgL⁻¹, respectively. The method can be easily extended with other banned substances of interest, as demonstrated by the addition of 21 β₂-agonists to the original analyte mixture in urine, without causing any interferences.

Keywords: Steroids; Liquid chromatography; Mass spectrometry; Urine; Veterinary control; Sports doping

Development of a method which discriminates between endogenous and exogenous β-boldenone by M.H. Blokland; H.J. van Rossum; S.S. Sterk; L.A. van Ginkel; R.W. Stephany (pp. 147-153).

One potential explanation for the presence of β-boldenone in calf urine is contamination of the sample with feces containing β-boldenone. It has been demonstrated that after oral and intramuscular administration of β-boldenone esters, several metabolites are formed and excreted in urine. One of the (minor) metabolites is 6β-hydroxy-17α-boldenone.This paper describes an analytical method that can discriminate between unconjugated boldenone, its glucuronide- and sulphate-conjugates, 6β-hydroxy-17α/β-boldenone and coprostanol, a marker for fecal contamination. The method was applied to all samples suspected to contain boldenone within the Dutch National Residue Control Plan. Approximately 10,000 samples of urine were screened (LC–MS) in 2004–2005 by VWA-East, one of the official Dutch control laboratories, from which 261 samples were suspected to contain boldenone. These samples were all analyzed for their conjugation state, 6β-hydroxy-17α/β-boldenone and for the presence of coprostanol.Alfa-boldenone, the major metabolite in bovine urine after boldenone-ester administration, was found in a large number of these samples. The presence of α-boldenone was proven also to be a result of fecal contamination. None of the samples tested contained residues of the metabolite 6β-hydroxy-17α/β-boldenone. Not finding this metabolite indicates that the origin of α-boldenone-conjugates is endogenous. The results confirm that the presence of unconjugated β-boldenone and α-boldenone conjugates next to α-boldenone are no indicators for illegal administration of boldenone-esters. No indications were obtained that conjugated β-boldenone can be of endogenous origin.

Keywords: Boldenone; Feces; Coprostanol; Gas chromatography–mass spectrometry; Conjugation; Marker; Growthpromotor

Development of a method which discriminates between endogenous and exogenous β-boldenone by M.H. Blokland; H.J. van Rossum; S.S. Sterk; L.A. van Ginkel; R.W. Stephany (pp. 147-153).

Keywords: Boldenone; Feces; Coprostanol; Gas chromatography–mass spectrometry; Conjugation; Marker; Growthpromotor

Development of a method which discriminates between endogenous and exogenous β-boldenone by M.H. Blokland; H.J. van Rossum; S.S. Sterk; L.A. van Ginkel; R.W. Stephany (pp. 147-153).

Keywords: Boldenone; Feces; Coprostanol; Gas chromatography–mass spectrometry; Conjugation; Marker; Growthpromotor

Development and validation of a liquid chromatography–tandem mass spectrometry method for the separation of conjugated and unconjugated 17α- and 17β-boldenone in urine sample by Mara Gasparini; Walter Assini; Eros Bozzoni; Nadia Tognoli; Guglielmo Dusi (pp. 154-162).

Natural occurrence or illegal treatment of boldenone (BOLD) presence in cattle urine is under debate within the European Union. Separation of conjugated and unconjugated forms of 17α-boldenone (α-BOLD) and 17β-boldenone (β-BOLD) and presence of related molecules as androsta-1,4-diene-3,17-dione (ADD) appear critical points for the decision of an illegal use. The aim of this study is a new analytical approach of BOLD and ADD confirmation in cattle urine. The separation between conjugated and unconjugated forms of BOLD was obtained by a preliminary urine liquid–liquid extraction step with ethyl acetate. In this step the organic phase extracts only unconjugated BOLD and ADD, while BOLD in conjugated form remain in urine phase. Afterwards the urine phase, contains conjugated BOLD, was subjected to an enzymatic deconjugation. Solid-phase extraction (OASIS-HLB Waters) was used for the purification and concentration of analytes in organic and urine phases and liquid chromatography ion electrospray tandem mass spectrometry (LC–MS–MS) was applied for the confirmation of BOLD and ADD, using deuterium-labelled 17β-boldenone (BOLD-d3) as internal standard. The method was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/CE. The results obtained demonstrate that the developed method show very high specificity, precision, trueness and ruggedness. Decision limits (CCα) smaller than 0.5ngmL⁻¹ were obtained for each analyte.

Keywords: Boldenone; Conjugate; Unconjugate; Mass spectrometry

Formation of boldenone and boldenone-analogues by maggots of Lucilia sericata by K. Verheyden; H. Noppe; V. Mortier; J. Vercruysse; E. Claerebout; F. Van Immerseel; C.R. Janssen; H.F. De Brabander (pp. 163-170).

Current evidence suggests that neo formation of the anabolic steroid boldenone (androsta-1,4-diene-17-ol-3-one) occurs in calves’ faecal material, making it difficult to distinguish between illegally administered boldenone and its potential endogenous presence. This strengthens the urgent need to elucidate the pathway leading to boldenone formation. In our laboratory, the invertebrate Neomysis integer ( Crustacea, Mysidacea) was used since 2004 as an alternative model for the partial replacement of vertebrate animals in metabolisation studies with illegal growth promotors and veterinary drugs, e.g. boldenone. The present study evaluates the metabolic capacity of other invertebrates, the brine shrimp Artemia franciscana and maggots of the greenbottle fly Lucilia sericata. The first results indicate that maggots of L. sericata are able to convert phytosterols and –stanols, nowadays in substantial amounts added to animal feed, into androsta-1,4-diene-3,17-dione (ADD), the precursor of boldenone, at a yield of 0.10–0.14% ( p<0.001, significance compared to endogenous excretion of maggots) but not to boldenone itself. Furthermore, β-testosterone, an endogenous hormone, was transformed into androst-4-ene-3,17-dione (AED), ADD and β-boldenone at a significant ( p<0.001, significance compared to endogenous excretion of maggots) yield of circa 13%, 0.80% and 2.2%, respectively. In future studies these results are of value to further evaluate the use of maggots of L. sericata as an invertebrate model in metabolisation studies.

Keywords: Boldenone; Anabolic steroids; Phytosterols; Metabolisation; Lucilia sericata

Excretion profile of boldenone in urine of veal calves fed two different milk replacers by R. Draisci; R. Merlanti; G. Ferretti; L. Fantozzi; C. Ferranti; F. Capolongo; S. Segato; C. Montesissa (pp. 171-176).

The residue profiles of 17α-/17β-boldenone conjugated (17α/β-Bol) and ADD were investigated by liquid chromatography–tandem mass spectrometry (LC–MS/MS) in urine of male veal calves fed two commercial milk replacers, with different content of cholesterol and phytosterols. The urine samples were collected within 4h after feeding and further from all the animals. Detectable amounts of 17α-Bol conjugated were measured in urine collected from all calves, but the concentrations of 17α-Bol were higher in urine from calves receiving the milk replacer with the greater amount of phytosterols. During the whole experiment, 17β-Bol and ADD were never detected in urine samples collected.

Keywords: Anabolic hormones; Alpha and beta boldenone metabolism; Milk replacer; Veal calves; Urine; Phytosterols; Liquid chromatography–tandem mass spectrometry

An in vitro study on metabolism of 17β-boldenone and boldione using cattle liver and kidney subcellular fractions by R. Merlanti; G. Gallina; F. Capolongo; L. Contiero; G. Biancotto; M. Dacasto; C. Montesissa (pp. 177-183).

17β-Boldenone (17β-BOLD) and Boldione (ADD) are steroid compounds with androgenic activity, likely to be used as growth promoters in cattle. Different studies still on-going aiming to distinguish between “natural” occurrence or illegal BOLD source had already indicated that their metabolism in cattle is of relevant significance. To identify metabolites as in vivo markers to support the thesis of exogenous administration, a further approach to the in vitro biotransformation of 17β-BOLD and ADD was performed using different subcellular fractions obtained from both liver and kidney of untreated cattle. Polar and non-polar metabolites obtained from incubated parent compounds were formerly separated by high performance liquid chromatography (HPLC) elution and successively identified by liquid chromatography tandem mass spectrometry (LC–MS/MS) detection.The bovine liver was the target tissue of the main metabolic reaction transforming 17β-BOLD to ADD and vice versa. The presence of 6β-hydroxy-17β-BOLD, produced from both compounds when NADPH was added as cofactors to liver post mitochondrial and microsomal fractions suggests that cytochrome P450-dependent enzymes could be involved in the biotransformation, as it occurs for 6β-hydroxylation of 17β-testosterone. The results indicated that the urinary excretion profile in vivo of 6β-hydroxy-17β-BOLD and 16α-hydroxy-17β-BOLD could be studied together with 17α- and 17β-BOLD as putative markers of BOLD treatment in cattle.

