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Analytica Chimica Acta (v.529, #1-2)

Outside front cover - Editorial Board (pp. co1).

Contents (pp. vii-ix).

Calendar of forthcoming meetings (pp. n1-n3).

Foreword by Rainer W. Stephany (pp. 1-1).

Impact of new legislation on the registration of veterinary drugs by Rick Clayton (pp. 3-6).

The European legal framework covering medicinal products was reviewed by the European institutions during the period from November 2001 to February 2004.The new legislation for registration of medicinal products was published on 30 April 2004 in the Official Journal of the European Union and will come into force towards the end of 2005. It seeks to reach a balance between encouraging innovation and facilitating the authorisation of generics. It also aims to reduce product maintenance costs (renewals) and strengthen pharmacovigilance. However, the overall regulatory burden is not reduced, and this affects the global competitiveness of the European industry. There will be greater emphasis on good manufacturing practice (e.g. for starting materials of active ingredients) and scientific advice. The role of the EMEA will be increased in the areas of pharmacovigilance and inspections, and its structures will be adapted to cope with EU enlargement. Transparency is increased in all areas. The mutual recognition procedure is reorganised to drive harmonisation and reduce the rate of withdrawal from some member states during the procedure (e.g. automatic arbitration of any unresolved issues).New variations regulations with harmonised and clearer procedures were published in June 2003. They will reduce the workload more for competent authorities than for companies. The legislation covering maximum residue limits and residue monitoring is currently being reviewed, with the objective of developing a more balanced and coordinated approach.

Trends in animal feed composition and the possible consequences on residue tests by Gianfranco Brambilla; Stefania De Filippis (pp. 7-13).

In the recent past years, some relevant changes have been introduced in the nutrition of farmed animals. To meet safety issues induced by Bovine Spongiform Encephalopathy (BSE) and ‘dioxins’ crises, new kinds of vegetal feed materials have been introduced in the diet, to replace animal proteins and fats. Moreover, the progressive withdrawal of some feed additives with anti-microbial activity, such as avoparcin, bacitracin, virginiamycin, tylosin, spiramycin, carbadox and olaquindox due to a renewed analysis about their toxicological and pharmacoresistance risk, could prompt the market to propose some other alternatives to farmers, such as the administration of immuno-stimulants (i.e. colostrum) and/or natural substances having a direct or indirect anti-bacterial effect (i.e. prebiotics, probiotics, herbal extracts). Last but not least, one of the fastest growing areas is represented by the study of feed formulations able to produce nutraceuticals and dietary foods of animal origin on large scale, thus boosting consumers perception about their overall quality, such as polyunsaturated fatty acids and Vitamin E enriched eggs, via the use of selected materials in feeds. This paper aims to illustrate some examples of such innovative trends in animal feeding, along with the discussion on the analytical challenges useful for a reliable assessment of a possible new risks/benefits ratio along the food chain.

Keywords: Animal hygiene; Feedingstuffs; Residues analysis; Nutraceuticals.

Sulfonamides residues analysis: evaluation of results dispersion at maximum residual limit by the expanded uncertainty and 2002/657/EC decision limit approaches by Ivan Pecorelli; Rita Bibi; Laura Fioroni; Arianna Piersanti; Roberta Galarini (pp. 15-20).

It is impossible to determine whether an analytical procedure is suitable to the purposes without some knowledge of its uncertainty. Several approaches for its estimation have already been proposed in the past years. Discussion on the estimation of uncertainty of chemical measurement has also influenced the ISO 17025 which explicitly refers to the bottom-up approach.On the other hand, the European Commission Decision 2002/657/EC concerning the performance of methods and the interpretation of results in the official control of residues in products of animal origin imposes the determination of some new parameters, such as decision limit (CCα) which represents an index of results dispersion. This decision also indicates that official laboratories must be accredited according to ISO 17025.In this work, the bottom-up approach together with in-house validation data is applied for the evaluation of measurement uncertainty associated with residue determination of 10 sulfonamides in muscle and, at the same time, the decision limit was calculated following the 2002/657/EC criteria.

Keywords: Sulfonamides; Uncertainty; Validation; 2002/657/EC

Rapid screening method for halofuginone residues in poultry eggs and liver using time-resolved fluorometry combined with the all-in-one dry chemistry assay concept by Virve Hagren; Lisa Connolly; Christopher T. Elliott; Timo L�vgren; Mika Tuomola (pp. 21-25).

The present study describes the development and validation of an immunoassay for the screening of coccidiostat halofuginone in poultry eggs and liver. The power of time-resolved fluorometry is utilised with a novel all-in-one dry chemistry assay concept, in which all the reagents needed for the competitive immunoassay are built into a single microtiter well in a dry, stable form. The entire immunoassay is performed by an automated immunoanalyser and the total assay time is only 18min. The assay protocol is simple: the extracted sample is added to the well and after the 15min sample incubation and wash steps are completed, the fluorescence signal is measured directly from the surface of the dry well in a time-resolved manner. The analytical limit of detection has been calculated as 0.02ngml⁻¹ ( n = 12) and the functional limit of detection as 1.7 and 1.0ngg⁻¹ for egg ( n = 6) and liver ( n = 6) samples, respectively. If needed, the assay sensitivity can be further improved simply by adjusting the dilution factor of the samples. The mean recovery has been determined as 90.5% for egg at concentration levels of 15 and 60ngg⁻¹, and as 106.0% for liver at concentrations of 7.5 and 30ngg⁻¹. The intra-assay variations have typically been below 10% and interassay variations have ranged between 7.7 and 11.6%. The halofuginone immunoassay has also been validated according to Commission Decision 2002/657/EC.

Keywords: Halofuginone; Residues; Screening; Immunoassay; Time-resolved fluorometry; Dry chemistry

Rapid screening of narasin residues in poultry plasma by time-resolved fluoroimmunoassay by Pekka Peippo; Timo L�vgren; Mika Tuomola (pp. 27-31).

A simple and rapid time-resolved fluoroimmunoassay (TR-FIA) method has been developed for the screening of narasin in poultry plasma. This method involves only a dilution of sample and competitive microwell plate immunoassay combined with the measurement of time-resolved fluorescence. The performance of the assay has been confirmed by validation according to Commission’s Decision 2002/657/EC. Decision limit and detection capability of the assay have been 1.2 and 1.5ngml⁻¹, respectively. Narasin recovery from plasma has ranged from 101.0 to 121.3%. In the feeding study broiler chickens have been fed narasin at a dose rate of 0, 3.5 and 70mgkg⁻¹ feed for 3-week period and the concentration of narasin in the plasma and muscle of broilers have been studied by the TR-FIA. Feeding with 70mgkg⁻¹ and no withdrawal period resulted in a very high narasin levels in blood, and the artificial contamination level of feed (3.5mgkg⁻¹) was also detected by the analysis of blood samples. A relationship was observed between the concentrations of narasin in plasma and breast muscle ( R² = 0.83) and leg muscle ( R² = 0.90). These results suggest that the analysis of poultry blood samples could be used as a predictor of narasin residues in muscle.

Keywords: Time-resolved fluoroimmunoassay; Plasma; Narasin

Potential application of gene expression fingerprinting for food safety screening by T. Buterin; C. Koch; H. Naegeli (pp. 33-39).

Drug residues or contaminants with unwanted adverse effects may include tens of thousands of synthetic or natural substances. Although most of these chemicals are usually present in the food at harmless concentrations, it is a tremendous task to identify those samples that pose a possible health hazard to the consumer. Current monitoring actions involve, in most cases, single-endpoint screening tests that detect only a unique chemical entity or a limited group of related substances. One major challenge in the area of food safety is, therefore, the development of multi-endpoint strategies that could increase the efficiency of high-throughput screening procedures. Here, we used human cell lines derived from breast epithelium (MCF-7 and T-47D) to explore the potential impact of microarray-based transcriptomic analyses in allowing the simultaneous detection of a large number of different residues or contaminants. Both cell lines yielded characteristic expression profiles upon exposure to representative chemicals that are often found in food products. Interestingly, this pilot study suggests that T-47D cells respond to treatment with different xenoestrogenic chemicals with distinct expression profiles. Thus, the use of oligonucleotide microarrays may considerably expand the range and improve the specificity of existing reporter bioassays.

Keywords: Microarrays; Transcriptomics; Genetic fingerprints; Dioxin; PCB; Genistein

Recombinant bovine somatotropin misuse in cattle by Gaud Pinel; Ronan Buon; Florence Aviat; Colette Larr�; Genevi�ve Andr�-Fontaine; Fran�ois Andr�; Bruno Le Bizec (pp. 41-46).

