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Analytica Chimica Acta (v.637, #1-2)

Editorial Board (pp. iii).

Preface by Hubert F. De Brabander; Leendert A. Van Ginkel; Saskia Sterk; Aldert Bergwerff (pp. 1-1).

Analysis of thyreostats: A history of 35 years by J. Vanden Bussche; H. Noppe; K. Verheyden; K. Wille; G. Pinel; B. Le Bizec; H.F. De Brabander (pp. 2-12).

Thyreostatic drugs (TS), illegally administrated to livestock for fattening purposes, are banned in the European Union since 1981 (Council Directive 81/602/EC). This paper reviews the trends in the analytical approaches for the determination of TS drugs in biological matrices. After a brief introduction on the different groups of compounds with a thyreostatic action, the most relevant legislation regarding the residue control of these compounds is presented. An overview of the analytical possibilities for the determination of TS in animal matrices, covering sample extraction, purification, separation techniques and detection methods is provided. Additionally, a brief outline of animal experiments is described that illustrates the excretion and distribution profiles of TS residues. Finally, the novel developments in TS analysis are highlighted. Also the possible semi-endogenous status of thiouracil is discussed.

Keywords: Thyreostatic drugs; EU-criteria; Residue analysis; Liquid chromatography; Gas chromatography; Mass spectrometry

Risks of antibiotic residues in milk following intramammary and intramuscular treatments in dairy sheep by A. Pengov; A. Kirbis (pp. 13-17).

Very few drugs on the market are approved for use in lactating ewes. Veterinarians in the European Union are allowed to prescribe drugs in an off-label manner but are then obligated to assure that residues do not enter the food chain. In case of mastitis treatment in dairy ewes antibiotic preparations designed and authorized for the bovine mammary gland are usually used. Due to inter-species differences, available bovine data cannot be accurately extrapolated for use in the dairy ewe. The objective of the study was therefore to determine appropriate withdrawal periods for ewe’s milk following mastitis treatment with two commercial lactating cow products. For the detection of all components standard agar plate diffusion techniques were used.Regardless of the therapy regime and the product used, residues of antibiotics in milk were detected up to 192h after the last infusion. These results indicate that the required withholding periods for ewe’s milk are considerably longer than recommended on the label for bovine milk.

Keywords: Mastitis; Treatment; Ewes; Withdrawal periods

Development and validation of a confirmatory method for the determination of sulphonamides in milk by liquid chromatography with diode array detection by Valentina Gamba; Chiara Terzano; Laura Fioroni; Simone Moretti; Guglielmo Dusi; Roberta Galarini (pp. 18-23).

A simple and rapid multiresidue method for the determination of seven sulphonamides residues (sulfadiazine, sulfapyridine, sulfamerazine, sulfamethazine, sulfamonomethoxine, sulfadimethoxine and sulfaquinoxaline) in milk samples was developed and validated. The drugs were extracted with a mixture chloroform/acetone and simply cleaned up on a cation exchange solid phase extraction column. The analytes determination was carried out using liquid chromatography with diode array detection (DAD). The procedure has validated as a quantitative confirmatory method according to the European Union (EU) Decision 2002/657/EC. The developed method shows good linearity, specificity, precision (repeatability and intra-laboratory reproducibility), ruggedness and is able to confirm each sulphonamide residue above 30μgkg⁻¹. Decision limits (CC α) around 110μgkg⁻¹ and recovery above 56% were obtained for all the analytes. The results of the validation process demonstrate that the method is suitable for application, as confirmatory method, in European Union statutory veterinary drug residue surveillance programmes. In addition, a hypothetical situation of sample judgement (compliance or not) in the case in which, at the same time, two different sulphonamides are found, is discussed.

Keywords: Sulphonamides; Milk; Diode array detector; Commission decision 2002/657/EC

Development, validation and implementation of a receptor based bioassay capable of detecting a broad range of β-agonist drugs in animal feedingstuffs by S. Boyd; H.H. Heskamp; T.F.H. Bovee; M.W.F. Nielen; C.T. Elliott (pp. 24-32).

A bioassay was developed for the detection of a broad range of β-agonist compounds in animal feeds. A solubilised β2-adrenoceptor was utilised as the binding protein in the assay. This protein was found to be highly stable when stored at 80°C. The assay was developed and initially validated to determine the sensitivity and relative selectivity against a panel of commonly used β-agonist compounds. It was also shown that when β-agonists were present as cocktails in samples a pronounced synergistic effect could be measured. The method was further validated according to EC Decision 2002/657 and proved capable of detecting 250ng clenbuterol equivalents per gram of sample. This is well below the quantities normally associated with β-agonist medicated feeds. The β2-adrenoceptor used in the study only failed to bind the compound zilpaterol, raising doubts as to whether this compound is a true β2-adrenergic drug.

Keywords: β-Agonist; β2-adrenoceptor; Receptor assay; Zilpaterol; Animal feeds

Concentrations of danofloxacin 18% solution in plasma, milk and tissues after subcutaneous injection in dairy cows by N. Mestorino; M.L. Marchetti; E. Turic; J. Pesoa; J. Errecalde (pp. 33-39).

Danofloxacin is a fluoroquinolone developed for use in veterinary medicine. Its concentrations and pharmacokinetic profile in plasma, milk and tissues of lactating dairy cows were determined, and its milk withdrawal time (WT) calculated.Twenty-one dairy cows received a single subcutaneous administration of 18% mesylate danofloxacin salt (6mgkg⁻¹). Plasma and milk samples were obtained at different times until 48h. Groups of three animals were sacrificed at different post-administration times and tissue samples (mammary gland, uterus, duodenum, jejunum, ileum, colon and mesenteric lymph nodes) obtained. Danofloxacin concentrations were determined by liquid chromatography with fluorescence detection. The milk WT was calculated by the Time to Safe Concentration method (Software WTM 1.4, EMEA).Danofloxacin was rapidly absorbed and its distribution from plasma to all sampled tissues and milk was extensive. Milk and tissues concentrations were several times above those found in plasma. Plasma area under the curve (AUCp) was 9.69μghmL⁻¹ and its elimination half life (Tβ1/2) was 12.53h. AUC values for the various tissues and milk greatly exceeded AUCp.Tβ1/2 from milk and tissues ranged between 4.57 and 21.91h and the milk withdrawal time was 73.48h.The reported results support the potential use of danofloxacin in the treatment of mastitis and other infections in milk cows with 3 days of withdrawal.

Keywords: Danofloxacin; Dairy cows; High pressure liquid chromatography; Milk; Pharmacokinetics; Plasma; Tissues

A confirmatory method for the determination of tetracyclines in muscle using high-performance liquid chromatography with diode-array detection by Elisa Cristofani; Chiara Antonini; Gloria Tovo; Laura Fioroni; Arianna Piersanti; Roberta Galarini (pp. 40-46).

Using high-performance liquid chromatography with diode-array detection (HPLC-DAD) technique, a confirmatory method for the determination of trace levels of tetracyclines (oxytetracycline, tetracycline, chlortetracycline and doxycycline) and their 4-epimers (4-epioxytetracycline, 4-epitetracycline and 4-epichlortetracycline) in animal tissues (muscle) was developed.The samples are extracted with a mixture of succinic acid 0.1M (pH 4) and methanol after the addition of metacycline as internal standard. The clean-up is carried out by metal chelate affinity chromatography with a following concentration step on an OASIS HLB polymeric reversed phase column. The chromatographic separation of the seven analytes is achieved in 10min on a short monolithic column (50mm×4.6mm i.d.) using a gradient elution.The method was validated in bovine muscle following the Commission Decision 2002/657/EC criteria: samples spiked at four concentration levels (0.25, 0.5, 1 and 1.5 times the maximum residue limit) were analysed. Method trueness and precision (repeatability and intra-laboratory reproducibility) as well as decision limits (CCα) and detection capabilities (CCβ) are reported.

Keywords: Tetracyclines; 4-Epimers; Muscle; High-performance liquid chromatography-diode-array detection; Validation; Commission Decision 2002/657/EC

Confirmatory method for the determination of resorcylic acid lactones in urine sample using immunoaffinity cleanup and liquid chromatography–tandem mass spectrometry by Guglielmo Dusi; Eros Bozzoni; Walter Assini; Nadia Tognoli; Mara Gasparini; Enrica Ferretti (pp. 47-54).