Keywords: 17β-Boldenone; Boldione; In vitro; metabolism; Cattle; Liver; Kidney; High performance liquid chromatography; Liquid chromatography tandem mass spectrometry

Quantitation of 17β-nandrolone metabolites in boar and horse urine by gas chromatography–mass spectrometry by Meritxell Roig; Jordi Segura; Rosa Ventura (pp. 184-195).

A method to quantify metabolites of 17β-nandrolone (17βN) in boar and horse urine has been optimized and validated. Metabolites excreted in free form were extracted at pH 9.5 with tert-butylmethylether. The aqueous phases were applied to Sep Pak C₁₈ cartridges and conjugated steroids were eluted with methanol. After evaporation to dryness, either enzymatic hydrolysis with β-glucuronidase from Escherichia coli or solvolysis with a mixture of ethylacetate:methanol:concentrated sulphuric acid were applied to the extract. Deconjugated steroids were then extracted at alkaline pH with tert-butylmethylether. The dried organic extracts were derivatized with MSTFA:NH4I:2-mercaptoethanol to obtain the TMS derivatives, and were subjected to analysis by gas chromatography mass spectrometry (GC/MS). The procedure was validated in boar and horse urine for the following metabolites: norandrosterone, noretiocholanolone, norepiandrosterone, 5β-estran-3α, 17β-diol, 5α-estran-3β, 17β-diol, 5α-estran-3β, 17α-diol, 17α-nandrolone, 17βN, 5(10)-estrene-3α, 17α-diol, 17α-estradiol and 17β-estradiol in the different metabolic fractions. Extraction recoveries were higher than 90% for all analytes in the free fraction, and better than 80% in the glucuronide and sulphate fractions, except for 17α-estradiol in the glucuronide fraction (74%), and 5α-estran-3β, 17α-diol and 17βN in the sulphate fraction (close to 70%). Limits of quantitation ranged from 0.05 to 2.1ngmL⁻¹ in the free fraction, from 0.3 to 1.7ngmL⁻¹ in the glucuronide fraction, and from 0.2 to 2.6ngmL⁻¹ in the sulphate fraction. Intra- and inter-assay values for precision, measured as relative standard deviation, and accuracy, measured as relative standard error, were below 15% for most of the analytes and below 25%, for the rest of analytes. The method was applied to the analysis of urine samples collected after administration of 17βN laureate to boars and horses, and its suitability for the quantitation of the metabolites in the three fractions has been demonstrated.

Keywords: Boar; Horse; Urine; 17β-Nandrolone metabolites; Gas chromatography/mass spectrometry

Quantitation of 17β-nandrolone metabolites in boar and horse urine by gas chromatography–mass spectrometry by Meritxell Roig; Jordi Segura; Rosa Ventura (pp. 184-195).

Keywords: Boar; Horse; Urine; 17β-Nandrolone metabolites; Gas chromatography/mass spectrometry

Quantitation of 17β-nandrolone metabolites in boar and horse urine by gas chromatography–mass spectrometry by Meritxell Roig; Jordi Segura; Rosa Ventura (pp. 184-195).

Keywords: Boar; Horse; Urine; 17β-Nandrolone metabolites; Gas chromatography/mass spectrometry

For almost two decades we have known that enzymatic hydrolysis of “normal” urine samples from the entire male horse using Escherichia coli ( E. coli) followed by solvolysis (ethyl acetate:methanol:sulphuric acid) results in the detection of significant amounts of estr-4-ene-3,17-dione (19-norandrost-4-ene-3,17-dione) along with estr-4-en-17β-ol-3-one (19-nortestosterone, nandrolone) in extracts of the hydrolysed urine and that both steroids are isolated from the solvolysis fraction. This solvolysis process is targeted at the steroid sulphates. Also we have shown that 19-norandrost-4-ene-3,17-dione and 19-nortestosterone are isolated from testicular tissue extracts.Subsequently, evidence was obtained that 19-nortestosterone detected in extracts of “normal” urine from male horses may not be derived from the 17β-sulphate conjugate. However, following administration of 19-nortestosterone based proprietary anabolic steroids to all horses (males, females and castrates), the urinary 19-nortestosterone arising from the administration is excreted primarily as the 17β-sulphate conjugate. Thus, if the 19-nortestosterone-17β-sulphate conjugate arises only following administration this has interesting implications for drug surveillance programmes to control administration of 19-nortestosterone based anabolic preparations to male horses.These results have led us to consider that the precursors to 19-nortestosterone and 19-norandrost-4-ene-3,17-dione, present in the urine prior to the hydrolysis steps, have the same basic structure except for the functionality at the 17-position. We have used preparative high pressure liquid chromatography (LC) and LC fractionation to separate these precursors from the high amounts of oestrogenic sulphates present in “normal” urine from the entire male horse. Purified fractions have then been studied by liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–mass spectrometry (GC–MS) to identify the precursors.

Keywords: Urinary steroidal 19-oic acids; 19-Nortestosterone; 19-Norandros-4-ene-3,17-dione; Male horses; Gas chromatography–mass spectrometry (GC–MS); Liquid chromatography–mass spectrometry (LC–MS); Decarboxylation

Metabolic studies of turinabol in horses by E.N.M. Ho; W.H. Kwok; D.K.K. Leung; T.S.M. Wan; A.S.Y. Wong (pp. 208-216).

Turinabol (4-chloro-17α-methyl-17β-hydroxy-1,4-androstadien-3-one) is a synthetic oral anabolic androgenic steroid. As in the case of other anabolic steroids, it is a prohibited substance in equine sports. The metabolism of turinabol in human has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of turinabol in racehorses with an objective to identify the most appropriate target metabolites for detecting turinabol administration.For the in vitro studies, turinabol was incubated with fresh horse liver microsomes. Metabolites in the incubation mixture were isolated by liquid–liquid extraction and analysed by gas chromatography–mass spectrometry (GC–MS) after trimethylsilylation. The results showed that the major biotransformation of turinabol was hydroxylation at the C6, C16 and C20 sites to give metabolites 6β-hydroxyturinabol (M1), 20-hydroxyturinabol (M2), two stereoisomers of 6β,16-dihydroxyturinabol (M3a, M3b) and 6β,20-dihydroxyturinabol (M4). The metabolite 6β-hydroxyturinabol was confirmed using an authentic reference standard. The structures of all other turinabol metabolites were tentatively identified by mass spectral interpretation.For the in vivo studies, two horses were administered orally with turinabol. Pre- and post-administration urine samples were collected for analysis. Free and conjugated metabolites were isolated using solid-phase extraction and analysed by GC–MS as described for the in vitro studies. The results revealed that turinabol was extensively metabolised and the parent drug was not detected in urine. Two metabolites detected in the in vitro studies, namely 20-hydroxyturinabol and 6β,20-dihydroxyturinabol, these were also detected in post-administration urine samples. In addition, 17-epi-turinabol (M5) and six other metabolites (M6a–M6c and M7a–M7c), derived fromD-ring hydroxylation andA-ring reduction, were also detected. Except for 17-epi-turinabol, none of these metabolites has ever been reported in any species. All in vivo metabolites were detected within 48h after administration.

Keywords: Metabolism; Horse; Turinabol; Steroids

Metabolic studies of turinabol in horses by E.N.M. Ho; W.H. Kwok; D.K.K. Leung; T.S.M. Wan; A.S.Y. Wong (pp. 208-216).

Keywords: Metabolism; Horse; Turinabol; Steroids

Metabolic studies of turinabol in horses by E.N.M. Ho; W.H. Kwok; D.K.K. Leung; T.S.M. Wan; A.S.Y. Wong (pp. 208-216).

Keywords: Metabolism; Horse; Turinabol; Steroids

Confirmatory analysis of acetylgestagens in plasma using liquid chromatography–tandem mass spectrometry by Sarah Kelly Mortensen; Mikael Pedersen (pp. 217-222).

A confirmatory method has been developed and validated for the determination of chlormadinone acetate (CMA), megestrol acetate (MGA), melengestrol acetate (MLA) and medroxyprogesterone acetate (MPA) in bovine and porcine plasma. Analytes are extracted from plasma samples using matrix-assisted liquid–liquid extraction (LLE) on Extrelut NT columns followed by C18 solid-phase extraction (SPE). Analytes were analysed using liquid chromatography–tandem mass spectrometry (LC–MS/MS), and quantification was performed using matrix-matched calibration standards in combination with deuterated internal standards. In accordance with Commission Decision 2002/657/EC, two ion transitions were monitored for each analyte. Decision limits (CCα) were estimated by analysing 20 blank plasma samples and ranged from 0.1 to 0.2ngmL⁻¹. Detection capabilities (CCβ) were estimated using 20 plasma samples fortified at 0.5ngmL⁻¹ and were <0.5ngmL⁻¹. In the range 0.5–2ngmL⁻¹, the mean intra-laboratory reproducibility of the analytes ranged from 6 to 18% (%R.S.D.). Analytes were shown to be stable in fortified plasma samples for >8 months when stored at −20°C.