Somatotropins (growth hormone, GH) which are used in cattle for growth and lactating performances are difficult to reliably detect since no direct method exists. Until now, the detection of GH in bovine milk, plasma and tissue has been based on radioimmunoassays and enzyme-linked immunosorbent assay (ELISA) tests that are unable to distinguish between endogenous and recombinant hormones. Attempt to directly detect recombinant GH administration is an analytical challenge which has never been successfully reported. The aim of the present work was to develop an original method, based on the detection in serum of antibodies raised against growth hormones as a consequence of their administration to animals. A second approach was developed with the characterisation of recombinant bovine somatotropin (rbST) in milk through the use of two-dimensional electrophoresis coupled to in-gel tryptic digestion followed by tandem mass spectrometry coupled to liquid chromatography (LC-ESI-MS/MS) measurements of the produced peptides.Results obtained showed the relevance of the two approaches. The Western blot one, carried out in this work on real biological blood samples collected on treated animals, would fit as a rapid and rather sensitive screening method, which can further more be achieved more over a long period after the administration of recombinant hormone. The second method, which consisted in a proteomic approach, dealt with the development of a confirmatory technique. Results on spiked milk samples were very promising and showed the need of further developments in this way.

Keywords: Growth hormones; Somatotropin; Bovine; Milk; Plasma; Western blot; 2D electrophoresis

Simple and rapid screening and confirmation of tetracyclines in honey and egg by a dipstick test and LC–MS/MS by G. Alfredsson; C. Branzell; K. Granelli; �. Lundstr�m (pp. 47-51).

The illegal use of antibiotics such as tetracyclines in honey production has recently been focused. The need for simple test methods has resulted in the development of a dipstick-test based on receptor binding, Tetrasensor^®, Unisensor, Belgium. In this paper, two types of the test, the kit for analysis of tetracyclines in honey and the kit for analysis of tetracyclines in egg, fish and tissues from several animal species, have been evaluated and the results have been confirmed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Different batches of Swedish honey and egg were analysed blank or spiked with oxytetracycline, tetracycline and chlortetracycline at 25 (honey) and 200μgkg⁻¹ (egg). The confirmation by LC–MS/MS was performed directly on diluted test extract without further purification (honey) or after a simple filtration (egg). This combination of a simple dipstick-test and LC–MS/MS proved to be a suitable and rapid way for the surveillance of tetracyclines in honey and egg.

Keywords: Liquid chromatography; Tetracycline; Mass spectrometry; Tetrasensor; Egg; Honey

Pharmacological characterization of new β-agonists using Huβ₁- and Huβ₂-adrenergic receptor binding assay in transfected HEK-293 cells by Cinzia Civitareale; Caterina Ambrosio; Maria Sbraccia; Maurizio Fiori; Gianfranco Brambilla; Cecilia Testa (pp. 53-56).

The continuous turn over of β-agonists molecules, may affect the reliability of screening tests. To overcome possible false negative results, a bioassay is under development to detect the presence of new β-agonists in feeds and biological matrices and so to provide a valid tool for a multi-analyte screening method. Preliminary study were focused on the pharmacological characterisation of new β-agonists, with the aim to combine both the screening results with a toxicological evaluation about the potential health risk for consumers. The interaction of G4, G5, G6, G8 adrenergic drugs with human β₁- and β₂-adrenergic receptors expressed separately in membranes of human embryonic kidney cells in culture (HEK-293-Huβ₁ and HEK-293-Huβ₂), were studied by a receptor binding assay and results compared with those from a well-known β-adrenergic agonist (clenbuterol). The specificity of the test was assured by the use of a specific radiolabel tracer ligand ([¹²⁵I]iodopindolol) as competitor.For compounds G4, G5 and G6 the affinity (IC₅₀) for β₁-adrenergic binding sites was of the same magnitude of that from clenbuterol. By contrast, G8 showed a 100-fold higher affinity. On β₂-adrenergic receptors the binding affinity was similar for G4 and G6, but about 10-fold higher for G5 and G8 with respect to that from clenbuterol.

Keywords: β-Adrenoceptor agonists; β-Adrenergic receptors; Receptor binding assay; Human embryonic kidney cells

Validation of a rapid yeast estrogen bioassay, based on the expression of green fluorescent protein, for the screening of estrogenic activity in calf urine by Toine F.H. Bovee; Henri H. Heskamp; Astrid R.M. Hamers; Ron L.A.P. Hoogenboom; Michel W.F. Nielen (pp. 57-64).

Previously we described the construction and properties of a rapid yeast bioassay stably expressing human estrogen receptor α (hERα) and yeast enhanced green fluorescent protein (yEGFP) in response to estrogens. In the present study, this yeast estrogen assay was validated as a qualitative screening method for the determination of estrogenic activity in calf urine. This validation was performed, according to EC decision 2002/657, which prescribes the determination of the detection capability ( CCβ), the specificity/selectivity and the stability/ruggedness/applicability. To determine these performance characteristics, twenty blank urine samples of 19-week-old calves were collected and spiked with 17β-estradiol (E2β) at 1ngml⁻¹, diethylstilbestrol (DES) at 1ngml⁻¹, 17α-ethynylestradiol (EE2) at 1ngml⁻¹, α-zearalanol at 30 and 50ngml⁻¹ or mestranol at 10ngml⁻¹. Following enzymatic deconjugation and solid phase extraction, 100μl equivalents of these blank and spiked urine samples were screened for estrogenic activity in a 96 well plate using the yeast estrogen bioassay. All of these low estrogen spiked urine samples could be distinguished from the blank samples as all spiked samples gave a signal above the determined decision limit CCα and the mean responses of the spiked samples were higher than the determined detection capability CCβ. As this CCβ criterion is met, these spiked samples have a lower than 5% probability to be classified as a false negative. The specificity of the method was determined with blank urine samples spiked with a high dose of testosterone or progesterone (1000ngml⁻¹). No response to these substances was detected in the yeast estrogen bioassay. There was also no interference of a high dose of testosterone or progesterone on the response of a low dose of the estrogens. Stability of urine samples was checked with spiked urine samples that were kept frozen for up to 90 days, showing that urine samples could be stored at −20°C for up to 60 days without changing the screening result of the assay. This method has been in routine use at RIKILT for more than one year.

Keywords: Specificity; Urine; Validation; Yeast Estrogen Bioassay

Confirmation of the identity of residues using quadrupole time-of-flight mass spectrometry by Jan F. Van Bocxlaer; Sofie R. Vande Casteele; Christof J. Van Poucke; Carlos H. Van Peteghem (pp. 65-73).

Tandem mass spectrometry (MS/MS) has become a very important tool in bio-analysis, including residue analysis. The combination of a quadrupole and a time-of-flight mass analyser (Q-TOF mass spectrometer) provides an instrument with particularly interesting characteristics, also for residue analysis, compared to the more common triple quadrupole instruments. Especially the accurate mass measurement capabilities, the full scan sensitivity of time-of-flight mass analysis and the possibility of information dependent acquisition are of interest. The main field of application therefore is qualitative analysis, i.e. the identification of unknowns. To illustrate the potential of a Q-TOF mass spectrometer regarding the analysis of residues, two examples from literature are given, which detail the detection of unknowns in illegal cocktails. In addition, a third new experimental study is described about the MS analysis of unknown mixtures of growth promoters using accurate mass. Nevertheless, Q-TOF mass spectrometric applications in the field of, especially, food residue analysis seem to be lagging behind. Residue analysis being a mainly quantitative type of work, the relatively limited quantitative reputation of a Q-TOF mass spectrometer system is probably the culprit.

Keywords: Quadrupole time-of-flight; Residue analysis; Accurate mass measurement; Information dependent acquisition

The analysis of beta-agonists in bovine muscle using molecular imprinted polymers with ion trap LCMS screening by P.R. Kootstra; C.J.P.F. Kuijpers; K.L. Wubs; D. van Doorn; S.S. Sterk; L.A. van Ginkel; R.W. Stephany (pp. 75-81).

A liquid chromatography–mass spectrometry (LC–MS) method for the analysis of beta-agonists in bovine muscle is developed. Sample clean-up is based on molecular imprinted polymers (MIPs). The LC–MS method was validated for screening purposes. The validation results show that eight compounds (cimaterol, cimbuterol, ractopamine, clenproperol, clenbuterol, brombuterol, mabuterol, mapenterol and isoxsuprine) meet the requirements for quantitative determination using MIPs for sample clean-up. Reliable screening is possible as low as 1μgkg⁻¹. The method was validated for bovine muscle but also has proven to be suitable for muscle samples from rabbit, duck and turkey, liver and various kinds of fish.