The presence of zeranol (α-zearalanol) in urine samples due to natural contamination or illegal treatment is under debate within the European Union. The simultaneous determination of zeranol, its epimer taleranol (β-zearalanol), zearalanone and the structurally related mycotoxin zearalenone with the corresponding α- and β-zearalenol metabolites appears to be critical in deciding whether an illegal use has occurred. The aim of this study is to develop and validate a simple analytical procedure applicable to bovine and swine urine samples for the determination of all six resorcylic acid lactones. After an enzymatic deconjugation, the urine was subjected to a one-step cleanup on a commercially available immunoaffinity chromatography cartridge. The analytes were detected by liquid chromatography–negative-ion electrospray tandem mass spectrometry using deuterium-labelled internal standards. The method was validated as a quantitative confirmatory method according to European Commission Decision 2002/657/EC. The evaluated parameters were: linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit, detection capability and ruggedness. The decision limits (CCα) obtained, were between 0.56 and 0.68μgL⁻¹; recovery above 66% for all the analytes. Repeatability was between 1.4 and 5.3% and within-laboratory reproducibility between 1.9 and 16.1% for the six resorcylic acid lactones.

Keywords: Zeranol; Resorcylic acid lactones; Urine; Immunoaffinity chromatography; Mass spectrometry

Multi-functional sample preparation procedure for measuring phytoestrogens in milk, cereals, and baby-food by liquid-chromatography tandem mass spectrometry with subsequent determination of their estrogenic activity using transcriptomic assay by Jean-Philippe Antignac; Isabelle Gaudin-Hirret; Hanspeter Naegeli; Ronan Cariou; Christopher Elliott; Bruno Le Bizec (pp. 55-63).

A method dedicated to the determination of a multiple range of phytoestrogens as endocrine disruptor compounds in infant food products was developed, with as double objective the specific measurement of 13 parameters and the evaluation of the estrogenic potency associated to this quantitative profile. A combined enzymatic and acidic chemical hydrolysis followed by a double purification on two successive C₁₈ and SiOH Solid Phase Extraction cartridges permitted to efficiently purify milk, cereals and baby-food samples while eliminating naturally occurring estrogen hormones. A specific liquid chromatography-tandem mass spectrometric measurement authorised unambiguous identification and quantification of the target compounds. The proposed methodology was fully validated and applied to a set of around 30 real samples, demonstrating the presence of phytoestrogens at levels globally ranging from several μgkg⁻¹ (ppb) to several tensmgkg⁻¹ (ppm). The prepared sample extracts were proven to be suitable and compatible with the evaluation of their induced biological transcriptional activity on MCF-7 cell lines. Because permitting to cope with difficult issues such as low-dose and mixture effects, this proposed methodology may appear of particular interest for further exposure assessment studies and hazard characterisation investigations related to this class of endocrine disruptor compounds.

Keywords: Phytoestrogens; Sample preparation; Mass spectrometry; Transcriptomics; Estrogenic potency; Endocrine disruptor

Determination of streptomycin residues in honey by liquid chromatography–tandem mass spectrometry by Rodrigo H.M.M. Granja; Alfredo M. Montes Niño; Roberto A.M. Zucchetti; Rosario E. Montes Niño; Raj Patel; Alessandro G. Salerno (pp. 64-67).

Streptomycin is an aminoglycoside antibiotic used in apiculture to protect bees against a variety of brood diseases. Brazilian authorities have included it in the National Regulatory Monitoring Program for honey production. A simple and reliable method using liquid chromatography–tandem mass spectrometry has been developed and validated for the determination of streptomycin in honey. The chromatography separation was performed on a Gemini 5μm C18 (50mm×2mm) column using 5mM heptafluorbutiric acid/acetonitrile (85:15) as the mobile phase at a flow rate of 0.2mLmin⁻¹. The detection of the analyte was achieved by positive ionization electrospray in multiple reaction-monitoring modes. Two characteristic transitions were monitored for streptomycin. Some analytical parameters were validated according to the guidelines laid down by European Commission Decision 2002/657/EC: decision limit, detection capability, recovery, precision and ruggedness. The recoveries of streptomycin from honey fortified at 2.5, 10, 15 and 20μgkg⁻¹ levels are around 100%. The decision limit and detection capability of streptomycin was 3μgkg⁻¹ and 4.7μgkg⁻¹ respectively.

Keywords: Honey; Streptomycin; Liquid chromatography–mass spectrometry; Residues

The development and validation of a multiclass liquid chromatography tandem mass spectrometry (LC–MS/MS) procedure for the determination of veterinary drug residues in animal tissue using a QuEChERS (QUick, Easy, CHeap, Effective, Rugged and Safe) approach by George Stubbings; Timothy Bigwood (pp. 68-78).

A novel rapid multiresidue/multiclass procedure with liquid chromatography tandem mass spectrometry (LC–MS/MS) detection has been developed to screen for the presence of veterinary drug residues in animal tissues. The method uses a new sample preparation procedure loosely based on QuEChERS (QUick, Easy, CHeap, Effective, Rugged and Safe) methodology. Validation to date has been restricted to chicken muscle and has been performed according to European Commission guidelines [COMMISSION DECISION of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results] for nitroimidazoles, sulphonamides, fluoroquinolones, quinolones, ionophores and dinitrocarbanilide. Recent work has shown that the method is also applicable to macrolide and lincosamide antibiotics, benzimidazoles, levamisole, avermectins and tranquillisers.

Keywords: Liquid chromatography tandem mass spectrometry; QUick, Easy, CHeap, Effective, Rugged and Safe; Ionophores; Sulphonamides; Fluoroquinolones; Quinolones; Nitroimidazoles; Dicarbanilide; Multiresidue; Multiclass; Dispersive solid phase extraction

Determination of 5-nitroimidazoles and corresponding hydroxy metabolites in swine kidney by ultra-performance liquid chromatography coupled to electrospray tandem mass spectrometry by Xi Xia; Xiaowei Li; Shuangyang Ding; Suxia Zhang; Haiyang Jiang; Jiancheng Li; Jianzhong Shen (pp. 79-86).

A rapid, sensitive and reliable multi-residue method for the simultaneous determination of four 5-nitroimidazoles (NIIMs) and their three corresponding metabolites in swine kidney was developed and validated. The compounds of interest were extracted from tissues with ethyl acetate. The crude extracts were subject to liquid–liquid partition with hexane followed by solid-phase extraction using mixed-mode strong cation-exchange column. Chromatographic separation was achieved on an AcQuity BEH C₁₈ column and was completed within 4min for each injection. Data acquisition under positive electrospray tandem mass spectrometry was performed by applying multiple reaction monitoring for both identification and quantification. Mean relative recoveries from fortified samples ranged from 83% to 111%, with coefficients of variation lower than 12%. The limits of detection and quantification for the NIIMs were in the range of 0.05–0.5 and 0.1–0.5μgkg⁻¹, respectively.

Keywords: Nitroimidazole; Ultra-performance liquid chromatography; Tandem mass spectrometry; Kidney

Rapid multi-residue analysis of antibiotics in muscle by liquid chromatography–tandem mass spectrometry by K. Granelli; Christina Elgerud; Åsa Lundström; Anita Ohlsson; Pernilla Sjöberg (pp. 87-91).

A way of carrying out simple and rapid multi-residue analysis of antibiotics in porcine and bovine muscle by liquid chromatography–tandem mass spectrometry (LC–MS/MS) is described. The method has previously been published as a screening method, but the scope has now been extended to quantification and confirmation.Nineteen compounds from five different classes of antibiotics, i.e. tetracyclines, sulfonamides, quinolones, (-lactams and macrolides, are included in the method. The samples are extracted by a single extraction using 70% methanol, diluted with water and injected in the LC–MS/MS. The run time is 7min per injection. About 60 samples can be analysed in 24h. By using this method the need for a separate screening step prior to confirmation is eliminated and consequently the total time from sampling to a confirmed result will be considerably reduced.Validation was performed according to Commission Decision 2002/657/EC.

Keywords: Antibiotics; Tetracyclines; Sulfonamides; Quinolones; β-Lactams; Macrolides; Muscle; Liquid chromatography–tandem mass spectrometry; Veterinary drugs; Multi-residue

Desorption electrospray ionisation mass spectrometry: A rapid screening tool for veterinary drug preparations and forensic samples from hormone crime investigations by M.W.F. Nielen; H. Hooijerink; F.C. Claassen; M.C. van Engelen; T.A. van Beek (pp. 92-100).

Hormone and veterinary drug screening and forensics can benefit from the recent developments in desorption electrospray ionisation (DESI) mass spectrometry (MS). In this work the feasibility of DESI application has been studied. Using a linear ion trap or quadrupole time-of-flight (TOF) MS instrument both full-scan and data-dependent collision-induced dissociation MSⁿ spectra were acquired in seconds without sample preparation. Preliminary data are presented for the rapid screening of (pro)hormone supplement samples, an illegal steroid cocktail and forensic samples from veterinary drug investigations. The potential of this DESI approach is clearly demonstrated since compounds observed could be independently confirmed by liquid chromatography/TOFMS with accurate mass measurement, and/or proton nuclear magnetic resonance spectroscopy. Specific concerns related to false-positive and false-negative findings due to limitations in quantification and memory-effects are briefly discussed. It is envisaged that DESI will achieve a prominent role in hormone and veterinary drug analysis in the near future.