Keywords: Gestagens; Liquid chromatography–tandem mass spectrometry (LC–MS/MS); Monitoring; Plasma; Residues; Steroids

Confirmatory analysis of acetylgestagens in plasma using liquid chromatography–tandem mass spectrometry by Sarah Kelly Mortensen; Mikael Pedersen (pp. 217-222).

Keywords: Gestagens; Liquid chromatography–tandem mass spectrometry (LC–MS/MS); Monitoring; Plasma; Residues; Steroids

Confirmatory analysis of acetylgestagens in plasma using liquid chromatography–tandem mass spectrometry by Sarah Kelly Mortensen; Mikael Pedersen (pp. 217-222).

Keywords: Gestagens; Liquid chromatography–tandem mass spectrometry (LC–MS/MS); Monitoring; Plasma; Residues; Steroids

Identification of a probable new adrenergic agonist by nuclear magnetic resonance and mass spectrometry by Gianpiero Boatto; Nicola Culeddu; Cecilia Testa; Bruno Neri; Gianfranco Brambilla; Jorge Barbosa; Clara Cruz (pp. 223-227).

In animal production, it is consolidated the synthesis and the illegal use of growth promoters of new generation, able to skip routine screening and confirmatory analysis. In this work it is reported the nuclear magnetic resonance (NMR) and the mass spectrometry identification of a probable new adrenergic drug found in a feed premix. The substance was selectively purified on alpha 1 acid glycoprotein affinity columns; then its structure was first achieved by recording the¹³C NMR spectrum that gave the total number of carbons of the molecule, successively sorted by DEPT experiments into quaternary, CH, CH₂, and CH₃ groups. However, the complete assignments of all resonances were derived from the bi-dimensional analysis and the crucial indications from the¹H–¹³C reverse experiments. Further characterisation was performed by atmospheric pressure chemical ionisation both in positive and negative ion mode, matching the molecular ion and the fragmentation pattern with those of most recently described new adrenergic agonists. After the loss of a ter-butylic group, the structure shows an internal symmetry along with the presence of Chlorine clusters. The proposed formula of the compound, the 8,8′-diamino-9,9′-dichloro-1-terbutyl-1,1′,4,4-tetrahydro-5 H,5′ H-2,2′-bi-1-benzazepine-5,5′-dione, partially resembles that of Zilpaterol for the presence of a heterocyclic ring; Further work is in progress to characterise the structure–activity relationship.

Keywords: Adrenergic agonist; Nuclear magnetic resonance; Mass spectrometry

Illicit treatments in cattle and urinary 6beta-hydroxycortisol/cortisol ratio by F. Capolongo; M. Tapparo; R. Merlanti; L. Ravarotto; E. Tealdo; G. Gallina; C. Montesissa; M. Dacasto (pp. 228-232).

Dexamethasone (DXM) is often illegally used as a growth promoter. To identify indirect biomarkers of illicit treatments, the urinary ratio between 6beta-hydroxycortisol (6beta-OHF) and cortisol (F) was measured in urines obtained from bulls experimentally treated per os and intramuscularly (im) with different DXM dosages.Dexamethasone, given per os at low doses elicited an early and lasting significant reduction of 6beta-OHF/F. No significant variations were seen in urines from bulls given DXM intramuscularly.These results suggest 6beta-OHF/F as a rapid, non-invasive, screening test for oral, low-dose, long-term corticosteroid treatment in cattle. Further studies are required to go deep inside the biochemical and molecular events underlying such an effect.

Keywords: Dexamethasone; Bull; Urine; 6Beta-hydroxycortisol/cortisol

Illicit treatments in cattle and urinary 6beta-hydroxycortisol/cortisol ratio by F. Capolongo; M. Tapparo; R. Merlanti; L. Ravarotto; E. Tealdo; G. Gallina; C. Montesissa; M. Dacasto (pp. 228-232).

Keywords: Dexamethasone; Bull; Urine; 6Beta-hydroxycortisol/cortisol

Illicit treatments in cattle and urinary 6beta-hydroxycortisol/cortisol ratio by F. Capolongo; M. Tapparo; R. Merlanti; L. Ravarotto; E. Tealdo; G. Gallina; C. Montesissa; M. Dacasto (pp. 228-232).

Keywords: Dexamethasone; Bull; Urine; 6Beta-hydroxycortisol/cortisol

Determination of gestagens in kidney fat by liquid chromatography tandem mass spectrometry by Madis Lõhmus; Tiia Kender (pp. 233-238).

The use of gestagens in animal fattening is prohibited within the European Union. Recently, the use of spectrometric methods for the detection and confirmation of banned substances was made obligatory. Therefore, conventional high-performance liquid chromatographic (HPLC) methods have been superseded. It has been possible to couple a previously described HPLC method for the determination of acetyl-gestagens in kidney fat to tandem mass spectrometry (LC-MS/MS). The decision limits CCα and the detection capability CCβ are found to be below the minimum required performance limit (MRPL) established for medroxyprogesterone acetate (MPA) at 1μgkg⁻¹. The calculated values for CCα are as follows: megestrol acetate (MGA)—0.15μgkg⁻¹, melengesterol acetate (MLA)—0.15μgkg⁻¹, chlormadinone acetate (CMA)—0.37μgkg⁻¹ and for medroxyprogesterone acetate (MPA)—0.24μgkg⁻¹. The CCβ values for these compounds have been determined as 0.19, 0.19, 0.47 and 0.32μgkg⁻¹, respectively.

Keywords: Liquid chromatography; Tandem mass spectrometry; Gestagens; Kidney fat; Acetyl-gestagens; Decision limit; Detection capability

Determination of gestagens in kidney fat by liquid chromatography tandem mass spectrometry by Madis Lõhmus; Tiia Kender (pp. 233-238).

Keywords: Liquid chromatography; Tandem mass spectrometry; Gestagens; Kidney fat; Acetyl-gestagens; Decision limit; Detection capability

Determination of gestagens in kidney fat by liquid chromatography tandem mass spectrometry by Madis Lõhmus; Tiia Kender (pp. 233-238).

Keywords: Liquid chromatography; Tandem mass spectrometry; Gestagens; Kidney fat; Acetyl-gestagens; Decision limit; Detection capability

A rapid surface plasmon resonance (SPR) biosensor immunoassay for screening of somatotropins in injection preparations by Tom H.J. Heutmekers; Maria G.E.G. Bremer; Willem Haasnoot; Michel W.F. Nielen (pp. 239-245).

The use of growth hormones (recombinant somatotropins (rSTs)) is approved in several countries, e.g. the USA, Brazil and Australia to enhance growth or lactating performances of livestock. Their use in the EU is banned, however, due to the widespread application, the illegal use within the EU cannot be excluded. To screen for rSTs in injection preparations, a biosensor immunoassay (BIA) using surface plasmon resonance (SPR) technology was developed. Compared to existing analysis methods for rSTs, like radio immunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), this technique provides a rapid (7min) alternative. A direct BIA was compared to an indirect (inhibition) BIA and the performances of several antibodies against (r)STs were compared in the indirect BIA. In the final inhibition assay, using rabbit anti-bovine rST, extracts from several injection preparations were shown to contain bovine rST (rbST). The limit of detection for rbST in the assay is 0.008μgmL⁻¹ which is far below the expected concentrations in injection preparations. Although the cross-reactivities for STs of other species were low, screening of injection preparations for porcine, equine and human ST was feasible through the analysis of less diluted extracts. Tryptic digestion followed by nano-electrospray liquid chromatography–ion trap tandem mass spectrometry (nano-LC–MS/MS) was used to identify STs.

Keywords: Growth hormones; Somatotropins; Surface plasmon resonance; Biosensor; Immunoassay; Injection preparations

The effect of long-term exposure to combinations of growth promoters in Long Evans rats by G. Silvan; M.M. Martínez-Mateos; A. Blass; L. Camacho; A. Gonzalez-Gil; P. Garcia-Partida; J.C. Illera (pp. 246-251).

The aim of the study was to investigate whether the chronic administration (45 days) of clenbuterol (CB) at a growth promoting dose (1mgkg⁻¹ bw) and/or dexamethasone (DEX: 0.1mgkg⁻¹ bw) may cause the disruption of rat endocrine adrenal function. Blood samples were taken weekly during the whole experiment (S0–S7), and at different days of withdrawal (W0, W5, W10, W15 and W20). Hormone profiles were determined by RIA (ACTH) or EIA (corticosterone and catecholamines). ACTH showed significantly elevated concentrations from S1 until W5 ( p<0.05) with CB administration. It began to decrease the day of DEX and CB-DEX administration. DEX showed significantly lowered ACTH concentrations from the day of drug injection ( p<0.05). Corticosterone showed significantly elevated levels until W10 ( p<0.01) with CB and CB+DEX. DEX showed lowered levels of corticosterone during the whole withdrawal period. Epinephrine presented significantly elevated plasma levels until W5 with CB and CB+DEX. With DEX, epinephrine was also elevated from W5 to W15 ( p<0.05). Norepinephrine also presented significantly elevated plasma levels until S7 with CB and CB+DEX ( p<0.001). With DEX no differences were found. Conclusion: Long-term administration of CB and/or DEX causes an endocrine adrenal disruption with changes in ACTH, glucocorticoid and catecholamine secretion.