Keywords: Molecular imprinted polymer; Beta-agonists; Growth promoter; Residues; Muscle

A new approach for detection of antimicrobial drugs in food by Sara Stead; S. Richmond; Matthew Sharman; Jacques Stark; Edith Geijp (pp. 83-88).

A rapid, high-throughput antimicrobial screening assay has been developed that combines either a physical fluid extraction or a solvent extraction technique with the commercially available Premi^®Test.In order to remove the subjectivity of the visual end-point measurement associated with this microbial inhibition assay, work has been conducted to couple the Premi^®Test to scanner technology. The use of the solvent extraction provides an enhanced detection capability for a wide range of drugs at or below one-half the maximum residue limit (MRL) concentrations in a variety of matrices, as demonstrated by dose response curve data. Secondary class-specific assays, for the identification of β-lactams and sulphonamides following the primary screen have been previously developed and recently validated using the scanner technology.Method validation using both fortified and incurred tissues has been undertaken to establish the ruggedness of the technique. The false-positive and -negative rates have been established at less than 5% for a range of drug/matrix combinations.The CCβ values determined for this qualitative screening assay are at concentrations less than the MRL. This integrated screening strategy provides a reliable tool for antimicrobial residue monitoring in surveillance programmes.

Keywords: Antimicrobial; Screening; Scanner; Premi; ^�; Test; Food safety

Simultaneous and rapid detection of five banned antibiotic growth promoters by immunoassay by C. Situ; C.T. Elliott (pp. 89-96).

As a result of increasing concerns over the transfer of resistance between different bacteria and between human and animals, a group of antimicrobial growth promoters including bacitracin, spiramycin, tylosin, virginiamycin and olaquindox have been banned in the EU since 1999. A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) with simple extraction procedures was developed for simultaneous screening of the five banned feed additives in animal feeds. Banned substances were extracted from animal feeds with 70% methanol in water followed by clean-up on OASIS^® HLB cartridges. Polyclonal (Pab) antibodies were generated and their specificities assessed by cross-reactivity studies with substantial numbers of antimicrobial agents that are administered to animals via feedingstuffs. The target minimum detectable concentrations (MDC) of 4mgkg⁻¹ for olaquindox and 1mgkg⁻¹ for bacitracin, spiramycin, tylosin and virginiamycin were set for the present study. Recoveries at the three levels of the target concentrations (4, 6 and 8mgkg⁻¹ for olaquindox, 1, 1.5 and 2.0mgkg⁻¹ for the other four compounds) ranged from 84 to 145% with coefficients of variation less than 18%. The detection capability for virginiamycin, bacitracin, spiramycin, tylosin and olaquindox were 0.2, 0.3, 0.6 and 1.5mgkg⁻¹, respectively.

Keywords: Antibiotic; Simultaneous; Growth promoter; Feed additive; Animal feedingstuff; ELISA

Detection of colistin in spiked and incurred milk samples by LC- and ELISA-technique by Gertraud Suhren; Karin Knappstein (pp. 97-101).

A liquid chromatography (LC) method for the determination of the polypeptide antibiotic colistin (COL) was developed and applied to incurred milk samples from cows treated by a drug containing COL and ampicillin. At maximum residue limit (MRL) concentration (50μgkg⁻¹) the recovery rate was 106.1% and the coefficient of variation 13.6%. Limits of detection and quantification were 9.3 and 14.3μgkg⁻¹, respectively. There was no indication that the analytical results were influenced either by elevated somatic cell count or by ampicillin. COL-ELISA proved to be a reliable screening method. All spiked and incurred samples with COL-concentrations ≥MRL-concentration were evaluated as positive by ELISA. Neither with ELISA nor with the LC-method concentrations ≥MRL were detected in samples of untreated cows. By ELISA-screening no car tanker milk sample ( n=416) was suspicious to contain COL.

Keywords: Colistin; Milk; LC-method; ELISA

Development and validation of screening and confirmatory methods for the detection of chloramphenicol and chloramphenicol glucuronide using SPR biosensor and liquid chromatography–tandem mass spectrometry by H.M. Ashwin; S.L. Stead; J.C. Taylor; J.R. Startin; S.F. Richmond; V. Homer; T. Bigwood; M. Sharman (pp. 103-108).

Biacore Q biosensor and liquid chromatography–tandem mass spectrometry (LC–MS/MS) based methods for the screening and confirmation of trace levels of chloramphenicol (CAP) and the mammalian metabolite chloramphenicol glucuronide (CAP-Glu) is reported. Both methods employ solvent extraction and clean-up by solid phase extraction (SPE) prior to analysis. The biosensor screening method utilises surface plasmon resonance (SPR) to determine chloramphenicol concentration in a range of matrices including honey and prawns. As the antibody used in the biosensor has a high cross-reactivity with CAP-Glu, direct detection of this metabolite is possible in matrices such as porcine kidney. LC–MS/MS is used in negative ion electrospray mode for the confirmatory procedure. Parent CAP is determined via the use of an internal standard. In the case of porcine kidney parent CAP is released from CAP-Glu following a short digestion with a β-glucuronidase. All methods have been validated to the latest EU requirements (Commission Decision 2002/657/EC). The calculated decision limits (CCα) and detection capabilities (CCβ) are less than 0.1 and 0.2μgkg⁻¹ respectively for the screening and confirmatory techniques. Incurred tissues were used to study and confirm the long-term reproducibility and agreement between methods. In addition, the stability of CAP and CAP-Glu has been investigated. The analytes are stable under nearly all storage conditions for at least 20 weeks.

Keywords: Chloramphenicol; Chloramphenicol glucuronide; Surface plasmon resonance; LC–MS/MS; Biosensor

Detection of chloramphenicol and chloramphenicol glucuronide residues in poultry muscle, honey, prawn and milk using a surface plasmon resonance biosensor and Qflex^� kit chloramphenicol by Julie Ferguson; Andrew Baxter; Paul Young; Glenn Kennedy; Chris Elliott; Stefan Weigel; Robert Gatermann; Helen Ashwin; Sara Stead; Matthew Sharman (pp. 109-113).

Immunochemical screening assays using surface plasmon resonance have been developed for chloramphenicol and chloramphenicol glucuronide residues in poultry muscle, honey, prawn and cows’ milk using a sensor chip coated with a chloramphenicol derivative and an antibody. The antibody cross-reacted with chloramphenicol glucuronide 73.8% (poultry), 69.2% (honey), 75.7% (prawn) and 84.8% (milk). There was no cross-reaction with similar drugs or other commonly used antibiotics. The assay allowed the direct analysis of bovine milk (fat content ∼3.5%). Poultry, honey and prawn samples were extracted with ethyl acetate followed by analysis on the biosensor. The decision limits (CCα) for each assay were determined as: poultry (0.005μgkg⁻¹), honey (0.02μgkg⁻¹), prawn (0.04μgkg⁻¹) and milk (0.04μgkg⁻¹) and the detection capabilities (CCβ) were 0.02, 0.02, 0.07 and 0.05μgkg⁻¹, respectively. Poultry muscle, honey and milk were spiked at 0.1μgkg⁻¹ and prawn at 0.15μgkg⁻¹ and the intra-assay precision ( n=10) calculated as 10.5, 5.0, 4.6 and 8.8%, respectively. Between run precision ( n=3) performed at the same levels yielded the following results: 3.0% (poultry), 4.7% (honey), 7.6% (milk) and 5.5% (prawn).

Keywords: Chloramphenicol; Chloramphenicol glucuronide; Residue; Surface plasmon resonance

Comparison of multi-sulfonamide biosensor immunoassays by Monique Bienenmann-Ploum; Teemu Korpim�ki; Willem Haasnoot; Fort�ne Kohen (pp. 115-122).

Three different group-specific anti-sulfonamide antibodies were compared in inhibition assay formats in an optical biosensor (BIACORE 3000) using CM5 sensor chips coated with three different sulfonamide derivatives. The antibodies used were an anti-sulfamethazine monoclonal antibody (Mab) 21C7, the sulfonamide binding protein (SBP) in the Qflex Kit Sulfonamides and a recently developed mutant antibody (M.3.4). Each of these antibodies showed interactions with all 17 sulfonamides tested and one (Mab 21C7) was sensitive for the N₄-acetyl metabolites also. The limits of detection of the different sulfonamides in chicken serum varied between 7 and >1000ngml⁻¹ (Mab 21C7), 15 and 340ngml⁻¹ (Qflex) and 4 and 82ngml⁻¹ (Mutant M.3.4). The mutant M.3.4 based assay was found to be the most sensitive towards most of the sulfonamides whereas the Qflex Kit Sulfonamides detected the five sulfonamides registered for application in poultry in The Netherlands within the narrowest measurement range.