Keywords: Mass spectrometry; Desorption electrospray ionisation DESI; Hormone; Veterinary drugs; Forensic

Determination of cyanuric acid residues in catfish, trout, tilapia, salmon and shrimp by liquid chromatography–tandem mass spectrometry by Christine M. Karbiwnyk; Wendy C. Andersen; Sherri B. Turnipseed; Joseph M. Storey; Mark R. Madson; Keith E. Miller; Charles M. Gieseker; Ron A. Miller; Nathan G. Rummel; Renate Reimschuessel (pp. 101-111).

In May 2007, investigators discovered that waste material from the pet food manufacturing process contaminated with melamine (MEL) and/or cyanuric acid (CYA) had been added to hog and chicken feeds. At this time, investigators also learned that adulterated wheat gluten had been used in the manufacture of aquaculture feeds. Concern that the contaminated feed had been used in aquaculture and could enter the human food supply prompted the development of a method for the determination of CYA residues in the edible tissues of fish and shrimp. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was employed as a sensitive technique for the analysis of CYA in catfish, tilapia, salmon, trout and shrimp tissue. CYA was extracted from ground fish or shrimp with an acetic acid solution, defatted with hexane, and isolated with a graphitic carbon black solid-phase extraction column. Residues were separated from matrix components using a porous graphitic carbon LC column, and then analyzed with electrospray ionization in negative ion mode on a triple quadrupole mass spectrometer. Selective reaction monitoring was performed on the [M−H]⁻ m/ z 128 ion resulting in the product ions m/ z 85 and 42. Recoveries from catfish, tilapia and trout fortified with 10–100μgkg⁻¹ of CYA averaged 67% with a relative standard deviation (R.S.D.) of 18% ( n=107). The average method detection limit (MDL) for catfish, tilapia and trout is 3.5μgkg⁻¹. An internal standard,¹³C₃-labeled CYA, was used in the salmon and shrimp extractions. Average recovery of CYA from salmon was 91% (R.S.D.=15%, n=18) with an MDL of 7.4μgkg⁻¹. Average recovery of CYA from shrimp was 85% (R.S.D.=10%, n=13) with an MDL of 3.5μgkg⁻¹.

Keywords: Cyanuric acid; Melamine; Liquid chromatography–tandem mass spectrometry; Fish; Shrimp

Development of a rapid method for the analysis of synthetic growth promoters in bovine muscle using liquid chromatography tandem mass spectrometry by E.M. Malone; C.T. Elliott; D.G. Kennedy; L. Regan (pp. 112-120).

A rapid liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the simultaneous identification, confirmation and quantitation of thirteen synthetic growth promoters in bovine muscle. The method was validated in accordance with the criteria defined in Commission Decision 2002/657/EC. A value of 1μgkg⁻¹ was chosen as the required performance level (RPL) for all analytes. The growth promoters investigated were α and β trenbolone, 16-β-OH stanozolol, methylboldenone, fluoxymesterone, methyltestosterone, medroxyprogesterone acetate, megestrol acetate, melengestrol acetate, dexamethasone, flumethasone, dienestrol and hexestrol. The method involved enzymatic hydrolysis, purification by solid phase extraction followed by analysis by UPLC–MS/MS using electrospray ionization operated in both positive and negative polarities with a total run time of 14min. The decision limit (CCα) values obtained, ranged from 0.09 to 0.19μgkg⁻¹ and the detection capability (CCβ) values obtained, ranged from 0.15 to 0.32μgkg⁻¹. The results of the inter-assay study, which was performed by fortifying bovine muscle samples ( n=18) on three separate days, show the accuracy calculated for the various analytes to range between 98% and 102%. The precision of the method, expressed as R.S.D. values for the inter-assay variation of each analyte at the three levels of fortification (1, 1.5 and 2.0μgkg⁻¹), ranged between 3.1% and 5.8%. A Day 4 assay was carried out to examine variations due to different animals and different muscle types.

Keywords: Growth promoters; Method validation; Mass spectrometry; Bovine muscle; Analysis

Elimination kinetic of recombinant somatotropin in bovine by Marie-Hélène Le Breton; Sandrine Rochereau-Roulet; Gaud Pinel; Nora Cesbron; Bruno Le Bizec (pp. 121-127).

Bovine somatotropin (bST), also called growth hormone is a protein hormone produced by the pituitary gland and responsible directly or indirectly for various effects on growth, development and reproductive functions. Its recombinant bovine somatotropin form (rbST) is used in dairy cattle to enhance milk production. Even if the effects of treatment with rbST have been largely studied, until now analytical methods able to detect rbST were limited to immunoassays, which suffer from the impossibility to distinguish between the endogenous and the recombinant form.In this study, a sample preparation procedure based on different precipitation steps, extraction on solid phase and enzymatic digestion was used to purify rbST from serum. The detection was performed by liquid chromatography coupled to tandem mass spectrometry in positive electrospray ionization mode (LC–ESI(+)−MS/MS) allowing the unambiguous identification and quantification of rbST in serum. Samples collected from a cow treated with recombinant bovine somatotropin were analysed and for the first time, the elimination kinetic specific to recombinant somatotropin has been characterized in serum. Detection of rbST was possible from 4h 30min to 4 days after administration and concentration was found up to 10ngmL⁻¹ during the kinetic.

Keywords: Bovine somatotropin; Growth hormone; Serum; Dairy cow; Pharmacokinetic; Liquid chromatography coupled to tandem mass spectrometry

A determinative and confirmatory method for residues of the metabolites of carbadox and olaquindox in porcine tissues by Joe O. Boison; Stephen C. Lee; Ron G. Gedir (pp. 128-134).

Carbadox (CBX) and olaquindox (OLQ) are used in swine feed for growth promotion, to improve feed efficiency, increase the rate of weight gain, control swine dysentery and bacterial enteritis in young swine. In 1991, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) recommended maximum residue limits (MRLs) of 30 and 5μgkg⁻¹ in liver and muscle tissues of pigs, respectively, based on the concentration of, and expressed as, quinoxaline-2-carboxylic acid (QCA) as marker residue. In 1998, the European Commission (EC) banned the use of CBX and OLQ in food animal production together with four other feed additives, following reports that CBX and desoxycarbadox (DCBX) are suspect carcinogens and mutagens. In 2001, the sale of CBX was halted in Canada. In 2003, JECFA recommended the withdrawal of the previously recommended acceptable daily intake (ADI) and MRLs and concluded that QCA was not a suitable marker residue for CBX, based on new sponsor studies reporting that DCBX, the suspect carcinogen, persisted in animal tissues much longer than had previously been thought. This paper presents a very sensitive LC–MS/MS method that was developed by CFIA scientists for the simultaneous determination and confirmation of DCBX residues at concentrations ≥0.050ngkg⁻¹ and QCA and mQCA residues at concentrations ≥0.50ngkg⁻¹in bovine muscle, pork liver and muscle tissues.

Keywords: Desoxycarbadox; Carbadox; Olaquindox; Quinoxaline-2-carboxylic acid; Methyl quinoxaline-2-carboxylic acid; Tandem LC mass spectrometry; Residues in pork tissues; Beef muscle

A generic method for the quantitative analysis of aminoglycosides (and spectinomycin) in animal tissue using methylated internal standards and liquid chromatography tandem mass spectrometry by Frédérique L. van Holthoon; Martien L. Essers; Patrick J. Mulder; Sara L. Stead; Marianne Caldow; Helen M. Ashwin; Matthew Sharman (pp. 135-143).

Aminoglycosides (AGs) are a large and diverse group of antibiotics. Although AGs may cause side effects of nephrotoxicity and ototoxicity, they are still occasionally being used for the treatment of serious infections. In this study the development of a method is described for the quantitative determination and confirmation of seven aminoglycosides (and relevant isomers) and spectinomycin in animal tissues. The extraction was based on an extraction followed by a concentration and clean-up step using weak cation exchange solid phase extraction. The separation was performed by ion-pair liquid chromatography on a C₁₈ column followed by mass spectrometric detection. The method was validated according to the EU requirements for a quantitative confirmatory method. Permethylated aminoglycosides (in-house synthesised internal standards) were used for accurate quantification. The accuracy of the analyses of AGs in kidney ranged from 94 to 111%, intra-day precision ranged between 2.5 and 7.4% (R.S.D._r) and inter-day precision ranged between 2.2 and 17.3% (R.S.D._RL, n=21, MRL level). Accuracy (muscle tissue) varied from 83 to 128% with an intra-day precision between 2.2 and 17.3% (R.S.D._r, n=7, MRL level). From the results it was concluded that the method was able to monitor MRL levels which ranged from 750 to 20,000μgkg⁻¹ for kidney and from 50 to 10,000μgkg⁻¹ for muscle tissue.

Keywords: Aminoglycosides; Permethylated internal standard; Validation; Ion-pair chromatography; Animal tissue; Liquid chromatography tandem mass spectrometry

Validation of multiresidue methods for veterinary drug residues; related problems and possible solutions by A. Kaufmann (pp. 144-155).