Keywords: Growth promoters; Clenbuterol; Dexamethasone; Adrenal endocrinology; Corticotropin (ACTH); Corticosterone; Catecholamines; Rats

The effect of long-term exposure to combinations of growth promoters in Long Evans rats by J.C. Illera; L. Peña; M.M. Martínez-Mateos; L. Camacho; A. Blass; P. Garcia-Partida; M.J. Illera; G. Silván (pp. 252-258).

The aim of the study was to investigate the effects of long-term exposure (45 days) to growth promoters: clenbuterol (CB: 1mgkg⁻¹bw) and/or dexamethasone (DEX: 0.1mgkg⁻¹bw), in adrenal gland morphology, and the possibility of recovery after the withdrawal of drug treatment. Animals were sacrificed at different days of withdrawal (W0, W5, W10, W15 and W20), and adrenal glands processed for histopathology and immunohistochemistry. Adrenals of CB treatment showed typical features of long-term administration of β-agonists at W0 such as capillary dilatation in the fasciculata-reticularis zone, and this feature was also presented at W20. Adrenals of CB+DEX treatments showed the same results of CB treatment at days W0 and W20. However, DEX treatment presented the typical results of the exposure to corticoids with the atrophy of adrenal cortex. Immunohistochemistry of adrenal cortex steroidogenic enzymes (P450: scc, 3β-HSD, aromatase) denoted that neither positive staining nor localization was affected by treatments. Aromatase enzyme was immunolocalized in adrenal medulla cells in controls as well as in treated groups. The immunolocalization of glucocorticoid receptors showed an increase in CB (+++) and CB+DEX (++) treatments compared to the control group (0) and DEX treatment (0). Histopathological and immunohistochemical results are closely related to those found for adrenal endocrine function. We can conclude that chronic administration of growth promoters influence adrenal morphology and glucocorticoid receptor expression.

Keywords: Growth promoters; Clenbuterol; Dexamethasone; Adrenal histopathology; Immunohistochemistry; Steroidogenic enzymes; Rats

Dual biosensor immunoassay-directed identification of fluoroquinolones in chicken muscle by liquid chromatography electrospray time-of-flight mass spectrometry by Gerardo R. Marchesini; Willem Haasnoot; Philippe Delahaut; Hüsniye Gerçek; Michel W.F. Nielen (pp. 259-268).

Fluoroquinolones (FQs) are synthetic antibiotics of broad-spectrum antibacterial activity widely used to treat infections in farmed fish, turkeys, pigs, calves and poultry. Monitoring these substances residues is therefore regulated by law.For the detection of FQs, we studied the feasibility of coupling the simultaneous screening of several FQs, using a dual surface plasmon resonance (SPR) biosensor immunoassay (BIA), in parallel, with an analytical chemical methodology for their identification.Six FQs were simultaneously screened at or below their maximum residue level (MRL) in chicken muscle using a multi-FQ BIA for norfloxacin, ciprofloxacin, enrofloxacin, difloxacin and sarafloxacin, and a specific BIA for flumequine. The two BIAs were serially coupled in a multi-channel SPR biosensor featuring a dual BIA in a competitive inhibition format.The samples non-compliant during the screening with the dual BIA were further concentrated and fractionated with gradient liquid chromatography (LC). The effluent was splitted toward two 96-well fraction collectors resulting in two identical 96-well plates. One was re-screened with the dual BIA to identify the immunoactive fractions and direct the identification efforts toward the relevant fractions in the second well-plate with high resolution LC-electrospray time-of-flight mass spectrometry (ESI-TOFMS). The system not only allows the possibility to screen and identify known FQs, but also to discover unknown chemicals of similar structure which show activity in the dual BIA.

Keywords: Fluoroquinolones; Biosensor immunoassays; Generic antibodies; Surface plasmon resonance; Time of flight mass spectrometry; High resolution liquid chromatography

Simultaneous multiresidue determination of tetracyclines and fluoroquinolones in catfish muscle using high performance liquid chromatography with fluorescence detection by Marilyn J. Schneider; Ahmed M. Darwish; Donald W. Freeman (pp. 269-274).

Efficient methods are needed for analysis of veterinary drug residues in food. A number of methods are available for single analytes. Multiresidue methods are now increasingly available. It is still rare, however, to find methods not involving mass spectrometry which allow for analysis of more than one class of drug residue. An efficient multiresidue method for the simultaneous determination of fluoroquinolones (FQs) and tetracyclines (TCs) in catfish muscle has now been developed. This method involves an extraction of the analytes with a mixture of acetonitrile and citrate buffer containing magnesium chloride. After centrifugation and evaporation of the supernatants, the residues are determined using high performance liquid chromatography with fluorescence detection. With this method, five fluoroquinolones and three tetracyclines were determined in fortified catfish muscle at levels of 20, 50, and 100ngg⁻¹. Average recoveries for ciprofloxacin (CIP), sarafloxacin (SAR), danofloxacin (DANO), enrofloxacin (ENRO), difloxacin (DIF), oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) were in the range of 60–92% with good relative standard deviations. The limits of quantitation ranged from 0.15 to 1.5ngg⁻¹. Utilization of the method to successfully analyze catfish muscle samples incurred with enrofloxacin and with oxytetracycline is described.

Keywords: High performance liquid chromatography-fluorescence; Fluoroquinolones; Tetracyclines; Fish

Experiences with an identification and quantification program for inhibitor-positive milk samples by Claudia Kress; Caroline Seidler; Bianca Kerp; Elisabeth Schneider; Ewald Usleber (pp. 275-279).

Beta-lactam antibiotics (penicillins, cephalosporins) are still the most commonly used antibiotics for dairy cows in Germany. In routine milk testing, according to the German milk quality regulation, a positive result obtained for bulk tank milk by microbiological inhibitor tests needs no further confirmation, but results in reduced milk payment of €0.05kg⁻¹ for one month. In some cases, however, further identification of the causative agent can be of interest, either if antimicrobial drugs have not knowingly been used recently, or if improper use of such drugs is denied. As a service for milk producers, our laboratory offers further analyses of violative milk samples, aiming at the identification and quantification of the inhibitor(s). In this program, a panel of microbiological inhibitor tests, receptor tests, and enzyme immunoassays (EIA) is used in a step-by-step analysis, which primarily focusses on β-lactams, but also includes other compounds such as sulfonamides or tetracyclines, respectively. Here we report results for violative milk samples ( n=63) analysed between 2003 and 2005. In most cases (95%), β-lactam antibiotics could be identified, although not always at levels exceeding the respective MRL values. Penicillin G (mostly together with benzylpenicilloyl metabolites) could be identified in 74.6% of all samples. Other compounds identified were, in decreasing order, ceftiofur (11%), ampicillin/amoxicillin (6.3%), isoxazolyl penicillins (3.2%), and sulfonamides (1.6%). The results indicate that penicillin G is still the predominant antibiotic responsible for violative bulk tank milk samples as detected during regulatory control.

Keywords: Milk; β-Lactum antibiotics; Pencillin G; Ceftiofur; Residue

Experiences with an identification and quantification program for inhibitor-positive milk samples by Claudia Kress; Caroline Seidler; Bianca Kerp; Elisabeth Schneider; Ewald Usleber (pp. 275-279).

Keywords: Milk; β-Lactum antibiotics; Pencillin G; Ceftiofur; Residue

Experiences with an identification and quantification program for inhibitor-positive milk samples by Claudia Kress; Caroline Seidler; Bianca Kerp; Elisabeth Schneider; Ewald Usleber (pp. 275-279).

Keywords: Milk; β-Lactum antibiotics; Pencillin G; Ceftiofur; Residue

Validation and comparison of the Copan Milk Test and Delvotest SP-NT for the detection of antimicrobials in milk by Marie-Hélène Le Breton; Marie-Claude Savoy-Perroud; Jean-Marc Diserens (pp. 280-283).

The Delvotest SP-NT and Copan Milk Test, two microbiological tests designed for screening antimicrobial substances in milk were compared and validated. The performance criteria described by the European Decision 2002/657/EC were used for the study. Both tests were evaluated with visual and automated reading (scanner) and the validation was performed on 10 different antibiotics (penicillin-G, cloxacillin, sulfamethazine, sulfadiazine, oxytetracycline, gentamicin, cephalexin, cefquinome, dihydrostreptomycin and trimethoprim). Both tests were found to detect penicillin, cloxacillin, sulfamethazine, sulfadiazine, cephalexin and gentamicin at or below the EU maximum residue limits (MRLs). Some other antibiotics such as oxytetracycline, dihydrostreptomycin, trimethoprim and cefquinome were not detected or only with a low sensitivity. Both tests were found easy to use, robust and fulfilled EU requirements.