Keywords: Biosensor immunoassay; Biacore; Monoclonal antibodies; Recombinant antibodies; Sulfonamides; Chicken serum

Multi sulfonamide screening in porcine muscle using a surface plasmon resonance biosensor by Terry McGrath; Andrew Baxter; Julie Ferguson; Simon Haughey; Peter Bjurling (pp. 123-127).

A binding protein displaying broad-spectrum cross-reactivity within the sulfonamide group was used in conjunction with a sulfonamide specific sensor chip and a surface plasmon resonance biosensor to develop a rapid broad spectrum screening assay for sulfonamides in porcine muscle. Results for 40 samples were available in just over 5h after the completion of a simple sample preparation protocol. Twenty sulfonamide compounds were detected. Acetylated metabolites were not recognised by the binding protein. Limit of detection (mean–three times standard deviation value when n=20) was calculated to be 16.9ngg⁻¹ in tissue samples. Intra-assay precision ( n=10) was calculated at 4.3 %CV for a sample spiked at 50ngg⁻¹ with sulfamethazine, 3.6 %CV for a sample spiked at 100ngg⁻¹ with sulfamethazine, 7.2 %CV for a sample spiked at 50ngg⁻¹ with sulfadiazine and 3.1 %CV for a sample spiked at 100ngg⁻¹ with sulfadiazine. Inter-assay precision ( n=3) was calculated at 9.7 %CV for a sample spiked at 50ngg⁻¹ with sulfamethazine, 3.8 %CV for a sample spiked at 100ngg⁻¹ with sulfamethazine, 3.5 %CV for a sample spiked at 50ngg⁻¹ with sulfadiazine and 2.8 %CV for a sample spiked at 100ngg⁻¹ with sulfadiazine.

Keywords: Generic sulfonamides assay; Screening test; SPR Biosensor; Pork muscle; Sulfamethazine sulfadiazine

The ion suppression phenomenon in liquid chromatography–mass spectrometry and its consequences in the field of residue analysis by Jean-Philippe Antignac; Katia de Wasch; Fabrice Monteau; Hubert De Brabander; Fran�ois Andre; Bruno Le Bizec (pp. 129-136).

During their first period of development, the liquid chromatography–mass spectrometry techniques were met with great enthusiasm from most end-users. An extended application range, the needlessness of derivatisation step prior to injection, the possibility of reduced sample preparation and high throughput analysis were some of the arguments given in favor of these techniques. Few years and more than thousands applications later, more attention is paid to their adverse aspects and limitations, especially regarding the existence of matrix effects. Such problems are well known for many years and may concern various detection techniques. But ion suppression appears as a kind of matrix effect specifically linked to mass spectrometry that probably represents one of the main source of pitfalls in liquid chromatography–mass spectrometry (LC–MSⁿ). In the actual tendency to promote these techniques for control purposes in the field of residue analysis, it was thought necessary to highlight one of their possible side-effect which may have critical consequences for the analytical results. In this context, the objectives of the present article, which is based on a literature review and additional experiments, were to present the origins and mechanisms of ion suppression, to expose several case studies illustrating its consequences in the field of residue analysis, and finally to propose and comment on some solutions that may overcome this problem.

Keywords: Ion suppression; Mass spectrometry; Liquid chromatography–mass spectrometry; Electrospray ionization; Atmospheric pressure chemical ionization; Residue analysis

Determination of tetracycline residues in shrimp and whole milk using liquid chromatography with ultraviolet detection and residue confirmation by mass spectrometry by Wendy C. Andersen; Jos� E. Roybal; Steve A. Gonzales; Sherri B. Turnipseed; Allen P. Pfenning; Laura R. Kuck (pp. 145-150).

Two methods have been developed for the simultaneous determination of tetracycline, oxytetracycline, and chlortetracycline in shrimp and in whole milk. These methods were designed to simplify sample extraction and clean-up steps and to be fast and convenient for routine testing in a regulatory environment. Both methods rely on a simple extraction of the shrimp or milk matrix with succinic acid followed by isolation on a copolymeric solid phase extraction column. Chromatographic separation was achieved using a polar end-capped C8 column with an isocratic mobile phase consisting of organic acid, acetonitrile, and methanol, where 0.1% formic acid or 0.01M oxalic acid was used as the acid. Formic acid allowed direct confirmation of the three residues by liquid chromatography–tandem mass spectrometry (LC–MS–MS). LC with ultraviolet absorbance at 370nm resulted in the quantitation of all three tetracycline residues from shrimp and milk samples fortified at 50, 100, 200, 300, and 400ngg⁻¹. Average recoveries were greater than 75% with R.S.D. values less than 10%. All three tetracycline residues were confirmed in shrimp (25–400ngg⁻¹) and milk (50–300ngg⁻¹) samples by LC–MS–MS.

Keywords: Tetracyclines; Shrimp; Milk

An analytical method to determine conjugated residues of ceftiofur in milk using liquid chromatography with tandem mass spectrometry by S. Makeswaran; I. Patterson; J. Points (pp. 151-157).

A new analytical method to identify and quantify ceftiofur and its metabolites in milk is described. The method uses solid phase extraction and liquid chromatography with tandem mass spectrometry (LC-MSMS).Samples are deproteinated with acetonitrile and cleaned up with a C₁₈ SPE column to reduce matrix interference. Conjugates of desfuroylceftiofur are deconjugated and converted to desfuroylceftiofur by treatment with dithioethrytol in a borate buffer. The desfuroylceftiofur that is released is unstable, so is stabilised by derivatisation to desfuroylceftiofur acetamide. Extracts are then purified by cation exchange SPE, and the derivative determined by LC-MSMS monitoring two selected transitions from the protonated molecular ion.The performance characteristics of the method have been calculated as specified in Commission Decision 2002/657/EC.

Keywords: Ceftiofur; β-Lactams; Milk; Residues analysis; LC-MSMS

Analysis of avermectin and moxidectin residues in milk by liquid chromatography–tandem mass spectrometry using an atmospheric pressure chemical ionization/atmospheric pressure photoionization source by Sherri B. Turnipseed; Jos� E. Roybal; Wendy C. Andersen; Laura R. Kuck (pp. 159-165).

The avermectins—ivermectin (IVR), doramectin (DOR), eprinomectin (EPR)—and also the milbemycin moxidectin (MOX) are anthelmintic compounds that may be administered to cattle. Different ionization techniques, including atmospheric pressure photoionization (APPI), were evaluated for the detection of these residues in milk. The ionization response of these compounds using APPI was compared with that obtained by atmospheric pressure chemical ionization (APCI), a combination of APPI and APCI, and electrospray. It was found that the relative response of these drugs with different ionization protocols varied depending on the compound and the mobile phase. When monitoring negative ions, the use of the UV lamp increased the MS response. However, the best response for these compounds was obtained by operating the APCI/APPI source in the positive ion mode without any discharge current applied to the corona needle, whether the UV lamp was on or not. Using this mode of ionization, an MS–MS method was established to monitor the product ion scans of the sodiated molecular ions with an ion trap instrument. Milk fortified with these compounds (0.5–20ngg⁻¹) and milk samples from dosed animals were analyzed after isolating the residues with a simple solid phase extraction method. EPR, DOR and IVR were confirmed in all of the extracts analyzed. MOX was confirmed in all samples fortified at 5ngg⁻¹ or higher. Acceptable recoveries (≥60%) and relative standard deviations (R.S.D. values ≤ 20%) were observed for the residues at the following levels: EPR and IVR (1–20ngg⁻¹); DOR and MOX (5–20ngg⁻¹).

Keywords: Avermectins; Moxidectin; No-discharge atmospheric pressure chemical ionization; Atmospheric pressure photoionization; Milk

Liquid chromatography–electrospray ionisation-mass spectrometry based method for the determination of estradiol benzoate in hair of cattle by H. Hooijerink; A. Lommen; P.P.J. Mulder; J.A. van Rhijn; M.W.F. Nielen (pp. 167-172).

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

Keywords: Estradiol benzoate; Hair; Liquid chromatography mass spectrometry

Determination of residues of malachite green in finfish by liquid chromatography tandem mass spectrometry by Peter Scherpenisse; Aldert A. Bergwerff (pp. 173-177).