This paper describes problems and possibly solutions encountered when validating a multiresidue method used for analyzing some 100 different veterinary drugs at trace levels in a number of matrices. The validation concept as proposed by the commission decision 2002/657/EC (CD) has been utilized. Slightly deviating approaches had to be developed in order to produce and access the data generated by a multiresidue method. This included concepts, which permit the common validation of banned and regulated substance within the same analytical method. In addition, an alternative calculation of performance parameters (e.g., CC α) was developed. This was done in order to ensure correct results even in the absence of measurable bank noise, as typically observed when using high resolution mass spectrometry techniques. The economical aspect of validation (number of required assays) as well as the relative importance of some performance criteria (e.g., CC β) have been critically discussed.

Keywords: Validation; Multiresidue method; Mass spectrometry; Commission decision 2008/657/EC; Performance parameters; Veterinary drugs

In-house validation and factorial effect analysis of a liquid chromatography–tandem mass spectrometry method for the determination of steroids in bovine muscle by Kathrin S. Schmidt; Carolin S. Stachel; Petra Gowik (pp. 156-164).

Anabolic steroids are banned from use in food-producing animals in the EU (Council Directive 96/22/EC). To control the zero-tolerance concept a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the screening and confirmation of most of the relevant natural and synthetic estrogenic and androgenic steroids in bovine muscle was developed and validated. The method permits to confirm and quantify almost all steroids below 1μgkg⁻¹. The validation was carried out according to Commission Decision 2002/657/EC, chapter 3.1.3 “alternative validation”, by applying a matrix-comprehensive in-house validation concept. Decision limit CCα, detection capability CCβ, recovery, repeatability, within-laboratory reproducibility and the uncertainty of measurement were calculated. Furthermore, a factorial effect analysis was carried out to identify factors that have a significant influence on the method. Factors considered to be relevant for the method in routine analysis (e.g., operator, storage duration of the extracts before measurement, different cartridge lots) were systematically varied on two levels. The factorial analysis showed that different cartridge lots and different storage durations of the extracts before measurement can exert a relevant influence on the method.

Keywords: Anabolic steroids; Bovine muscle; Liquid chromatography–tandem mass spectrometry; Matrix-comprehensive in-house validation; Multivariate effect analysis; Residue control

Determination of hormonal growth promoters in bovine hair: Comparison of liquid chromatography–mass spectrometry and gas chromatography–mass spectrometry methods for estradiol benzoate and nortestosterone decanoate by Eleanor Duffy; Lauriane Rambaud; Bruno Le Bizec; Michael O’Keeffe (pp. 165-172).

The detection of steroid residues in hair is a powerful strategy to demonstrate long-term administration of these growth promoters in meat production animals. Analysis of the ester form of administered steroids is an unambiguous approach to prove the illegal use of natural hormones. For detection, gas chromatography–mass spectrometry (GC–MS/MS) was generally used. However, recent advances in liquid chromatography–tandem mass spectrometry (LC–MS/MS) technology have improved the robustness and potential sensitivity of this method. This paper describes development and validation, according to Commission Decision 2002/657/EC, of LC–MS/MS and GC–MS/MS methods, in two separate laboratories, for determination of steroid esters in bovine hair. Bovine hair samples taken from animals treated with estradiol-3-benzoate and nortestosterone decanoate, as well as from an untreated animal, were used to evaluate the comparability of the results of the two validated methods. The results of the inter-comparison demonstrate that both the LC–MS/MS and the GC–MS/MS methods are fit for purpose and capable of determining steroid esters in hair samples from treated bovine animals.

Keywords: Steroid hormones; Estradiol-3-benzoate; Nortestosterone decanoate; Bovine hair; Gas chromatography–mass spectrometry; Liquid chromatography–mass spectrometry

Depletion of chloramphenicol in trout after a hypothetic therapeutic treatment by G. Biancotto; L. Contiero; C. Benetti; M. Calligaris; E. Tibaldi; L. Cerni; M. Francese (pp. 173-177).

The study was intended to evaluate the depletion of chloramphenicol (CAP) in rainbow trout (about 300–550g body weight), after 10 days treatment with fish feedstuff containing chloramphenicol.A total of 60 animals were separated in two groups: one was fed with CAP containing feedstuff in order to have a dosage of about 80mgkg⁻¹day⁻¹, while a second group of fishes was fed with feedstuff not containing any CAP formulation (negative controls).The treatment was maintained for 10 days. After this period, groups of 2–5 animals were sacrificed at different withdrawal times up to a maximum of 31 days.Muscle tissues of each group of animals were then analysed for quantitative residual CAP determination both by enzyme linked immunoassay (ELISA) and liquid chromatography coupled to mass spectrometry (HPLC/MSMS).The methods applied were in house validated according to the guidelines laid down by the European Decision 657/2002/EC.Results and considerations are presented.

Keywords: Chloramphenicol; Trouts; Depletion study; Liquid chromatography–mass spectrometry (HPLC/MSMS)

Detection and identification of 20-hydroxyecdysone metabolites in calf urine by liquid chromatography-high resolution or tandem mass spectrometry measurements and establishment of their kinetics of elimination after 20-hydroxyecdysone administration by Blandine Destrez; Gaud Pinel; Fabrice Monteau; René Lafont; Bruno Le Bizec (pp. 178-184).

Ecdysteroids, which are steroid hormones in invertebrates, but are also present in plants, could be potentially used as anabolic agents in food-producing animals because of their growth-promoting properties. In this context, the metabolism of 20-hydroxyecdysone (20E) has been investigated in cattle in order to efficiently control its potential misuse. The analytical procedure involves purification on two solid-phase extraction cartridges (SPE octadecylsilyl and SPE silica) prior to detection based on liquid chromatography coupled to high resolution mass spectrometry in negative electrospray ionization mode (LC-(ESI−)-HRMS). Each new signal appearing on full-scan HRMS (30,000) during the analysis was investigated by tandem mass spectrometry (MS/MS). Comparison of the mass spectra pattern between 20E and potential metabolites has given informations on the chemical structures of the metabolites. This targeted approach, combining HRMS and MSⁿ measurements on a linear trap in tandem with an orbital trap, allowed us to elucidate the structure of several 20E metabolites in calf urine: 14-deoxy-20-hydroxyecdysone, 20,26-dihydroxyecdysone and 14-deoxy-20,26-dihydroxyecdysone, the last of which had never previously been reported in bovine. The kinetics of elimination of these metabolites were investigated, and two of them were monitored by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) measurements over 6 days after 20E administration to calves, thus increasing the time-window for detection after 20E administration and thereby allowing for more efficient control of its misuse.

Keywords: Ecdysteroids; 20-Hydroxyecdysone; Urine; Liquid chromatography; High-resolution mass spectrometry; Kinetics of elimination

Rapid method for the determination of tranquilizers and a beta-blocker in porcine and bovine kidney by liquid chromatography with tandem mass spectrometry by Kamila Mitrowska; Andrzej Posyniak; Jan Zmudzki (pp. 185-192).

A fast and simple liquid chromatography with tandem mass spectrometry method for detection and confirmation of tranquilizers (chlorpromazine, propionylpromazine, acepromazine, triflupromazine, promazine, azaperone and its metabolite, azaperol) and beta-blocker (carazolol) in porcine and bovine kidney has been presented. The method relies on the extraction with acetonitrile followed by centrifugation. After evaporation of acetonitrile, the residue was reconstituted in a mobile phase and filtrated. The separation of analytes was performed on a C18 column using a mobile phase of acetonitrile and ammonium formate buffer (0.05M, pH 4.5) with gradient elution. The electrospray ionization was used to obtain the protonated molecules [M+H]⁺ and two product ions were monitored for each compound. For quantification deutered internal standards were used. The whole method has been validated according to the European Union requirements. Specificity, decision limit (CCα), detection capability (CCβ), trueness and precision were determined. The results showed good trueness ranged from 73.2% to 110.6% with a good R.S.D., less than 13.0% under within-laboratory reproducibility conditions. The calculated critical concentrations of CCα for phenothiazines were between 5.8 and 6.6μgkg⁻¹ while for azaperone CCα was 105.5μgkg⁻¹ and for azaperol was 121.4μgkg⁻¹. CCα for carazolol was 16.7μgkg⁻¹ in bovine and 21.9μgkg⁻¹ in porcine kidney. CCβ for phenothiazines were between 6.3 and 7.6μgkg⁻¹, for azaperone was 119.0μgkg⁻¹ and for azaperol was 140.0μgkg⁻¹. For carazolol in bovine kidney CCβ was 18.6μgkg⁻¹ whereas in porcine kidney was 24.4μgkg⁻¹.