Keywords: Milk; Antibiotics; Screening; Inhibitory test; Validation

Stability of frozen stock solutions of beta-lactam antibiotics, cephalosporins, tetracyclines and quinolones used in antibiotic residue screening and antibiotic susceptibility testing by Lieve Okerman; Johan Van Hende; Lieven De Zutter (pp. 284-288).

The stability of frozen stock solutions of antibiotics belonging to three different families was evaluated using an agar diffusion test, with Bacillus subtilis as a test strain. Diameters of inhibition zones were measured at monthly intervals during 6 months, and the decline in active substance was calculated. Penicillin and amoxicilline lost nearly half of their potency, the cephalosporins ceftiofur and cefapirin one quarter, but ampicillin was more stable. The quinolones flumequine, enrofloxacin and marbofloxacin were relatively stable; the loss of activity was less than 10% after 6 months of preservation at −20°C. This was also the case for doxycycline and chlortetracycline, while oxytetracycline and tetracycline lost about 25% of their potency. When used in microbiology, i.e. for residue testing or for determination of minimum inhibitory concentrations, a diminution of activity less than 25% will not be noticed. For these applications, the four tetracyclines and three quinolones tested can be kept for 6 months at −20°C, while the beta-lactam antibiotics should be discarded after 3 months. Standard stock solutions of beta-lactam antibiotics and cephalosporins should preferably be used the same day when they are intended for quantitative residue analysis.

Keywords: Stock solutions; Microbiology; Tetracyclines; Beta-lactam; Fluoroquinolones

Rapid multi-residue screening of antibiotics in muscle and kidney by liquid chromatography-electrospray ionization–tandem mass spectrometry by K. Granelli; C. Branzell (pp. 289-295).

A new approach for simple and rapid multi-residue screening of antibiotics in muscle and kidney by liquid chromatography–tandem mass spectrometry (LC–MS/MS) is described.Nineteen analytes from five classes of antibiotics, i.e. tetracyclines, sulfonamides, quinolones, β-lactams and macrolides were included in the method. The recently registered drug acetylisovaleryltylosin and its metabolite 3- ortho-acetyltylosin, for which no methods of analysis have been published so far, are among the analytes.The samples were extracted by a single extraction using 70% methanol, diluted with water and injected in the LC–MS/MS. The total run time for each sample was 15min. At least 60 samples could be analysed and evaluated in 24h. The accuracy of the method is sufficiently good for screening of antibiotics at the maximum residue limit (MRL) in both muscle and kidney from pig, cattle, sheep, deer, horse and reindeer. Validation was performed according to Commission Decision 2002/657/EC.In addition, a possible way of rapid and simple confirmation of the analytes in muscle is suggested.

Keywords: Antibiotics; Enrofloxacin; Ciprofloxacin; Danofloxacin; Difloxacin; Sulfadiazine; Sulfathiazole; Sulfametazine; Sulfadoxine; Amoxicillin; Ampicillin; Penicillin G; Tylosin; Spiramycin; Acetylisovaleryltylosin; 3-; Ortho; -acetyltylosin; Oxytetracycline; Chlortetracycline; Tetracycline; Doxycycline; Muscle; Kidney; liquid chromatography–tandem mass spectrometry (LC–MS/MS); Veterinary drugs

Rapid multi-residue screening of antibiotics in muscle and kidney by liquid chromatography-electrospray ionization–tandem mass spectrometry by K. Granelli; C. Branzell (pp. 289-295).

Development of a receptor-based microplate assay for the detection of beta-lactam antibiotics in different food matrices by Janine Lamar; Michael Petz (pp. 296-303).

The penicillin-binding protein PBP 2x^* from Streptococcus pneumoniae has been utilised to develop a novel microplate assay for the detection and determination of penicillins and cephalosporins with intact beta-lactam structure in milk, bovine and porcine muscle juice, honey and egg. In the assay, the receptor protein is immobilised to a microplate in the first step. To each sample a bifunctional reagent is added, with ampicillin and digoxigenin as functional groups (DIG-AMPI). The amount of bifunctional reagent, which is bound via its ampicillin part to the receptor protein, decreases with increasing beta-lactam concentration in the sample. The detection step uses anti-digoxigenin F_ab fragments marked with horseradish peroxidase. The more bifunctional reagent is bound to the receptor protein, the more antibody fragments are bound via the digoxigenin part of the reagent. A maximum colour development with tetramethylbenzidine as chromogen for the peroxidase reaction is achieved, when no beta-lactam residues are present.A fractional factorial design was applied to detect chemometrically effects and interactions of the assay parameters. For optimisation of the significant parameters a Box-Behnken design was used.The assay has been developed for various food matrices as screening test with the option for a quantitative assay, when the identity of the residual beta-lactam is known (e.g. elimination studies). Cefoperazon, cefquinome, cefazolin, cloxacillin, ampicillin and benzylpenicillin could be detected at levels corresponding to 1/2EU maximum residue limit (MRL) in milk, meat juice from muscle tissue of different species, egg and honey (where applicable) without needing lengthy and elaborate sample pre-treatment. Matrix calibration curves are presented, which show that quantitative analyses are possible.

Keywords: Penicillins; Cephalosporins; Penicillin-binding protein; Chemometric optimisation; Residues; Food

Development of a receptor-based microplate assay for the detection of beta-lactam antibiotics in different food matrices by Janine Lamar; Michael Petz (pp. 296-303).

Keywords: Penicillins; Cephalosporins; Penicillin-binding protein; Chemometric optimisation; Residues; Food

Development of a receptor-based microplate assay for the detection of beta-lactam antibiotics in different food matrices by Janine Lamar; Michael Petz (pp. 296-303).

Keywords: Penicillins; Cephalosporins; Penicillin-binding protein; Chemometric optimisation; Residues; Food

Degradation of incurred tylosin to desmycosin—Implications for residue analysis of honey by Thomas S. Thompson; Stephen F. Pernal; Donald K. Noot; Adony P. Melathopoulos; Johan P. van den Heever (pp. 304-311).

As a result of the application of tylosin to honey bee colonies for the control of American foulbrood disease, antibiotic residues may exist in honey destined for human consumption. It has been recognized that the parent compound, tylosin A, degrades in acidic media such as honey to yield the antimicrobially active degradation product, desmycosin. Data is presented documenting levels of incurred tylosin and desmycosin in honey resulting from simulated therapeutic applications of a commercial formulation of tylosin during the fall. It is demonstrated that honey destined for human consumption should be analyzed for both tylosin A and desmycosin (tylosin B) rather than the parent antibiotic alone. An analytical method that permits the simultaneous determination of tylosin A and desmycosin in honey using liquid chromatography–tandem mass spectrometry is also presented.

Keywords: Tylosin; Desmycosin; Antibiotics; Honey; Residues; Liquid chromatography–tandem mass spectrometry (LC–MS/MS); American foulbrood (AFB); Paenibacillus larvae

Biosensor immunoassay for flumequine in broiler serum and muscle by Willem Haasnoot; Hüsniye Gerçek; Geert Cazemier; Michel W.F. Nielen (pp. 312-318).

Flumequine (Flu) is one of the fluoroquinolones most frequently applied for the treatment of broilers in The Netherlands. For the detection of residues of Flu in blood serum of broilers, a biosensor immunoassay (BIA) was developed which was fast (7.5min per sample) and specific (no cross-reactivity with other (fluoro)quinolones). This inhibition assay was based on a rabbit polyclonal anti-Flu serum and a CM5 biosensor chip coated with Flu which could be detected in the range of 15–800ngmL⁻¹.For the detection of Flu in muscle, an easy extraction procedure in buffer was selected and the measuring range was from 24 to 4000ngg⁻¹. Average recoveries of 66 till 75% were found with muscle samples spiked at 0.5, 1 and 2 times the maximum residue limit (MRL in muscle=400ngg⁻¹) and the decision limit (CCα) and the detection capability (CCβ) were determined as 500 and 600ngg⁻¹, respectively.Incurred muscle samples were analysed by the BIA and by LC-MS/MS and a good correlation was found ( R²=0.998). Serum and muscle samples from with Flu treated broilers were analysed and the concentrations found in serum were always higher than those found in muscle (average serum/muscle ratio was 3.5) and this proved the applicability of the BIA in serum as predictor of the Flu concentration in muscle.

Keywords: Biosensor immunoassay; Surface plasmon resonance; Flumequine; Broiler; Serum; Muscle

Biosensor immunoassay for flumequine in broiler serum and muscle by Willem Haasnoot; Hüsniye Gerçek; Geert Cazemier; Michel W.F. Nielen (pp. 312-318).

Keywords: Biosensor immunoassay; Surface plasmon resonance; Flumequine; Broiler; Serum; Muscle

Biosensor immunoassay for flumequine in broiler serum and muscle by Willem Haasnoot; Hüsniye Gerçek; Geert Cazemier; Michel W.F. Nielen (pp. 312-318).