A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of leuco-malachite green (LMG) in various fish tissues is described. LMG, which is the primary metabolite of the parasiticide and fungicide malachite green (MG), is the targeted analyte to reveal abuse of this veterinary drug in fish. After extraction using McIlvaine buffer and acetonitrile, the extract was purified on an aromatic sulphonic acid solid-phase extraction column. After conversion of LMG into MG by post-column oxidation with PbO₂, the effluent was analysed by LC-MS/MS in the multiple reaction monitoring (MRM) mode. The MS–MS trace m/z 329 → m/z 313 was used for quantification of LMG and, for salmon, gave an averaged decision limits (CC α; α 1%) and detection capability (CC β; β 5%) of 0.11 and 0.18μgkg⁻¹, respectively, with a measurement each of three consecutive days. The last values were comparable for those for MG. Other traces were used to collect sufficient identification points to establish the identity of this prohibited veterinary drug, which was achieved at CC β and higher. These values were comparable for other tested species, including pangasius, tilapia, trout and Victoria perch. Recoveries ranged from 66% in trout at 0.4μgkg⁻¹ to 112% in pangasius at 0.1μgkg⁻¹. Three out of nineteen samples including pangasius, salmon, shrimps and trout bought in local shops, revealed detectable amounts of residues, i.e. in excess of CC α, and were considered non-compliant. The findings demonstrate the suitability of the presented analytical method to detect residues of malachite green in various aquatic species at relatively low residue levels.

Keywords: Residue analysis; Veterinary public health; Food safety; Veterinary drugs; Mariculture; Aquaculture

Quantitative determination of salicylic acid and metabolites in animal tissues by liquid chromatography–tandem mass spectrometry by Siska Croubels; An Maes; Kris Baert; Patrick De Backer (pp. 179-187).

In order to study the excretion pattern of salicylic acid and its metabolites in animals, a liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed. The method was used to study the biotransformation of salicylic acid in poultry (chicken and pigeon) after therapeutic use, i.e. in matrices such as plasma, excreta (=combined urine and faeces), kidney and liver. Thereafter, the method was also adopted for the analysis of residues of salicylic acid in porcine tissues, such as muscle, kidney, liver and skin + fat.For the biotransformation study, salicylic acid, the oxidation and amino acid conjugation metabolites such as gentisic acid, salicyluric acid, and a double conjugated ornithine metabolite, together with the internal standard phenoxyacetic acid were extracted from 1.0g of tissue or 100μl of plasma into ethyl acetate, after acidifying using 1M HCl. After centrifugation and for kidney and liver tissue, the analytes were back-extracted and subjected to a solid-phase clean-up on SAX columns. For the analysis of excreta, the sample preparation included only a dilution step, performed before LC–MS/MS analysis.For the residue study, salicylic acid and the internal standard phenoxyacetic acid were extracted from 1.0g of porcine muscle, liver, kidney and skin + fat with 6ml of diethyl ether, after acidifying using 3ml of 1M HCl. After extraction and centrifugation, the ether phase was evaporated under N₂ at 40°C. The residue was redissolved with 500μl of 0.1% acetic acid in water. For skin + fat and when needed for the other tissues, a supplemental extraction of the redissolved residue with 500μl of hexane was performed.The analysis of the extracts was done on a Nucleosil 100-5 C18 column, using a gradient elution with 0.1% acetic acid in water and methanol. A Quattro Ultima^® triple quadrupole instrument was used, equipped with an ESI z-spray source, which was operated in the negative ion MS/MS mode.The methods were validated for the linearity (100–1000ngml⁻¹ and 10–50μgml⁻¹ for plasma; 5–250μgg⁻¹ for excreta; 100–1000ngg⁻¹ and 5–25μgg⁻¹ for the biotransformation study in poultry kidney and liver; 25–1000ngg⁻¹ for the residue study in porcine tissues); trueness and precision; specificity; limit of detection and limit of quantification. The limit of quantification for the residue analysis of salicylic acid in porcine tissues was set at 50ngg⁻¹.

Keywords: Salicylic acid; Metabolites; Amino acid conjugates; Poultry; Porcine; Residues; LC–MS/MS

Detection of zilpaterol (Zilmax^®) in calf urine and faeces with liquid chromatography–tandem mass spectrometry by N. Van Hoof; R. Schilt; E. van der Vlis; P. Boshuis; M. Van Baak; A. Draaijer; K. De Wasch; M. Van de Wiele; J. Van Hende; D. Courtheyn; H. De Brabander (pp. 189-197).

Zilpaterol is a new powerful beta-agonist, which is officially registered for fattening purposes in cattle in Mexico and South Africa. Its chemical structure is different from the well-known beta-agonists. Therefore, the routinely used screening methods are not likely to be suited for the analysis of zilpaterol. Also gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry (LC–MS) methods need to be adapted to enable detection of zilpaterol.In this study, a LC–MS³ confirmatory method was developed for the simultaneous detection of zilpaterol and di-aromatic beta-agonists in urine samples. A LC–MS² method was optimised for the detection of zilpaterol in faeces. To study the excretion profile in urine and faeces, a male veal calf was orally treated with daily doses of Zilmax^® during 2 weeks. Zilpaterol was mainly excreted via urine.

Keywords: Zilpaterol; Beta-agonists; LC–MS; ⁿ; Excretion profile

Determination of clenbuterol, ractopamine and zilpaterol in liver and urine by liquid chromatography tandem mass spectrometry by Jos� Blanca; Patricia Mu�oz; Miguel Morgado; Nely M�ndez; Angela Aranda; Thea Reuvers; Henny Hooghuis (pp. 199-205).

Beta-agonists have been misused as growth promoting agents in meat producing animals over 20 years. Clenbuterol (CLB) was the main beta-agonist substance used illegally for this purpose. But other beta-agonist substances have been developed to be used for zootechnical purposes, such as ractopamine (RCT), licensed for this application in the United States and currently zilpaterol (ZIL), licensed in South Africa and Mexico. However, these compounds are banned in the European Union (EU) Council Directive 96/22/EC. An analytical method able to identify these three beta-agonists by liquid chromatography tandem mass spectrometry (LC–MS–MS), after clean-up with mixed-mode SPE Bond Elut Certify cartridges (6mL, 300mg) was developed and validated according to the Commission Decision 2002/657/EC. Decision limit (CCα) ranged from 40 to 110pgmL⁻¹ and from 80 to 150pgg⁻¹ (ppt) for urine and liver, respectively. Detection capability (CCβ) ranged from 180 to 270pgmL⁻¹ and 270 to 520pgg⁻¹ (ppt) for urine and liver respectively. This method appeared suitable for the control of these beta-agonists residues in liver and urine.

Keywords: Beta-agonists; Analysis; Residue; Multiresidue; LC–MS–MS

Evaluation of two different clean-up steps, to minimise ion suppression phenomena in ion trap liquid chromatography–tandem mass spectrometry for the multi-residue analysis of beta agonists in calves urine by Maurizio Fiori; Cinzia Civitareale; Sabrina Mirante; Eugenia Magar�; Gianfranco Brambilla (pp. 207-210).

In an analytical strategy, the selectivity of the steps from the extraction to the detection is a key factor to assure both reliability and forensic validity to the results. The availability of MS/MS ion trap facilities could induce the risk to maximise the relevance of MS/MS technique, despite the choice of a more selective clean-up step. In this work, we reported about the evaluation of two different purification procedures, as requisite to minimise both matrix-induced ion suppression phenomena and progressive loss of sensitivity in LC–MS/MS multi-residue beta agonists routine analysis. To this purpose, calves urine extracts from SPE C18 not endcapped (NE) and molecular imprinted polymers (MIPs) columns were tested according to the following procedure: 20 blank samples were analysed and then spiked with pooled beta agonists before and after the clean-up step. Background noise and analytes signals were compared with those from pure standards injected at the same nominal spiking concentration. In such a way, both recovery factors and matrix phenomena were evaluated. Results indicate that MIPs clean-up is effective to reduce ion suppression phenomena below 10%, with no detectable loss of sensitivity after 20 runs. The same results are not achievable with conventional SPE C18, even if absolute recoveries are found to be better.

Keywords: LC–MS/MS; Ion suppression; MIPs; SPE; Beta agonists

Detection of banned antibacterial growth promoters in animal feed by liquid chromatography–tandem mass spectrometry: optimisation of the extraction solvent by experimental design by C. Van Poucke; F. Dumoulin; C. Van Peteghem (pp. 211-220).