Keywords: Tranquilizers; Beta-blocker; Liquid chromatography; Tandem mass spectrometry; Kidney

How to decrease ion suppression in a multiresidue determination of β-agonists in animal liver and urine by liquid chromatography–mass spectrometry with ion-trap detector by Francisco Moragues; Carmen Igualada (pp. 193-195).

A new liquid chromatography–mass spectrometry with ion-trap detection method for the determination of clenbuterol, ractopamine, clenpenterol, brombuterol, hydroxyclenbuterol, mapenterol and mabuterol in animal liver and urine is described to decrease ion suppression in both matrices.The developed method consists of an aqueous extraction and a two-step clean-up. Final extract was injected in a liquid chromatograph with ion trap mass spectrometer optimized to obtain MS³ ions which provided more specificity and better signal to noise ratio in the investigated analytes.Those steps have been essential to reach decision limits (CCα) values below the proposed MRPL values for each substance. The CCα were determined and their values were 0.05μgkg⁻¹ for clenbuterol, 0.2μgkg⁻¹ for ractopamine and 0.1μgkg⁻¹ for the other analytes in liver and the same values in μgL⁻¹ for urine samples.

Keywords: Ion-suppression; β-Agonist; Liver; Urine; Mass spectrometry

New method for the analysis of flukicide and other anthelmintic residues in bovine milk and liver using liquid chromatography–tandem mass spectrometry by Brian Kinsella; Steven J. Lehotay; Katerina Mastovska; Alan R. Lightfield; Ambrose Furey; Martin Danaher (pp. 196-207).

A liquid chromatographic-tandem mass spectrometric (LC–MS/MS) multi-residue method for the simultaneous quantification and identification of 38 residues of the most widely used anthelmintic veterinary drugs (including benzimidazoles, macrocyclic lactones, and flukicides) in milk and liver has been developed and validated. For sample preparation, we used a simple modification of the QuEChERS method, which was initially developed for pesticide residue analysis. The method involved extracting sample (10g) with acetonitrile (10mL), followed by phase separation from water (salting out) with MgSO₄:NaCl (4:1, w/w). After centrifugation, an aliquot of the extract (1mL) was purified by dispersive solid-phase extraction with MgSO₄ (150mg) and C₁₈ (50mg), prior to LC–MS/MS analysis. Two injections of the same extract were required with the LC–MS/MS instrument to cover the 30 electrospray positive and 8 electrospray negative analytes. The limit of quantitation of the method was 5μgkg⁻¹ for 37 analytes (and 10μgkg⁻¹ for dichlorvos). The method was successfully validated according to the 2002/657/EC guidelines. Recovery of analytes was typically in the 70–120% range, with repeatabilities and reproducibilities typically <15% in milk and <20% in liver.

Keywords: Anthelmintics; Flukicides; Residues; Food; Liquid chromatography–tandem mass spectrometry; QuEChERS

Simple and rapid determination of enrofloxacin and ciprofloxacin in edible tissues by turbulent flow chromatography/tandem mass spectrometry (TFC–MS/MS) by Ralph Krebber; Franz-Jürgen Hoffend; Frank Ruttmann (pp. 208-213).

A simple and rapid method for the determination of residues of enrofloxacin and its metabolite ciprofloxacin in edible tissues of farm animals using turbulent flow chromatography/tandem mass spectrometry (TFC–MS/MS) is described.The tissue samples were extracted with a mixture of acetonitrile, water and formic acid. After addition of internal standard solution, an aliquot of the extract was injected into the turbulent flow chromatography system. Matrix components contained in the injected sample were separated from the retained analytes on a polymer-based extraction column suited for pretreatment of samples at high flow rates. The analytes were eluted to an analytical column and the quantitative determination was performed using a tandem mass spectrometric detector. The run time for the analysis was 4min.The limit of quantitation (LOQ) was 25μgkg⁻¹ for each analyte and tissue material. Validation was performed in edible tissues of cattle, pig, turkey and rabbit in the range from 25μgkg⁻¹ to at least twice the MRL. Mean recovery rates for the tissues of the different species were in the range from 72 to 105% with a coefficient of variation (CV) between 4.3 and 18%.

Keywords: On-line solid phase extraction; Enrofloxacin; Turbulent flow chromatography; Liquid chromatography–tandem mass spectrometry

Determination of ten sulphonamides in egg by liquid chromatography–tandem mass spectrometry by A.F. Forti; G. Scortichini (pp. 214-219).

A precise and reliable method for the determination of 10 sulphonamide antibiotics (sulfadiazine, sulfathiazole, sulfamerazine, sulfamethazine, sulfamethoxypyridazine, sulfachloropyridazine, sulfamethoxazole, sulfamonomethoxine, sulfadimethoxine and sulfaquinoxaline) in egg by liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed.Drugs were extracted using a mixture of dichloromethane/acetone (50:50, v/v), acidified with acetic acid and then cleaned-up on a cation-exchange solid-phase extraction (SPE) cartridge. The chromatographic separation was performed by gradient on a C₁₈ column with a mobile phase of methanol–water containing 0.1% formic acid and 5mM ammonium acetate, then sulphonamides were detected in a triple-quadrupole mass spectrometer operated in positive electrospray ionization mode (ESI⁺).The method was validated at 15, 30 and 45μgkg⁻¹. These levels were much lower than the corresponding maximum residue limit of 100μgkg⁻¹ set for sulphonamides in several matrices but not in eggs, where the presence of such residues is not permitted.Results were quantitated against the selected internal standard¹³C₆-sulphamethazine and also according to the matrix-matched approach. The within-laboratory reproducibility, expressed as a relative standard deviation, never exceeded 21%. All decision limit (CCα) values lied in the range between 16.1 and 20.5μgkg⁻¹ and the corresponding results for detection capability (CCβ) were 16.9 and 25.7μgkg⁻¹. Ruggedness was estimated according to the Youden robustness test.

Keywords: Sulphonamides; Egg; Liquid chromatography–tandem mass spectrometry; Decision 2002/657/EC

Determination of dapsone in meat and milk by liquid chromatography tandem mass spectrometry by M. Hadjigeorgiou; Ch. Papachrysostomou; Z. Theodorou; P. Kanari; S. Constantinou (pp. 220-224).

Within the EU the use of dapsone (4,4-diaminodiphenylsulfone) is prohibited in food-producing animals and consequently it's included in the Annex IV of the Directive 90/2377/EC. A quantitative confirmatory method has been developed and validated according to the criteria defined in the Commission Decision 2002/657/EC, for the determination of dapsone in meat and milk. Samples, after homogenization in alkaline conditions and organic solvent extraction, were purified on silica gel solid phase extraction cartridges. The eluate was evaporated and redissolved in mobile phase and was analysed by liquid chromatography tandem mass spectrometry (LC–MS/MS) in positive electrospray ionisation (ESI) using deuterium labelled Sulphadimidine-d7 as internal standard. The calculated value for, decision limit, CCα is 0.12μgkg⁻¹, and the detection capability; CCβ value is 0.16μgkg⁻¹.

Keywords: Dapsone; Meat; Milk; Validation; Decision 2002/657/EC

Validation and application of a yeast bioassay for screening androgenic activity in calf urine and feed by Toine F.H. Bovee; Gerrit Bor; Henri H. Heskamp; Johan J.P. Lasaroms; Marieke B. Sanders; Michel W.F. Nielen (pp. 225-234).

Bioassays are valuable tools for combating the illegal use of steroids in cattle fattening. Previously we described the construction and properties of a rapid and robust yeast androgen bioassay stably expressing the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. In the present study this yeast androgen bioassay was validated as a qualitative screening method for the determination of androgenic activity in calf urine and animal feed. This validation was performed according to EC Decision 2002/657. 20 blank samples were spiked with testosterone, 17α-methyltestosterone, 19-nortestosterone, 17β-trenbolone, 17β-boldenone or 17α-methylboldenone at 2 or 15ngmL⁻¹ in urine and 50 or 100ngg⁻¹ in feed. All blank and spiked samples fulfilled the CCα and CCβ criterions, meaning that all 20 blank samples gave signals below the determined decision limits CCα and were thus classified as compliant ( α=1%). For each component, at least 19 out of the 20 spiked samples gave a signal above the CCα and were thus classified as suspect ( β=5%). The method was specific, and high amounts of dexamethasone did not interfere with the outcome of the test. Although high levels of 17α-ethynylestradiol can significantly inhibit the response obtained with low amounts of androgens, that situation is not relevant in veterinary practice. When stored at their specific conditions, the androgens in feed were stable for at least 91 days. Real urine samples from a national control program were screened and a representative part of the compliant and suspect samples were confirmed by gas chromatography–tandem mass spectrometry.

Keywords: Androgens; Bioassay; Hormone abuse; Steroids; Validation

Biosensor-based detection of reduced sex hormone-binding globulin binding capacities in response to growth-promoter administrations by Mark H. Mooney; Aldert A. Bergwerff; Jeroen A. van Meeuwen; Peter B. Luppa; Chris T. Elliott (pp. 235-240).