Keywords: Biosensor immunoassay; Surface plasmon resonance; Flumequine; Broiler; Serum; Muscle

Quantitative determination of amoxicillin in animal feed using liquid chromatography with tandem mass spectrometric detection by S. De Baere; Patrick De Backer (pp. 319-325).

A liquid chromatographic tandem mass spectrometric (LC-MS/MS) method for the determination of amoxicillin (AMO) in animal feed was developed and validated. The method was used to examine the quality requirements for products intended for incorporation into animal feedingstuffs (medicated premixes), as documented in the EMEA/CVMP/080/95-Final guideline.After addition of the internal standard (ampicillin), the medicated feed samples were extracted using a 0.01M potassium dihydrogenphosphate buffer solution (pH 4.5), followed by a centrifugation and filtration step. An appropriately diluted aliquot of the extract was analysed on a PLRP-S polymeric column (150mm×2.1mm i.d., 100Å) using a mixture of 0.1% formic acid in water and acetonitrile as the mobile phase. Gradient elution was performed at a flow-rate of 0.2mLmin⁻¹. The mass spectrometer was used in the positive electrospray ionization MS/MS mode. The LC-MS/MS method was validated for linearity, trueness, precision, limit of quantification, limit of detection and specificity. The results fell within the ranges specified. The method was used for the homogeneity and stability testing of AMO in a commercial medicated feed. Some extracts were also injected onto a LC-UV and LC-fluorescence instrument (after pre-column derivatization with a formaldehyde reagent). These experiments showed that the LC-MS/MS method was superior with regard to speed of analysis, selectivity and sensitivity.

Keywords: Amoxicillin; Animal feed; Liquid chromatography-tandem mass spectrometry (LC-MS/MS); Quality requirements

Quantitative determination of amoxicillin in animal feed using liquid chromatography with tandem mass spectrometric detection by S. De Baere; Patrick De Backer (pp. 319-325).

Keywords: Amoxicillin; Animal feed; Liquid chromatography-tandem mass spectrometry (LC-MS/MS); Quality requirements

Quantitative determination of amoxicillin in animal feed using liquid chromatography with tandem mass spectrometric detection by S. De Baere; Patrick De Backer (pp. 319-325).

Keywords: Amoxicillin; Animal feed; Liquid chromatography-tandem mass spectrometry (LC-MS/MS); Quality requirements

Oxytetracycline as environmental contaminant in arable lands by Gianfranco Brambilla; Michele Patrizii; Stefania Paola De Filippis; Giuseppe Bonazzi; Paolo Mantovi; Davide Barchi; Luciana Migliore (pp. 326-329).

Oxytetracycline (OXY) is a broad-range antimicrobial routinely used in pig production, at doses in the range of few g/kg of medicated feed, during the weaning period. It could persist at ppm level in pig liquid manure that routinely is used for organic fertilisation. In the present work we describe a methodology to study OXY environmental fate in arable land where crops are cultivated for animal feeding purposes. A liquid-liquid extraction followed by metal chelate affinity chromatography was applied to environmental samples of manures and soils drawn within a case-control study. Extracts were then analysed by high performance liquid chromatography with UV/DAD detection, using a reverse phase column, and expressing the results as 4-epioxytetracycline epimer. Results indicate OXY is well retained at mgkg⁻¹ levels in soil exposed to contaminated pig manure fertilisation. Such compartment could constitute an abiotic reservoir for the systemic and/or for the external contamination of corn.

Keywords: Oxytetracycline; Manure contamination; Soil contamination

Determination of phoxim residues in eggs by using high-performance liquid chromatography diode array detection after treatment of stocked housing facilities for the poultry red mite ( Dermanyssus gallinae) by G. Hamscher; B. Prieß; H. Nau (pp. 330-335).

The poultry red mite Dermanyssus gallinae is the most important ectoparasite of poultry in several European countries. Phoxim is a well-known antiparasitic agent in wide use. Initial studies indicated that this compound could successfully be applied to eliminate D. gallinae in egg-laying birds and in henhouses by treating the cages and the equipment with it. In order to investigate whether phoxim residues are present in eggs from laying hens, we developed a selective and sensitive high-performance liquid chromatography method employing a simple water/acetonitrile gradient system. The amount of phoxim was determined by UV detection at 281nm, and the presence of the residue was confirmed by diode array detection. The eggs were homogenized for sample pretreatment and extracted with acetonitrile and partitioned with n-hexane. The acetonitrile extract was further purified with silica gel column chromatography. Recovery rates (performed at the 5–120μgkg⁻¹ level) were in the range of 86.0–92.1% with relative standard deviations between 3.1% and 16.3%. Based on a signal to noise ratio of 3, the limit of detection of the assay was approximately 2μgkg⁻¹. The day-to-day variation in the concentration of phoxim in four contaminated eggs (5.7–51.6μgkg⁻¹) was generally less than 20%. The decision limit (CCα) and the detection capability (CCβ) were 62.0 and 68.7μgkg⁻¹, respectively. The applicability of the method was demonstrated in eggs from three clinical trials and from a field study. In these investigations, all animals were kept in conventional battery cages. No sample was found containing more than the maximum residue level of 60μgkg⁻¹ for phoxim in eggs as given in Annex I of Council Regulation (EEC) No. 2377/90.

Keywords: Battery cages; Dermanyssus gallinae; Diode array detection; High-performance liquid chromatography; Laying hens; Phoxim

Multi-residue monitoring for the simultaneous determination of five nitrofurans (furazolidone, furaltadone, nitrofurazone, nitrofurantoine, nifursol) in poultry muscle tissue through the detection of their five major metabolites (AOZ, AMOZ, SEM, AHD, DNSAH) by liquid chromatography coupled to electrospray tandem mass spectrometry—In-house validation in line with Commission Decision 657/2002/EC by Eric Verdon; Pierrick Couedor; Pascal Sanders (pp. 336-347).

Following the ban of four nitrofurans in the mid-90s (furazolidone, furaltadone, nitrofurantoine, nitrofurazone), the nifursol, a veterinary drug from the nitrofuran class of antibacterials which has been used prophylactically as feed additive for treating turkeys against histomoniasis (blackhead disease) was also declared in Annex IV of the European Union Directive no. 90/2377/EC in 2002 according to the Regulation no. 1756/2002/EC. As for the four other nitrofurans, nifursol disappears from tissues within a few days after treatment of food-producing animals. But toxic metabolites are still present for longer periods (several weeks or even months). The major metabolite that can readily be monitored in the tissues following nifursol abuse is the 3,5-dinitro-salicylic acid hydrazine (DNSAH). This article displays some improvements and the revalidation of the analytical method by liquid chromatography coupled to electrospray tandem mass spectrometry (LC-esiMS/MS) already in use in our laboratory for monitoring nitrofuran metabolites but also including the nifursol metabolite at the confirmatory minimum required performance level (MRPL) of 1μgkg⁻¹. The validation is applied both to artificially and to naturally incurred turkey muscle.

Keywords: Nitrofuran metabolites; Furazolidone; Furaltadone; Nitrofurantoine; Nitrofurazone; Nifursol; Turkey muscle; LC–MS/MS

Validation of a confirmatory method for the determination of residues of four nitrofurans in egg by liquid chromatography–tandem mass spectrometry with the software InterVal by C. Bock; C. Stachel; P. Gowik (pp. 348-358).

A method for the detection and determination of nitrofuran derivatives in egg by liquid chromatography–tandem mass spectrometry (LC–MS/MS) was validated with the software InterVal and can be applied for the confirmation of nitrofuran metabolites in fresh or lyophilised eggs. The validation study comprises variations in operator, storage condition, breeding, equipment and duration of sample preparation. A comprehensive overview of the robustness of the method is obtained by analysing eight samples at six concentration levels. First results of short- and medium-term investigations for stability of analytes in solution show that standard solutions of nitrofuran metabolites are stable for at least 1 year when stored at +4°C in the dark. The decision limit CC_α expressed for the underivatised metabolite is 0.05μgkg⁻¹ for 3-amino-5-methyl-morpholino-2-oxazolidinone, 0.03μgkg⁻¹ for 3-amino-2-oxazolidinone, 0.20μgkg⁻¹ for semicarbazide and 0.22μgkg⁻¹ for 1-amino-hydantoin.

Keywords: Nitrofurans; Matrix-comprehensive; InterVal; Decision limit; Detection capability; Liquid chromatography–tandem mass spectrometry (LC–MS/MS)

Determination of nitrofurans in animal feeds by liquid chromatography-UV photodiode array detection and liquid chromatography-ionspray tandem mass spectrometry by Jorge Barbosa; Sara Moura; Rita Barbosa; Fernando Ramos; Maria Irene Noronha da Silveira (pp. 359-365).