Because of the risk that residues of antibacterial growth promoters in edible tissues could lead to allergic reactions and development of resistant strains of bacteria in humans, the European Commission decided to ban bacitracin, olaquindox, spiramycin, tylosin and virginiamycin as feed additives. To control the compliance of this ban, a single multi-analyte liquid chromatography–tandem mass spectrometry method was developed in 2003. Starting from this method, we further tried to optimise the response with the aid of experimental design. Using a central composite design, we searched for the optimal extraction solvent for each of the five components separately. As expected, this optimal composition was different for each of these antibacterial growth promoters. Two groups of compounds could be distinguished. The first group contained those compounds (spiramycin, tylosin and virginiamycin) that had the highest response (peak area) when the extraction solvent consisted of pure methanol (with or without the addition of formic acid). The second group contained the components (bacitracin and olaquindox) that preferred between 50 and 70% (v/v) methanol as extraction solvent. To find a compromise between these two groups, we created weighed utility functions and analysed these data with linear regression ( α < 0.05). The extraction solvent that gave the overall best response for the type of feed used during this experiment was very similar to the extraction solvent used during the validation of the multi-analyte method (70% (v/v) methanol and 2% (v/v) formic acid) [C. Van Poucke, K. De Keyser, A. Baltusnikiene, J.D.G. McEvoy, C. Van Peteghem, Anal. Chim. Acta 483 (2003) 99]. Finally, a recovery experiment was set up using different types of feed to identify if the outcome of this experiment was valid for all types of feedstuffs.

Keywords: Animal feed; Antibacterial growth promoters; Experimental design

Quantitative liquid chromatographic–mass spectrometric analysis of amoxycillin in broiler edible tissues by S. De Baere; P. Wassink; S. Croubels; S. De Boever; K. Baert; Patrick De Backer (pp. 221-227).

A sensitive and specific method for the quantitative determination of amoxycillin (AMO) in broiler tissues (kidney, liver, muscle, fat and skin+fat) using liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) is presented.A liquid extraction using an aqueous 0.01M potassium dihydrogen phosphate solution as the extraction solvent, was performed for a preliminary sample clean-up. After ultrafiltration, the extracts were further purified by solid-phase extraction using an octadecyl (C18) column. Ampicillin was used as the internal standard for AMO analysis.Chromatographic separation of the analytes of interest was achieved on a PLRP-S polymeric column (150mm×2.1mm i.d., 100Å) using a mixture of 0.1% of 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 MS/MS mode.The method was fully validated according to European Guidelines (linearity, precision, trueness, detection capability, decision limit, specificity and ruggedness) and the results fell within the ranges specified.Biological samples from broiler chickens that were treated orally with an amoxycillin formulation, were analyzed using the described method. Some results are presented.

Keywords: Amoxycillin; Residue analysis; LC–MS/MS; Edible tissues

Determination of the coccidiostat diclazuril in poultry feed and meat by liquid chromatography–tandem mass spectrometry by Leen Mortier; Els Daeseleire; Carlos Van Peteghem (pp. 229-234).

A liquid chromatographic-tandem mass spectrometric method (LC–MS/MS) for the detection of the coccidiostat diclazuril in poultry meat and feed was developed. After an appropriate extraction with an organic solvent, the samples were injected into the LC–MS/MS system on a C₁₈ column. A gradient with acetonitrile and water, each containing 0.1% formic acid, was applied. A structure analogue of diclazuril was used as internal standard. The precursor ions produced by negative electrospray ionisation were selected for collisional dissociation with argon. For diclazuril, two product ions were recorded with multiple reaction monitoring. Validation of the methods was performed based on commission decision 2002/657/EC . The methods were applied to real-life samples: meat of a home-bred chicken and its feed were analysed.

Keywords: Coccidiostats; Diclazuril; Liquid chromatography–tandem mass spectrometry; Residues

Determination of phenylbutazone and oxyphenbutazone in animal urine by ion trap liquid chromatography–mass spectrometry by Carmen Igualada; Francisco Moragues (pp. 235-238).

The purpose of this study was to develop and validate a method to determine oxyphenbutazone and phenylbutazone residues in urine of several animal species.Extraction from urine was performed using liquid extraction with chloroform after chemical hydrolysis. Final extract was analysed by reversed phase liquid chromatography–electrospray ionisation–mass spectrometry using an ion trap selective detector and phenylbutazone-D10 as internal standard.The method was validated according to the requirements of the 2002/657/EC European decision and the calculated decision limit (CCα) and detection capability (CCβ) were 2 and 3ngml⁻¹, respectively.

Keywords: Phenylbutazone; Oxyphenbutazone; Non-steroidal anti-inflammatory drug; LC–ESI–MS; ⁿ; Mass spectrometry; Ion trap

Boldenone and related hormones quantification by liquid chromatography–mass spectrometry in urine and faeces after calf administration of boldenone undecanoate by Emanuele Sangiorgi; Valentina Polignano; Silvia Gardini (pp. 239-248).

A 6-month calf was treated with 1mgkg⁻¹ wet weight of 17-β-boldenone undecanoate and urine and faeces were collected for a period of 30 days after the treatment to follow the depletion of the different substances related to boldenone. Hair was collected for just a few days. Multiresidue extraction and purification methods were developed for the different matrices. Separation was carried on a RP-C18 LC column and liquid chromatography–mass spectrometry (LC–MS/MS) detection was performed using an ion trap mass spectrometer equipped with electrospray source for urine and with atmospheric pressure chemical ionisation for faeces. The concentration found for the different analytes was discussed compared to a control animal.

Keywords: Boldenone; Hormones; Metabolism; Liquid chromatography–mass spectrometry

Determination of chloramphenicol residues in rainbow trouts by gas chromatography–mass spectometry and liquid chromatography–tandem mass spectometry by L�cia Santos; Jorge Barbosa; M. Concei��o Castilho; Fernando Ramos; Carlos A. Fontes Ribeiro; M. Irene Noronha da Silveira (pp. 249-256).

A methodology for the identification and quantification of chloramphenicol (CAP) residues was developed and validated. The method is based on gas chromatography–mass spectrometry (GC–MS) for screening, in electron impact (EI) mode, after a solid phase extraction (SPE) step using C18 columns, and data acquired in selective ion monitoring (SIM) mode with the following ions: m/ z 225, m/z 208 and m/ z 242. Confirmatory method consists on liquid chromatography–tandem mass spectrometry (LC–MS/MS), in ionspray-negative mode, after the same extraction and clean-up procedure. The m/ z 321 was selected as a parent ion and m/ z 152 and m/ z 194 as daughter ions. The data were acquired in the negative multiple reaction monitoring (NMRM) mode, by monitoring the transitions 321 > 152 and 321 > 194. The method gave a decision limit (CCα) and a detection capability (CCβ) of 0.267 and 0.454μgkg⁻¹, respectively.The described methodology was applied on 40 samples of rainbow trout, collected in supermarkets of the Centre Region of Portugal, in order to evaluate the presence of CAP residues.Positive results were confirmed in 9 of the 15 suspected samples, which correspond to 22.5% of the whole samples collected.

Keywords: Chloramphenicol; Rainbow trout; GC–MS; LC–MS/MS

Determination of chloramphenicol in honey by liquid chromatography–tandem mass spectrometry by A.F. Forti; G. Campana; A. Simonella; M. Multari; G. Scortichini (pp. 257-263).

An effective liquid chromatographic method with tandem mass spectrometric (LC–MS/MS) detection and identification is presented for the determination of chloramphenicol (CAP) in honey. After a preliminary dissolution in water, samples were extracted with a mixture of dichloromethane/acetone and evaporated to dryness; the following clean up was carried out on an octadecyl (C₁₈) SPE cartridge. CAP was determined by LC–MS/MS, using electrospray ionization in the negative ion mode (ESI⁻). The column was a LUNA Phenomenex with a mixture of methanol-aqueous ammonium acetate (60:40, v/v) as a mobile phase. Honey samples were fortified at CAP levels 0.30–0.45–0.60μgkg⁻¹ with 5D-CAP as internal standard. At these levels, trueness ranged between 98.7 and 102.0% and within-laboratory reproducibility was lower than 6.2%, expressed as relative standard deviation. The limit of decision (CCα) was 0.07μgkg⁻¹ and detection capability (CCβ) was 0.10μgkg⁻¹.

Keywords: Chloramphenicol; Honey; Solid-phase extraction; Liquid chromatography–tandem mass spectrometry; Validation

Validation of a liquid chromatography–tandem mass spectrometric method for the quantification of eight quinolones in bovine muscle, milk and aquacultured products by N. Van Hoof; K. De Wasch; L. Okerman; W. Reybroeck; S. Poelmans; H. Noppe; H. De Brabander (pp. 265-272).

Quinolones are a group of structurally related antibacterial agents. Over the present decade there has been a significant and progressive increase in the use of this class of antibiotics in animal production. As a consequence the increased use of quinolones can promote the resistance of bacteria. To protect the consumers health, Maximum Residue Limits (MRL) have been established in edible animal matrices by the European Union.A liquid chromatography–tandem mass spectrometric (LC–MS²) method was developed and validated for the simultaneous quantification of eight quinolones at MRL level in bovine muscle, milk and aquacultured products. The studied quinolones were enrofloxacin, ciprofloxacin, sarafloxacin, danofloxacin, oxolinic acid, flumequine, difloxacin and marbofloxacin. The method involved a single solid-phase extraction followed by the analysis of all quinolones in a single chromatographic run using LC–ESI–MS². Quinine was selected as internal standard. This paper consists of two parts: the discussion of the analytical method and the discussion of the different validation parameters according to Commission Decision 2002/657/EEC.