Growth-promoting agents are illicitly used during animal rearing processes and the detection of their use is limited by new compounds and dosing practices that limit the efficiency of current testing which is based on residue analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and gas chromatography–mass spectrometry (GC–MS) methodology. An alternative approach is to use indirect biological evidence as a screening tool to identify growth-promoter treated animals thus improving the effectiveness of residue testing through the targeted sampling of these animals. Sex hormone-binding globulin (SHBG) is a glycoprotein which binds and controls the levels of sex-hormones within the circulation. Using a biosensor assay based on measurement of binding to an immobilised 1α-dihydrotestosterone (1α-DHT) derivative, reduced SHBG binding capacities were detected in growth-promoter treated animals. During the course of a veal treatment regime based on repeated oestradiol benzoate, nortestosterone decanoate and dexamethasone administrations, treated male and female calves were shown to have significantly lower SHBG capacities. To assess the effectiveness of using SHBG binding capacities as a biomarker of treatment and to investigate the role of individual growth-promoter components to the SHBG capacity lowering effects, adult heifer animals were subjected to repeated doses of nortestosterone decanoate. These animals also demonstrated a reduction in SHBG capacity levels at Day 39 of the study, in contrast to oestradiol benzoate treated adult steers who were found to have unaltered levels. These findings suggest that the measurement of SHBG binding capacities using a biosensor assay has potential in the identification of illegally treated animals, particularly those exposed to androgens.

Keywords: Growth-promoters; Sex hormone-binding globulin; Biomarker; Biosensor

A rapid microbial inhibition-based screening strategy for fluoroquinolone and quinolone residues in foods of animal origin by Helen Ashwin; Sara Stead; Marianne Caldow; Matthew Sharman; Jacques Stark; Angelique de Rijk; Brendan J. Keely (pp. 241-246).

A rapid, high-throughput antimicrobial screening assay for the detection of fluoroquinolone and 4-quinolone residues in foods of animal origin has been developed in ampoule format. The assay employs a single Escherichia coli species sensitive to those Gram-negative inhibitiory antimicrobial compounds and is presented in a comparable format to the existing commercially available Premi^®Test and Delvotest^® ampoule-based microbial inhibition tests (DSM, Delft, The Netherlands). In the novel E. coli assay the microorganism, in vegetative state, is inoculated into a nutrient agar pellet containing a pH sensitive acid–base indicator dye. A simple extraction protocol that is selective for fluoroquinolone and quinolone compounds was developed to recover, cleanup and concentrate the target analyte(s) from a variety of tissue types and matrices prior to screening analysis. The method detected 16 target compounds at concentrations equal to or below the maximum residue limits (where applicable). The method has been validated using the prototype assay in accordance with the 2002/657/EC guidelines for the validation of qualitative screening assays. False positive and false negative responses rates for the procedure have been determined as less than 5%. The stability of a selection of representative target analytes has been demonstrated for a 20-week period under a variety of storage conditions both in tissue and in extract.

Keywords: Fluoroquinolone; 4-Quinolone; Antimicrobial inhibition; Screening and food safety

Detection of anabolic androgenic steroid abuse in doping control using mammalian reporter gene bioassays by Corine J. Houtman; Saskia S. Sterk; Monique P.M. van de Heijning; Abraham Brouwer; Rainer W. Stephany; Bart van der Burg; Edwin Sonneveld (pp. 247-258).

Anabolic androgenic steroids (AAS) are a class of steroid hormones related to the male hormone testosterone. They are frequently detected as drugs in sport doping control. Being similar to or derived from natural male hormones, AAS share the activation of the androgen receptor (AR) as common mechanism of action. The mammalian androgen responsive reporter gene assay (AR CALUX^® bioassay), measuring compounds interacting with the AR can be used for the analysis of AAS without the necessity of knowing their chemical structure beforehand, whereas current chemical–analytical approaches may have difficulty in detecting compounds with unknown structures, such as designer steroids. This study demonstrated that AAS prohibited in sports and potential designer AAS can be detected with this AR reporter gene assay, but that also additional steroid activities of AAS could be found using additional mammalian bioassays for other types of steroid hormones. Mixtures of AAS were found to behave additively in the AR reporter gene assay showing that it is possible to use this method for complex mixtures as are found in doping control samples, including mixtures that are a result of multi drug use. To test if mammalian reporter gene assays could be used for the detection of AAS in urine samples, background steroidal activities were measured. AAS-spiked urine samples, mimicking doping positive samples, showed significantly higher androgenic activities than unspiked samples. GC–MS analysis of endogenous androgens and AR reporter gene assay analysis of urine samples showed how a combined chemical–analytical and bioassay approach can be used to identify samples containing AAS. The results indicate that the AR reporter gene assay, in addition to chemical–analytical methods, can be a valuable tool for the analysis of AAS for doping control purposes.

Keywords: Mammalian reporter gene assay; Luciferase; Sport doping; Androgen; Urine; Mixture

Improved screening method for the detection of a range of nitroimidazoles in various matrices by optical biosensor by Colin S. Thompson; Imelda M. Traynor; Terence L. Fodey; Steven R.H. Crooks (pp. 259-264).

An immunobiosensor assay was developed for the multi-residue screening of a range of nitroimidazole compounds in various species and sample types including porcine, bovine and ovine kidney, avian liver, serum and eggs and bovine milk. A polyclonal antibody which binds at least seven of the major nitroimidazoles and their metabolites was raised in a sheep after inoculation with a metronidazole protein conjugate. Sample homogenates were extracted into acetonitrile and subjected to micro-centrifugation prior to biosensor analysis. Validation data obtained from the analysis of 20 fortified samples has shown that the method has a detection capability (CCβ) of less than 1μgkg⁻¹ (or μgL⁻¹) for dimetridazole (DMZ) in all species and matrices investigated. In addition, cross-reactivity data and the analysis of a small number of fortified samples have shown that the method will also detect a range of other major parent nitroimidazoles and their metabolites including ronidazole (RNZ), ipronidazole (IPZ), metronidazole (MNZ), hydroxymetronidazole (MNZOH), hydroxydimetridazole (DMZOH) and hydroxyipronidazole (IPZOH). The cross-reactivity profile and validation data for the detection of these nitroimidazoles are presented together with the results obtained following the analysis of a small number of incurred samples using the developed method.

Keywords: Screening; Detection; Nitroimidazoles; Optical biosensor

Inter-laboratory comparison of a yeast bioassay for the determination of estrogenic activity in biological samples by Toine F.H. Bovee; Gerrit Bor; Ilse Becue; Frieda E.J. Daamen; Majorie B.M. van Duursen; Sylvi Lehmann; Gϋnter Vollmer; Raffaella De Maria; Jennifer E. Fox; Hilda Witters; Silke Bernhöft; Karl-Werner Schramm; Ron L.A.P. Hoogenboom; Michel W.F. Nielen (pp. 265-272).

An inter-laboratory exercise was performed with a yeast estrogen bioassay, based on the expression of yeast enhanced green fluorescent protein (yEGFP), for the determination of estrogenic activity in extracts of calf urine samples. Urine samples were spiked with 1 and 5ngmL⁻¹ 17β-estradiol and 17α-ethynylestradiol, 10 and 50ngmL⁻¹ mestranol, and 100ngmL⁻¹ testosterone and progesterone. Sample extracts of blank and spiked urine samples were prepared at our laboratory and sent to seven laboratories together with a reagent blank, a DMSO blank, and eight 17β-estradiol stock solutions in DMSO ranging in concentration from 0 to 545ngmL⁻¹. Sample extracts and standards were coded and tested blindly. A decision limit (CCα) was determined based on the response of seven blank urine samples. Signals of the negative controls, e.g. urine samples spiked with 100ngmL⁻¹ testosterone or progesterone, were all below the determined CCα and were thus screened as compliant. Positive controls, i.e. the urine samples spiked at two levels with 17β-estradiol, 17α-ethynylestradiol and mestranol, were almost all screened as suspect, i.e. gave signals above the determined CCα. Determined EC₅₀ values calculated from the 17β-estradiol dose–response curves obtained by the seven laboratories ranged from 0.59 to 0.95nM.

Keywords: Bioassay; Estrogens; Transfer validation; Gas chromatography tandem mass spectrometry

Validation of an enzyme-linked immunosorbent assay screening for quinolones in egg, poultry muscle and feed samples by Giampiero Scortichini; Loredana Annunziata; Valeria Di Girolamo; Roberta Buratti; Roberta Galarini (pp. 273-278).