Within the EU, the use of nitrofurans is prohibited in food production animals. For this reason detection of these compounds in feedingstuffs, at whatever limit, constitutes an offence under EU legislation. This detection generally involves the use of analytical methods with limits of quantification lowers than 1mgkg⁻¹. These procedures are unsuitable for the detection and confirmation of trace amounts of nitrofurans in feedingstuffs due to contamination. It is well known that very low concentrations of these compounds can be the source of residues of nitrofuran metabolites in meat and other edible products obtained from animals consuming the contaminated feed. The present multi-compound method was capable of measuring very low concentrations of nitrofurantoin (NFT), nitrofurazone (NFZ), furazolidone (FZD) and furaltadone (FTD) in animal feed using nifuroxazide (NXZ) as internal standard. Following ethyl acetate extraction at mild alkaline conditions and purification on NH₂ column, the nitrofurans are determined using liquid chromatography with photodiode-array detection (LC-DAD). It was observed a CCα ranged from 50 to 100μgkg⁻¹. The liquid chromatography-tandem mass spectrometric (LC-MS/MS) procedure was used to confirm the identity of the suspected presence of any of the nitrofuran compounds.

Keywords: Nitrofurans; Feeds; Liquid-chromatography; Photo-diode array detection; Tandem mass spectrometry

The determination of biurea: A novel method to discriminate between nitrofurazone and azodicarbonamide use in food products by P.P.J. Mulder; B. Beumer; J.A. Van Rhijn (pp. 366-373).

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 45mgkg⁻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 10μgkg⁻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.

Keywords: Azodicarbonamide; Biurea; Semicarbazide; Nitrofurazone; Breaded products

The determination of biurea: A novel method to discriminate between nitrofurazone and azodicarbonamide use in food products by P.P.J. Mulder; B. Beumer; J.A. Van Rhijn (pp. 366-373).

Keywords: Azodicarbonamide; Biurea; Semicarbazide; Nitrofurazone; Breaded products

The determination of biurea: A novel method to discriminate between nitrofurazone and azodicarbonamide use in food products by P.P.J. Mulder; B. Beumer; J.A. Van Rhijn (pp. 366-373).

Keywords: Azodicarbonamide; Biurea; Semicarbazide; Nitrofurazone; Breaded products

Determination of residues of azaperone in the kidneys by liquid chromatography with fluorescence detection by Vesna Cerkvenik-Flajs (pp. 374-382).

An analytical method has been developed for the quantitative determination of residues of the tranquillizer azaperone (AZN) in the kidneys of slaughtered animals. Samples were extracted with acetonitrile, extracts were acidified and further purified with solid-phase extraction (SPE) on a polymeric mixed-mode cation-exchange sorbent, Oasis^®. AZN and its main metabolite azaperol (AZL) were eluted by alkaline methanol (MeOH), the eluate was evaporated, re-dissolved and analysed by gradient high performance liquid chromatography (LC) on reversed and deactivated phase LiChrospher 60-RP select B at excitation and emission wavelengths of 245 and 345nm, respectively. The method was validated according to the requirements of European Commission Decision 2002/657/EC, using fortified porcine kidneys. The method proved to be selective, specific against carazolol (CAR) and linear over a concentration range 10–150μgkg⁻¹ ( r²>0.99). Over a concentration range 50–150μgkg⁻¹, mean recovery of AZN and AZL was 88.2 and 91.2%, respectively, with intra-laboratory reproducibility of <11.0 and <9.0%, respectively. The decision limit (CCα) of AZN and AZL was 112 and 111μgkg⁻¹, respectively, and the limit of quantification (LOQ) was 10 and 5μgkg⁻¹, respectively. The procedure was also applied to bovine, poultry and horse kidneys, giving similar results, and has been successfully implemented in statutory residue monitoring control in food of animal origin in Slovenia.

Keywords: Azaperone; Azaperol; Residues; Kidneys; Analysis; Liquid chromatography-fluorescence

Determination of residues of azaperone in the kidneys by liquid chromatography with fluorescence detection by Vesna Cerkvenik-Flajs (pp. 374-382).

Keywords: Azaperone; Azaperol; Residues; Kidneys; Analysis; Liquid chromatography-fluorescence

Determination of residues of azaperone in the kidneys by liquid chromatography with fluorescence detection by Vesna Cerkvenik-Flajs (pp. 374-382).

Keywords: Azaperone; Azaperol; Residues; Kidneys; Analysis; Liquid chromatography-fluorescence

Validation of a method for the detection and confirmation of nitroimidazoles and the corresponding hydroxy metabolites in pig plasma by high performance liquid chromatography–tandem mass spectrometry by Stéphanie Fraselle; Veerle Derop; Jean-Marie Degroodt; Joris Van Loco (pp. 383-393).

Nitroimidazoles (Ronidazole, Dimetridazole, Metronidazole, Ipronidazole) and their hydroxy metabolites are banned substances with antibiotic and anticoccidial activity. They are suspected to be carcinogenic and mutagenic. Since nitroimidazoles showed an inhomogeneous distribution and a rapid degradation in incurred muscle samples, plasma is the preferred target matrix for residue analysis. The analytical method of Polzer et al. [J. Polzer, C. Stachel, P. Gowik, Anal. Chim. Acta 521 (2004) 189] was adapted for liquid chromatography–tandem mass spectrometry detection and was validated in house according to the Commission Decision 2002/657/EC. The method is specific for all nitroimidazole except for Ipronidazole and its metabolite, due to interferences at their retention times in chromatograms of blank plasma and reagents samples. The absence of a matrix effect enables the use of a (linear) calibration curve in solution for quantitation. The apparent recovery (obtained after correction with a deuterated internal standard) is between 93% and 123%, except for the metabolite of Metronidazole (58–63%). The repeatability (CVr=2.49–13.39%) and intralaboratory reproducibility (CVRW=2.49–16.38%) satisfy the Horwitz equation. The obtained values for the detection capacity (CCβ) range from 0.25 to 1μgL−1, while values obtained for the decision limit (CCα) are below CCβ.

Keywords: Nitroimidazoles; Plasma; Liquid chromatography–tandem mass spectrometry; Validation; Commission Decision 2002/657/EC

Confirmation of four nitroimidazoles in porcine liver by liquid chromatography–tandem mass spectrometry by X. Xia; X. Li; S. Zhang; S. Ding; H. Jiang; J. Shen (pp. 394-398).

A sensitive and reliable multiresidue method is described for analysis of ronidazole, metronidazole, dimetridazole and the common metabolite of ronidazole and dimetridazole, 2-hydroxymethyl-1-methyl-5-nitroimidazole in swine liver. The sample preparation procedure was based on liquid–liquid extraction and mixed mode cation exchange/reverse phase solid-phase extraction. The compounds of interest were determined by reverse phase gradient liquid chromatography separation and tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) mode. The limits of confirmation were 0.1–0.5μgkg⁻¹ for the analytes.

Keywords: Nitroimidazole; Liquid chromatography–tandem mass spectrometry; Liver

Confirmation of four nitroimidazoles in porcine liver by liquid chromatography–tandem mass spectrometry by X. Xia; X. Li; S. Zhang; S. Ding; H. Jiang; J. Shen (pp. 394-398).

Keywords: Nitroimidazole; Liquid chromatography–tandem mass spectrometry; Liver

Confirmation of four nitroimidazoles in porcine liver by liquid chromatography–tandem mass spectrometry by X. Xia; X. Li; S. Zhang; S. Ding; H. Jiang; J. Shen (pp. 394-398).

Keywords: Nitroimidazole; Liquid chromatography–tandem mass spectrometry; Liver

Multiwalled carbon nanotubes as a solid-phase extraction adsorbent for the determination of three barbiturates in pork by ion trap gas chromatography–tandem mass spectrometry (GC/MS/MS) following microwave assisted derivatization by Haixiang Zhao; Liping Wang; Yueming Qiu; Zhiqiang Zhou; Weike Zhong; Xiang Li (pp. 399-406).

A new method was developed for the rapid screening and confirmation analysis of barbital, amobarbital and phenobarbital residues in pork by gas chromatography–tandem mass spectrometry (GC/MS/MS) with ion trap MSD. The residual barbiturates in pork were extracted by ultrasonic extraction, cleaned up on a multiwalled carbon nanotubes (MWCNTs) packed solid phase extraction (SPE) cartridge and applied acetone–ethyl acetate (3:7, v/v) mixture as eluting solvent and derivatized with CH₃I under microwave irradiation. The methylated barbiturates were separated on a TR-5MS capillary column and detected with an ion trap mass detector. Electron impact ion source (EI) operating MS/MS mode was adopted for identification and external standard method was employed for quantification. One precursor ion m/ z 169 was selected for analysis of barbital and amobarbital and m/ z 232 was selected for phenobarbital. The product ions were obtained under 1.0V excitation voltage. Good linearities (linear coefficient R>0.99) were obtained at the range of 0.5–50μgkg⁻¹. Limit of detection (LOD) of barbital was 0.2μgkg⁻¹ and that of amobarbital and phenobarbital were both 0.1μgkg⁻¹(S/N≥3). Limit of quatification (LOQ) was 0.5μgkg⁻¹ for three barbiturates (S/N≥10). Satisfying recoveries ranging from 75% to 96% of the three barbiturates spiked in pork were obtained, with relative standard deviations (R.S.D.) in the range of 2.1–7.8%.