Keywords: Quinolones; Validation; Bovine muscle; Aquacultured products and milk

Results of a European proficiency test for the detection of streptomycin/dihydrostreptomycin, gentamicin and neomycin in milk by ELISA and biosensor methods by Val�rie Gaudin; Nathalie Cadieu; Pascal Sanders (pp. 273-283).

An interlaboratory study was proposed to the European Community and Third Countries National Reference Laboratories (NRL) for the analysis of aminoglycoside residues in bovine milk by ELISA. This test was intended to allow the participants to control their aminoglycoside ELISA methods and also to compare the performance of various ELISA kits (neomycin, gentamicin, streptomycin and DHS) in bovine milk.Twelve random coded frozen samples were sent in dried ice (including 2 blank samples and 10 spiked milk samples). Each sample had to be analysed in triplicate (that means three different extractions for each sample) with the ELISA kits of their choice (commercial or in-house), if possible looking for streptomycin/DHS, gentamicin and neomycin. If not, it was recommended to test only streptomycin/DHS twice, with two different batches of the same kit.The 14 participants performed the analyses with ELISA kits (or biosensor methods) for the detection of streptomycin/DHS. Seven participants tested the samples for the detection of neomycin and six for the detection of gentamicin with ELISA kits.Then qualitative and quantitative analyses of the results were performed. Four parameters were calculated according to a reference publication for the validation of screening qualitative methods: false non-compliant and false compliant rates, sensitivity and specificity. A quantitative exploitation was also performed by calculating the assigned value for each material. Then the evaluation of laboratory’s performance was carried out by calculating the accuracy and repeatability Z-scores.

Keywords: Interlaboratory study; Aminoglycosides; Residues; Milk; ELISA; Biosensor

Multi-residue method for the determination of benzimidazoles in bovine liver by Geraldine Dowling; Helen Cantwell; Michael O’Keeffe; Malcolm R. Smyth (pp. 285-292).

A method has been developed to analyse for 12 benzimidazole drug residues in bovine liver. Liver samples were extracted with ethyl acetate, sample extracts were defatted with hexane and cleaned-up by automated SPE on C₁₈ solid phase extraction cartridges. Aliquots of the extracts were analysed by LC with UV detection (298nm). The method was validated in bovine liver, according to the criteria defined in Commission Decision 2002/657/EC. The decision limit (CC_α) was between MRL + 12% and MRL + 25% and the detection capability (CC_β) was between MRL + 25% and MRL + 45% for the range of benzimidazoles investigated. The results of the inter-assay study, which was performed by fortifying bovine liver samples ( n = 6) in three separate assays, show the mean recovery to be between 60% and 100% for albendazole sulphoxide, albendazole sulphone, thiabendazole, oxfendazole/fenbendazole sulphoxide, hydroxy-mebendazole, fenbendazole sulphone, oxibendazole, mebendazole and flubendazole. Lower mean recovery was obtained for amino-flubendazole and albendazole (approximately 50%) and amino-albendazole sulphone (approximately 25%). The precision of the method, expressed as R.S.D. values for the within-laboratory repeatability, was generally below 25%.

Keywords: Benzimidazoles; Automated solid phase extraction; Residue analysis; Liver; Method validation

Stability of β-agonist methyl boronic derivatives before gas chromatography–mass spectrometry analysis by Milagro Reig; Natalia Batlle; Jos� Luis Navarro; Fidel Toldr� (pp. 293-297).

A method for the confirmatory analysis of clenbuterol, mabuterol and salbutamol in bovine urine and feeds by gas chromatography–mass spectrometry (GC–MS) is described. β-Agonist residues were extracted and further purified and concentrated by immunoaffinity chromatography. Methyl boronic acid (MBA) was used for the derivatisation of β-agonist residues and time and temperature of derivatisation were optimised. These derivatives allowed the use of shorter columns while improving the chromatographic resolution. Methyl boronic derivatives were stored either at room temperature or at −20°C and then injected into the gas chromatograph–mass spectrometer at specific time intervals. Methyl boronic derivatives showed a slight decrease for clenbuterol and a more marked loss for mabuterol when stored at −20°C up to 3 days and then remained stable up to 7 days. However, the stability for salbutamol was poorer as it rapidly decreased at 2 days. When derivatives were stored at room temperature, clenbuterol and internal standard (IS) showed good stability up to 6h while the stability of mabuterol was progressively reduced. Salbutamol dropped to very low values at just the first hour. Under these conditions it is clear that the analysis should be performed as soon as possible after derivatisation. The combination of immunoaffinity chromatography, MBA derivatisation and gas chromatography–mass spectrometry has shown a high sensitivity, good reproducibility and short chromatography time for the control of the studied β-agonists.

Keywords: Agonists; Clenbuterol; Derivatives stability; Urine; Frozen storage

Nitroimidazoles in turkey muscle—the influence of sampling conditions on measurement results in residue control by J. Polzer; P. Gowik (pp. 299-303).

Nitroimidazoles in incurred muscle samples showed a considerable inhomogeneity in their distribution. A possible explanation for this phenomenon were the sampling conditions. In an animal study, the influence of sampling after slaughtering was investigated by applying four different ways of treating muscle samples prior to freezing at −24°C. The muscle samples were analysed using gas chromatography-negative chemical ionisation/mass spectrometry after silylation. The results reaffirmed an inhomogeneous distribution of ipronidazole and ronidazole in muscle samples, irrespective of the sampling procedure. Thermal stress caused a significant loss of analytes, even at +4°C.

Keywords: Residue control; Nitroimidazoles; Homogeneity; Sampling conditions; Turkey; Muscle

Effect of various heat treatments and cold storage on sulphamethazine residues stability in incurred piglet muscle and cow milk samples by E.P. Papapanagiotou; D.J. Fletouris; E.I. Psomas (pp. 305-309).

The stability of sulphamethazine (SMZ) residues in incurred piglets muscle tissue and incurred cow milk, under various heat treatments (pasteurizing, boiling, autoclaving and microwaving) was studied. Moreover, the storage stability of SMZ residues at −20 and −75°C as well as the stability of SMZ standard solutions under boiling and autoclaving conditions was also investigated. The results showed that in the pasteurized milk samples SMZ residues concentration remained unchanged, while in the boiled and autoclaved milk samples, a decrease in their concentration was observed. Regarding the heating treatments of piglet muscle, after allowance for weight loss and migration of SMZ into the surrounding fluid has been made, a considerable reduction in the quantity of SMZ in piglet muscle during boiling, autoclaving and microwaving, was confirmed. The decrease was higher in the autoclaved milk and muscle tissue samples than in the boiled ones, while the microwaved muscle tissue samples showed the lowest decrease. Since SMZ standard solutions were stable under boiling and autoclaving conditions, the decrease of SMZ measured concentrations in muscle and milk samples might be attributed to the binding of SMZ with proteins, which was caused by the various heat treatments. SMZ residues were stable at −20 and −75°C in all samples examined for at least 3 and 5 months, respectively.

Keywords: Sulphamethazine; Stability; Residues; Piglet muscle; Cow milk

The study of some new anabolic drugs by metabolism experiments with Neomysis integer by S. Poelmans; K. De Wasch; D. Courtheyn; N. Van Hoof; H. Noppe; C. Janssen; H.F. De Brabander (pp. 311-316).

New illegal veterinary drugs are produced and distributed on the black market continuously. The anabolic drugs described here were found on the black market for body-builders, but are probably also available for growth promoting purposes in cattle.The residue laboratories are obligated to develop extraction and detection methods to identify and/or quantify the metabolites of these continuously emerging new drugs. In practice, the formation of the metabolites is investigated with animal experiments in which vertebrate animals are treated with the illegal compound. Different matrices of the animal are collected and examined. Because of the complexity and duration of the animal experiment and the method development, a lot of time and money is consumed. Some of these vertebrate experiments can be replaced by invertebrate metabolism experiments [K. De Wasch, S. Poelmans, T. Verslycke, C. Janssen, N. Van Hoof, H. De Brabander, Anal. Chem. Acta 473 (2002) 59; T. Verslycke, K. De Wasch, H. De Brabander, C. Janssen, Gen. Comp. Endocr. 126 (2002) 190; S. Poelmans, K. De Wasch, Y. Martelé, R. Schilt, N. Van Hoof, H. Noppe, T. Verslycke, C.R. Janssen, D. Courtheyn, H.F. De Brabander, Proceedings of the Euro Food Chem XII Strategies for Safe Food, September 24–26, Brugge, Belgium, 2003, p. 74]. By using an invertebrate for the metabolism studies there is a reduction in time and money in comparison with vertebrate animal experiments.In this study an invertebrate model, the mysid crustacean Neomysis integer ( Crustacea, Mysidacea) was used as an alternative model for metabolism of some new anabolic drugs.The investigated analytes were dehydroepiandrosterone (DHEA, (3β)-3-hydroxy-androst-5en-17one), maxterone (ADL: 5α-androstan-3β,17β-diol), 5-androstenedione (5AED, 5-androstene-3,17-dione), 5α-androstenedione (5αAED, 1-androstene-3,17-dione) and 1-testosterone (A1T, 1-(5α)T, 1-(5α)-androsten-17β-ol-3-one).