Quinolones are a group of chemotherapeutic agents with an excellent efficiency against poultry pathogens. Two commercial enzyme-linked immunosorbent assay (ELISA) tests have been applied in parallel for the qualitative screening analysis of several quinolones in eggs, poultry muscle and feeds at the required levels. During the validation study, carried out according to the Commission Decision 2002/657/EC criteria, two different sample treatments were compared in foods: the simple and fast procedure suggested by the kit producer (Euro-Diagnostica) and a more complex solid-phase extraction (SPE) sample preparation. The results demonstrated that the method based on SPE clean up exhibited better characteristic performances, particularly in eggs for which lower detection levels are required. Despite the fact that screening methods should be rapid and cheap, the use of non-chromatographic techniques such as ELISA for multiresidual detection of a class of substances involves some additional attention to sample preparation.

Keywords: Quinolones; Enzyme-linked immunosorbent assay; Eggs; Muscles; Feeds; Validation

Multiresidue method for the triphenylmethane dyes in fish: Malachite green, crystal (gentian) violet, and brilliant green by Wendy C. Andersen; Sherri B. Turnipseed; Christine M. Karbiwnyk; Rebecca H. Lee; Susan B. Clark; W. Douglas Rowe; Mark R. Madson; Keith E. Miller (pp. 279-289).

Liquid chromatographic methods are presented for the quantitative and confirmatory determination of crystal violet (CV; also known as gentian violet), leucocrystal violet (LCV), brilliant green (BG), and leucobrilliant green (LBG) in catfish. LCV and LBG were oxidized to the chromic CV and BG by reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, and residues were measured as the combined CV±LCV and BG±LBG. These methods are extensions of published methods for malachite green (MG) analysis to allow simultaneous determination of MG, CV, and BG. Residues were extracted from muscle with ammonium acetate buffer and acetonitrile, and extracts cleaned up using dichloromethane partitioning and solid-phase extraction. Extracts were analyzed by liquid chromatography with visible detection (LC-VIS). The method was validated for catfish fortified with LCV over the range 0.25–10ngg⁻¹ and CV at 2ngg⁻¹. Average recoveries were 90.6% (±8.1% R.S.D., n=45) for LCV and 84.4% (±4.2% R.S.D., n=6) for CV. The average recovery for samples fortified with BG or LBG over the range 0.5–10ngg⁻¹ was 67.2% (±14.8% R.S.D., n=31). CV and BG were confirmed in fish extracts by ion trap LC–mass spectrometry (LC–MSⁿ) with no discharge-atmospheric pressure chemical ionization. Average LC–MSⁿ recoveries were 96.5, 96.6, and 70.2% for samples fortified with CV, LCV, and BG or LBG. The limits of detection for CV, BG, and MG were in the range of 0.07–0.24ngg⁻¹ (ppb) for the two different instrumental methods. This methodology was applied to the analysis of catfish treated with CV and BG.

Keywords: Crystal violet; Brilliant green; Malachite green; Leuco metabolites; Catfish

Comparison of screening methods for antibiotics in beef kidney juice and serum by Marilyn J. Schneider; Katerina Mastovska; Steven J. Lehotay; Alan R. Lightfield; Brian Kinsella; Craig E. Shultz (pp. 290-297).

Rapid screening tests can be used as part of an efficient program designed to monitor veterinary drug residues in cattle. In this work, three rapid tests designed to screen samples for the presence of antibiotic residues, the Fast Antimicrobial Screen Test (FAST), Premi^® and Kidney Inhibition Swab (KIS™) tests, were compared using beef kidney juice and serum samples. In order to provide a realistic assessment, potentially incurred samples of beef kidney juice and serum were obtained from 235 carcasses which had been retained by inspectors in a processing plant for further testing. In addition, liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis was conducted on these samples to identify what antibiotics were present, if any, and their levels. The comparison of the three rapid screening test results with those from LC–MS/MS analysis allowed for a more complete comparison of the relative sensitivity of these analytical methods, as well as valuable information on false positive and negative response rates.

Keywords: Antibiotics; Screening; Microbial inhibition assays; Liquid chromatography–mass spectrometry

Comparison of three microbial screening methods for antibiotics using routine monitoring samples by Mariël G. Pikkemaat; Michel L.B.A. Rapallini; Sabrina Oostra-van Dijk; J.W. Alexander Elferink (pp. 298-304).

Monitoring large numbers of slaughter animals for the presence of antimicrobial residues is preferably carried out using microbiological screening methods, because of their high cost-effectiveness. An evaluation of the Nouws antibiotic test (NAT) was performed on routine monitoring samples and the performance of the method was compared with two other microbial screening methods: Screening test for antibiotic residues (STAR) and Premi^®Test. Analysis of 591 samples yielded four MRL violations. Three of them concerned tetracyclines that were only detected with the NAT and the STAR method. The fourth, 172μgkg⁻¹ Sulfadiazine, was detected by all three methods. Additionally, 156μgkg⁻¹ Tulathromycin was found in porcine meat, while for this residue no MRL in muscle has been established.

Keywords: Antibiotic residues; Microbial screening method; Premi; ^®; Test; Nouws antibiotic test; Screening test for antibiotic residues

Detection of anabolic steroids in dietary supplements: The added value of an androgen yeast bioassay in parallel with a liquid chromatography–tandem mass spectrometry screening method by Jeroen C.W. Rijk; Toine F.H. Bovee; Si Wang; Christof Van Poucke; Carlos Van Peteghem; Michel W.F. Nielen (pp. 305-314).

Recently we constructed a recombinant yeast cell that expresses the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. When exposed to testosterone, the concentration where half-maximal activation is reached (EC₅₀) was 50nM. Eighteen different dietary supplements, already analysed by a liquid chromatography–tandem mass spectrometry method (LC–MS/MS) for the presence of anabolic steroids, were screened for androgenic activity. Eleven samples containing at least one anabolic steroid, with a concentration that was around or above 0.01mgunit⁻¹ according to LC–MS/MS, were also positive in the bioassay. Seven samples did not contain any of the 49 compounds screened for in LC–MS/MS. In contrast two of them were positive in the bioassay. Bioassay-directed identification, using the bioassay as an off-line LC-detector and LC–time of flight-MS with accurate mass measurement was carried out in these two samples and revealed the presence of 4-androstene-3β,17β-diol and 5α-androstane-3β,17β-diol in the first and 1-testosterone in the second supplement, showing the added value of the bioassay in comparison with a LC–MS/MS screening method alone.

Keywords: Anabolic steroids; Androgen receptor; Bioassay; Doping; Supplements

Study of the depletion of lincomycin residues in honey extracted from treated honeybee ( Apis mellifera L.) colonies and the effect of the shook swarm procedure by Stuart J. Adams; Richard J. Fussell; Mike Dickinson; Selwyn Wilkins; Matthew Sharman (pp. 315-320).

Bee colonies were treated with 1.2g lincomycin hydrochloride per hive (single treatment in sucrose solution) and samples of honey were then collected at intervals over a 41-week period. The samples were analysed for lincomycin using Liquid Chromatography–Mass Spectrometry/Mass Spectrometry (LC–MS/MS). The highest mean concentration of lincomycin (pooled analytical results for brood and super honey) was 24μgg⁻¹ 3 days after treatment, a mean of 3.5μgg⁻¹ after 129 days. The shook swarm procedure was investigated and resulted in a lincomycin concentration of 34μgg⁻¹ in honey (pooled results for brood and super honey) 3 days after treatment, declining to 0.38μgg⁻¹ 129 days after treatment. Lincomycin was persistent in the hive and detected in all over winter (290 days after dosing) samples of honey collected from both non-shook swarmed and shook swarmed colonies. The results overall indicate that lincomycin parent is a suitable marker compound to detect lincomycin misuse in apiculture.

Keywords: Lincomycin; Honey; Veterinary drug residues; Apiculture

Detection of glucocorticoid bioactivity in bovine urine samples using a reporter gene assay by Lisa Connolly; Kai Cai; Edwige Van der Heiden; Marie-Louise Scippo; Marc Muller; Jonathan Tarbin; Chris Elliott (pp. 321-327).

The illegal use of anabolic substances in the meat producing industry is an ongoing problem due to the continual production of new synthetic compounds and/or the practice of low-level cocktail administration to avoid detection by the surveillance schemes of EU member states National Plan surveillance systems.We present a highly sensitive reporter gene assay and sample extraction procedure based on a two step solid phase extraction and high performance liquid chromatography, developed for the detection of glucocorticoid abuse in bovine urine. The assay is capable of detecting compounds with glucocorticoid bioactivity and is extremely sensitive with an EC₅₀ of 0.79ngmL⁻¹ for dexamethasone. New or unknown compounds with glucocorticoid bioactivity and low-level cocktail mixtures are detectable by this assay.Cross-reactivity data for a range of 11β-hydroxyglucocorticoids has been provided. This assay shows low interference from the 11-keto prohormones and other steroidal hormones. The assay may be suitable for application in other matrices such as hair. In conclusion this screening assay offers advantages over existing analytical techniques.