Keywords: Barbiturates; Ultrasonic extraction; Multiwalled carbon nanotubes; Microwave assisted derivatization; Gas chromatography–tandem mass spectrometry; Pork

Determination of residues of tricaine in fish using liquid chromatography tandem mass spectrometry by Peter Scherpenisse; Aldert A. Bergwerff (pp. 407-410).

A liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the determination of residues of the anaesthetic tricaine mesilate (MS222) in fish tissues is described. Residues were extracted from homogenized tissues with McIllvaine buffer/methanol and purified over a C₁₈ solid-phase extraction column followed by LC–MS/MS analysis. In the multiple-reaction monitoring mode of the mass spectrometer, chromatograms were recorded by monitoring the m/ z 166→ m/ z 138 and m/ z 166→ m/ z 94 transitions for quantification and confirmation of the residues in the finfish matrix, respectively. Recoveries were in the range of 67%±10% ( n=6) for tilapia at 2μgkg⁻¹, 95%±7% ( n=6) at 2μgkg⁻¹ in salmon and 92%±3% ( n=5) for trout at 2.5μgkg⁻¹. The limits of detection were 0.5, 0.6 and 0.6μgkg⁻¹ in trout, salmon and tilapia, respectively. No residues of tricaine were found in eight sampled aquacultured fish (salmon and trout) bought from the local market.

Keywords: Residue analysis; Veterinary public health; Chemical contaminant; Food quality assurance; Food safety; Aquaculture

Determination of residues of tricaine in fish using liquid chromatography tandem mass spectrometry by Peter Scherpenisse; Aldert A. Bergwerff (pp. 407-410).

Keywords: Residue analysis; Veterinary public health; Chemical contaminant; Food quality assurance; Food safety; Aquaculture

Determination of residues of tricaine in fish using liquid chromatography tandem mass spectrometry by Peter Scherpenisse; Aldert A. Bergwerff (pp. 407-410).

Keywords: Residue analysis; Veterinary public health; Chemical contaminant; Food quality assurance; Food safety; Aquaculture

Confirmatory analysis of malachite green, leucomalachite green, crystal violet and leucocrystal violet in salmon by liquid chromatography–tandem mass spectrometry by Geraldine Dowling; Patrick P.J. Mulder; Conor Duffy; Liam Regan; Malcolm R. Smyth (pp. 411-419).

A method has been developed to analyse for malachite green (MG), leucomalachite green (LMG), crystal violet (CV) and leucocrystal violet (LCV) residues in salmon. Salmon samples were extracted with acetonitrile:McIIIvain pH 3 buffer (90:10 v/v), sample extracts were purified on a Bakerbond strong cation exchange solid phase extraction cartridge. Aliquots of the extracts were analysed by LC–MS/MS. The method was validated in salmon, according to the criteria defined in Commission Decision 2002/657/EC. The decision limit (CC_α) was 0.17, 0.15, 0.35 and 0.17μgkg⁻¹, respectively, for MG, LMG, CV and LCV and for the detection capability (CC_β) values of 0.30, 0.35, 0.80 and 0.32μgkg⁻¹, respectively, were obtained. Fortifying salmon samples ( n=6) in three separate assays, show the accuracy to be between 77 and 113% for MG, LMG, LCV and CV. The precision of the method, expressed as RSD values for the within-laboratory reproducibility, for MG, LMG and LCV at the three levels of fortification (1, 1.5 and 2.0μgkg⁻¹), was less than 13%. For CV a more variable precision was obtained, with RSD values ranging between 20 and 25%.

Keywords: Malachite green; Leucomalachite green; Crystal violet; Leucocrystal violet; Salmon; Method validation

The effects of cooking on residues of malachite green and leucomalachite green in carp muscles by Kamila Mitrowska; Andrzej Posyniak; Jan Zmudzki (pp. 420-425).

The effects of various cooking methods (boiling, baking and microwaving) on residues of malachite green (MG) and its major metabolite, leucomalachite green (LMG), in incurred carp muscles were investigated. Moreover, the stability of MG and LMG standard solutions under boiling in water and in oil was examined. The MG and LMG residues in cooked meat were determined by liquid chromatography with visible and fluorescence detectors. The results showed that in muscles cooked by boiling or baking MG concentration was reduced by 54% in 15min while LMG was stable in these conditions. By microwave cooking MG residues were reduced by 61% after 1min. Microwaving was the only method of cooking when a loss of LMG was observed (40% in 1min). Both MG and LMG standard solutions were stable in boiling water at 100°C. In cooking oil, MG was reduced by 49% after 10min and less than 3% of the original MG remains after 90min at 150°C. No losses of LMG were observed over a time period of 120min in cooking oil at 150°C. Upon increasing the temperature to 210°C and holding for 120min, MG was rapidly reduced by 97% after 10min. LMG under the same conditions was reduced by 18% after 10min. No further loses of MG and LMG were observed after 120min.The findings of this investigation show that the high temperature does not guarantee a full breakdown of residue of MG and LMG which may occur in carp muscles.

Keywords: Malachite green; Leucomalachite green; Residues; Carp; Cooking effects

The effects of cooking on residues of malachite green and leucomalachite green in carp muscles by Kamila Mitrowska; Andrzej Posyniak; Jan Zmudzki (pp. 420-425).

Keywords: Malachite green; Leucomalachite green; Residues; Carp; Cooking effects

The effects of cooking on residues of malachite green and leucomalachite green in carp muscles by Kamila Mitrowska; Andrzej Posyniak; Jan Zmudzki (pp. 420-425).

Keywords: Malachite green; Leucomalachite green; Residues; Carp; Cooking effects

Ex vivo formation of gastric metabolites of clenbuterol: Preliminary characterisation of their chemical structure by Gianfranco Brambilla; Simone di Bez; Donatella Pietraforte; Maurizio Minetti; Luigi Campanella; Alberto Loizzo (pp. 426-431).

The epidemiology of clenbuterol food-borne intoxication outbreaks indicates a possible discrepancy between the severity and long duration of clinical symptoms, and the presumed dose ingested as parent compound residue. In this work, we explore the possibility that clenbuterol could undergo to a biological transformation, in presence of salivary nitrites, at gastric pH (<3). Human salivary specimens were drawn before and after meal, accounting for the different physiological nitrite content (40 and 400μmolL⁻¹, respectively, as average). Clenbuterol (10μmolL⁻¹) was then incubated within the pH range 2–6 and possible products monitored by liquid chromatography–mass spectrometry (LC–MS), drawing at regular intervals serial aliquots of the incubation mixture. With respect to controls, two differential peaks were noted along with a quantitative bio-transformation of the parent compound, at pH values ≤3. Under pre-meal conditions, a 4 mono-nitro compound was identified as main metabolite, whereas under post-meal condition a second metabolite, showing a complete de-chlorination, along with the probable presence of three nitro groups on the aromatic ring, was revealed. The reaction was highly reproducible and the kinetics suggested the involvement of nitrogen-related free radicals. The results are discussed in the light of the possible formation of pharmacological active tissue-bound residues as cause of symptoms severity.

Keywords: Clenbuterol; Saliva; Nitrites; Bio-transformation

Rapid method for the determination of non-steroidal anti-inflammatory drugs in animal tissue by liquid chromatography–mass spectrometry with ion-trap detector by Carmen Igualada; Francisco Moragues; Jorge Pitarch (pp. 432-439).

A rapid and new liquid chromatography–mass spectrometry with ion-trap detection method for the determination of meloxicam (MLX), flunixin meglumine (FLU), carprofen (CPF), and tolfenamic acid (TOLF) in animal tissue is described.MRLs between 10 and 500μgkg⁻¹ in muscle and between 65 and 1000μgkg⁻¹ in liver, from different animal species have been established in the EU for these compounds.After chemical hydrolysis, an organic extraction from homogenised tissue was performed. Final extract was injected in a liquid chromatograph with an ion-trap mass spectrometer with electrospray interface.Four identification points (one precursor and two product ions) and a minimum of one ion ratio was monitored for each compound.For quantitative purposes flunixin-D3 (FLU-D3) was used as internal standard.The method was validated using fortified blank muscle and liver from different animal species according to the 2002/657/EC European decision criteria.The decision limits (CCα) and detection capabilities (CCβ) were determined and their values were at concentrations near the MRL for each substance.

Keywords: Meloxicam; Carprofen; Flunixin meglumine; Tolfenamic acid; Non-steroidal anti-inflammatory drug; Mass spectrometry

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Analytica Chimica Acta (v.586, #1-2)

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Analytica Chimica Acta (v.586, #1-2)