Keywords: Glucocorticoid; Bovine urine; Reporter gene assay; Endocrine disruptor

Antibody production: Low dose immunogen vs. low incorporation hapten using salmeterol as a model by Terence L. Fodey; Nuala M. Greer; Steven R.H. Crooks (pp. 328-332).

Haptens are low molecular weight compounds that are non-immunogenic and so must be conjugated to carrier molecules to elicit an immune response.Doses of 50–1000μg protein conjugate have been suggested for immunisation of rabbits with hapten–protein immunogens. Although larger doses may give a faster response, lower doses may result in higher affinity antibodies. The amount of hapten presented to the host's immune system can be controlled by varying either the amount of immunogen administered or the quantity coupled to a fixed amount of protein. This study compares the two approaches for the production of antibodies to the β-agonist salmeterol as a model.A range of salmeterol–HSA conjugates was prepared, with varying molar ratios of hapten:protein (80:1, 15:1, 7.5:1, 4:1), for use as immunogens. The 80:1 immunogen was administered to different animals at four concentrations (0.5, 0.2, 0.1 and 0.05mgdose⁻¹) while the remaining three immunogens were administered at 0.5mgdose⁻¹. The effects of immunogen dose and hapten incorporation on the titre and affinity of the antibodies produced to salmeterol were investigated.It was found that both approaches resulted in the production of more sensitive antibodies, although reducing the degree of hapten incorporation brought about a larger reduction in antibody titre. Reducing the degree of hapten incorporation also produced a more consistent pattern of results regarding antibody sensitivity (after the sixth immunisation in this study) which may make it easier to predict the most suitable time for antibody harvesting.

Keywords: Abbreviations; ELISA; enzyme linked immunosorbent assay; HRP; horseradish peroxidase; HSA; human serum albuminAntibody production; Immunogen dose; Hapten incorporation; Affinity; Titre

A National Residue Control Plan from the analytical perspective—The Brazilian case by Angelo de Q. Mauricio; Erick S. Lins; Marcelo B. Alvarenga (pp. 333-336).

Food safety is a strategic topic entailing not only national public health aspects but also competitiveness in international trade. An important component of any food safety program is the control and monitoring of residues posed by certain substances involved in food production. In turn, a National Residue Control Plan (NRCP) relies on an appropriate laboratory network, not only to generate analytical results, but also more broadly to verify and co-validate the controls built along the food production chain. Therefore laboratories operating under a NRCP should work in close cooperation with inspection bodies, fostering the critical alignment of the whole system with the principles of risk analysis. Beyond producing technically valid results, these laboratories should arguably be able to assist in the prediction and establishment of targets for official control. In pursuit of analytical excellence, the Brazilian government has developed a strategic plan for Official Agricultural Laboratories. Inserted in a national agenda for agricultural risk analysis, the plan has succeeded in raising laboratory budget by approximately 200%, it has started a rigorous program for personnel capacity-building, it has initiated strategic cooperation with international reference centres, and finally, it has completely renewed instrumental resources and rapidly triggered a program aimed at full laboratory compliance with ISO/IEC 17025 requirements.

Keywords: Residues; National Residue Control Plan; Brazil; Official control; Laboratories network

Food flavonoid aryl hydrocarbon receptor-mediated agonistic/antagonistic/synergic activities in human and rat reporter gene assays by Edwige Van der Heiden; Nathalie Bechoux; Marc Muller; Thérèse Sergent; Yves-Jacques Schneider; Yvan Larondelle; Guy Maghuin-Rogister; Marie-Louise Scippo (pp. 337-345).

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor mediating the adverse effects of dioxins and polycyclic aromatic hydrocarbons (PAHs). In this study, we investigated the genetic-, time-, dose-, species- and tissue-dependent AhR-mediated agonistic/antagonistic activities of three food flavonoids: quercetin, chrysin and genistein. To that end, four stably transfected cell lines were used in cell-based luciferase reporter gene assays: three lines were transformed with the ptKLuc vector harbouring four dioxin-responsive elements (DREs) upstream of the thymidine kinase promoter and the luciferase gene (HepG2-Luc, T-47D-Luc and H4IIE-ULg). The fourth is a patented cell line transformed with a different construct: H4IIE DR-CALUX^®. Both H4IIE cells were compared for their genetic construction. Human hepatoma (HepG2-Luc) and human breast tumour (T-47D-Luc) cells were compared for tissue-dependent effects. Rat hepatoma (H4IIE-ULg) and human hepatoma (HepG2-Luc) cells were compared for species-dependent activities. We concluded that quercetin, chrysin and genistein act in a time-, dose-, species- and tissue-specific way. For example, genistein displayed agonistic activities when exposed to rat hepatoma cells during 6h but not after 24h. Flavonoids displayed agonistic/antagonistic activities in human breast tumour cells, depending on the exposure time, while in human hepatoma cells, only antagonistic activities of flavonoids were measured. In addition, we report, in all the cells, a synergy between an isoflavone and two food contaminants; the 2,3,7,8-tetrachlorodibenzo- p-dioxin and 3-methylcholanthrene, a PAH. In rat cells, this synergy occurred when cells were exposed to flavonoids and contaminant for 6h, while it was observed in human cells only after 24h.

Keywords: Flavonoids; Luciferase reporter gene; Contaminants; Aryl hydrocarbon receptor (AhR); Synergy; (ant)-Agonist

In-house reference materials: 5-hydroxyflunixin and meloxicam in cow milk—preparation and evaluation by Piotr Jedziniak; Teresa Szprengier-Juszkiewicz; Małgorzata Olejnik (pp. 346-350).

Reference materials are helpful to evaluate the performance of laboratories as well as being useful for the quality control of analytical procedures. Certified reference materials and other reference materials containing non-steroidal anti-inflammatory drugs in milk are however, not available. Therefore, production and evaluation of in-house reference materials with incurred residues of 5-hydroxyflunixin (5OHFLU) and meloxicam (MEL) in cow milk has been performed. The milk was collected 12h after dosing from cows which received meloxicam (0.5mgkg⁻¹ b.w., i.v., single dose) or flunixin meglumine (2.2mgkg⁻¹ b.w., i.v. during three days). The concentrations of analytes were checked in the milk samples. The milk was diluted with milk free from NSAIDs residues, homogenised, put into sterile 20mL vials, frozen and lyophilised. The vials were weighed before and after lyophilisation, in order to calculate the amount of water necessary for reconstitution, and were stored at a temperature of −20±2°C. For the homogeneity study, 10 random samples were analysed in duplicate and the results were interpreted using Cochran's test, Horwitz standard deviation and the test for a sufficient homogeneity. The assigned values, calculated from the results of the homogeneity test were 54.3μgkg⁻¹ for 5OHFLU and 46.4μgkg⁻¹ for MEL. The samples were tested for their stability every 14 days for 2 months and after 9 months. It has been confirmed that an appropriate homogeneity and stability of the produced in-house reference material has been obtained.

Keywords: Reference material; Flunixin; Meloxicam; Liquid chromatography

Effect of growth-promoting 17β-estradiol, 19-nortestosterone and dexamethasone on circulating levels of nine potential biomarker candidates in veal calves by Giuseppe Cacciatore; Susanne W.F. Eisenberg; Chen Situ; Mark H. Mooney; Philippe Delahaut; Sjoerd Klarenbeek; Anne-Catherine Huet; Aldert A. Bergwerff; Christopher T. Elliott (pp. 351-359).

The use of screening methods based on the detection of biological effects of growth promoters is a promising approach to assist residue monitoring. To reveal useful effects on protein metabolism, male and female veal calves at 10 weeks of age were treated thrice with a combination of 25mg 17β-estradiol 3-benzoate and 150mg 19-nortestosterone decanoate with 2 weeks intervals and finally once with 4mg dexamethasone. Hormone-treated calves showed a significant accelerated growth rate over 6 weeks. Plasma samples of treated and control calves were analysed for immunoreactive inhibin (ir-inhibin), osteocalcin, insulin-like growth factor 1 (IGF-1), insulin-like growth factor-binding protein 2 (IGFBP-2), IGFBP-3, luteinzing hormone (LH), follicle-stimulating hormone (FSH) and prolactin using immunoaffinity assays. Hormone treatment did not affect levels of IGF-1, IGFBP-2, IGFBP-3, LH, FSH and prolactin. The concentration of circulating ir-inhibin decreased, however, significantly ( P<0.05) in bull calves upon administration of the sex steroids, whereas it remained unchanged in the female animals. Dexamethasone treatment decreased significantly ( P<0.05) circulating levels of osteocalcin in both female and male animals. Ir-inhibin and osteocalcin were, therefore, considered as candidates for a protein biomarker-based screening assay for detection of abuse of estrogens, androgens and/or glucocorticoids in cattle fattening, which is being developed in the framework of EU research project BioCop (www.biocop.org).

Keywords: Anabolics; Biomarker; Illegal growth-promotion; Proteomics; Residue analysis; Veterinary public health

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Analytica Chimica Acta (v.637, #1-2)