Keywords: Metabolism; Neomysis integer; Androgens; DHEA; ADL; 5AED; 5αAED; A1T

Metabolism of methyltestosterone, norethandrolone and methylboldenone in a heifer by M.H. Blokland; H.J. van Rossum; H.A. Herbold; S.S. Sterk; R.W. Stephany; L.A. van Ginkel (pp. 317-323).

In this study, metabolism and excretion of 17α-methyltestosterone, norethandrolone, methylboldenone and metabolites in a heifer were studied. The heifer was treated with these steroids by intra-muscular injection. Samples of urine were collected and analysed using gas-chromatography coupled to a mass-spectrometer. It was concluded that 17α-methyltestosterone, norethandrolone and methylboldenone show similar metabolic pathways. The main route was hydroxylation of the parent compound. The formation of oxygenated products was observed, but this was a minor pathway. The sequential treatment of the heifer possibly caused the formation of different metabolites after treatment with methylboldenone. The concentration of the metabolites in urine was higher than the concentration of the parent compound. The use of the marker metabolites in routine control programs increase the possibility of tracking bovine animals treated with 17α-methyl or ethyl substituted anabolic steroids.

Keywords: Methylboldenone; Methyltestosterone; Norethandrolone; Metabolism; Excretion rate; Cattle; Steroid

Sulphamethazine residue depletion study in piglets after oral administration by E.P. Papapanagiotou; D.J. Fletouris; I.E. Psomas (pp. 325-329).

The depletion profile of sulphamethazine (SMZ) was studied in healthy piglets after oral administration of a premix, containing 100gSMZ/kg, at a rate of 1kg/t of feed for 30 consecutive days. A total of 50 piglets were used in the experiment, 42 of which received the medication and the remaining 8 acted as control animals. The piglets were of 42 days of age and of a mean bodyweight of 28kg at the beginning of the experiment. Six medicated and one control piglet were sacrificed at days 5, 10, 15, 20, 25, 30 and 35 after the cessation of the treatment and muscle, liver, kidney and fat tissues were sampled and stored at −75°C pending analysis. The samples were analysed using a liquid chromatographic method, which was fully validated for SMZ residue analysis prior to use. Quite high mean concentrations of SMZ residues of 3.84mgkg⁻¹ for muscle, 8.14mgkg⁻¹ for liver, 6.42mgkg⁻¹ for kidney and 2.88mgkg⁻¹ for fat tissues were attained 5 days post-medication. SMZ residues were still detected even 20 days post-medication at levels higher than 1.12mgkg⁻¹ in all tissues examined. In descending order, the SMZ residues concentrations found in all examined tissues were liver>kidney>muscle>fat. The time needed for the concentration of SMZ to drop below the EU established MRL of 100μg/kg, was 30 days. The statistically estimated withdrawal period was calculated to be 41 days.

Keywords: Sulphamethazine; Depletion; Residues; Piglet tissues; Oral administration

Deposition and depletion of the coccidiostats toltrazuril and halofuginone in eggs by P.P.J. Mulder; P. Balzer-Rutgers; E.M. te Brinke; Y.J.C. Bolck; B.J.A. Berendsen; H. Ger�ek; B. Schat; J.A. van Rhijn (pp. 331-337).

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

Keywords: Toltrazuril; Ponazuril; Halofuginone; Egg; Deposition; Depletion

Metabolism and depletion of nifursol in broilers by T. Zuidema; P.P.J. Mulder; J.A. van Rhijn; N.G.M. Keestra; L.A.P. Hoogenboom; B. Schat; D.G. Kennedy (pp. 339-346).

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

Keywords: Nifursol; Nitrofurans; Metabolism; Depletion; DNSH; Tissue-bound residue; Broilers

Distribution of chloramphenicol residues in lactating cows following an external application by Andrej Pengov; Vesna Cerkvenik Flajs; Tomaž Zadnik; Janez Marinšek; Milan Poga�?nik (pp. 347-351).

The aim of the present investigation was to demonstrate the presence of eventual chloramphenicol residues in blood plasma, urine and milk of dairy cows after topical application of a product, which contains 5% chloramphenicol in an alcohol solution and was commercially used as a spray registered for animals not intended for human consumption. The experiment was performed on two (A + B) Holstein–Frisian cows in the early lactation period. The indication for treatment with the described antibiotic solution was in the first case (cow A) a teat lesion and in the second case (cow B) skin lesions on the shoulders and the claws. Four consecutive dermal administrations inside first 41h of the experiment were performed for both animals at the same time points. Milk, blood and urine samples were taken immediately before first treatment and in regular intervals inside applications and up to 12 days after the last application. All investigated matrices were analyzed using capillary GC-ECD after chloramphenicol derivatization. The outcome of our study confirmed our hypothesis that illegal and violative treatment (food-producing animals) of wounds with a spray containing chloramphenicol results in considerable residues of the active substance in milk, blood plasma and urine of the injured animals.

Keywords: Chloramphenicol; Residues; Milk; Blood plasma; Urine; Topical application

Pharmacokinetics of doramectin in lactating dairy sheep and suckling lambs by Vesna Cerkvenik Flajs; Iztok Grabnar; Nevenka Kožuh Eržen; Irena Marc; Urška Požgan; Mitja Gomba�?; Lucija Kolar; Milan Poga�?nik (pp. 353-359).

The aim of the present study was to estimate permeation of doramectin (DOR) into sheep's milk by following its time course in blood plasma and milk. Six Istrian Pramenka sheep in the early lactation period, each having a suckling lamb, were administered DOR in a single subcutaneous dose of 0.2mgkg⁻¹ body weight. Blood plasma and milk samples were taken from days 1 to 42 following drug administration. Mean maximal DOR concentration observed ( c_max) in ewes’ blood plasma and milk were 22.8 and 31.1μgl⁻¹, respectively, at day 3 ( t_max) following drug administration. Mean elimination half-lives ( t_1/2) and mean residence times (MRT) were 3.4 and 6.2 days for plasma data and 4.6 and 6.9 days for milk data, respectively. Transfer of DOR residues to suckling lambs was evaluated by determination of DOR concentration time courses in lambs’ blood plasma. Mean maximal DOR concentration 2.1μgl⁻¹ ( c_max), was observed at 5.5 days ( t_max) following drug administration to ewes, while t_1/2 and MRT were 3.8 and 9.1 days, correspondingly. Mean time in which concentrations fell below the limit of detection was >35 days for ewes’ blood plasma and >37 days for milk, while residual DOR concentration in lambs’ blood plasma fell below the limit of detection on day 20 following drug administration to ewes in only one out of six lambs. DOR extensively permeated into sheep's milk. Mean milk to plasma concentration ratio was 1.4. It was estimated that 1.6% of the DOR dose was excreted into milk and ingested by suckling lambs.As DOR use during lactation is prohibited, its long lasting presence of residues in milk merits proper veterinary sanitary control. The results reported contribute to further understanding of the DOR persistence and excretion patterns in lactating sheep and implementation of evidence-based guidelines to anti parasitic treatment of dairy animals.

Keywords: Doramectin; Sheep; Residues; Pharmacokinetics; Milk; Suckling lambs

Quantitative determination of dexamethasone in bovine plasma and tissues by liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry to monitor residue depletion kinetics by Marc Cherlet; Siegrid De Baere; Siska Croubels; Patrick De Backer (pp. 361-369).

Dexamethasone (DXM) is a synthetic glucocorticoid that is authorized for therapeutic use in veterinary medicine. The European Community (EC) fixed a maximum residue limit (MRL) at 2ngg⁻¹ for liver, 0.75ngg⁻¹ for muscle and kidney tissues and 0.3ngml⁻¹ for milk, while its use as a growth-promotor is completely banned. We developed earlier a LC–APCI–MS/MS method capable of quantifying such low MRL levels in milk. Minor modifications—concerning only the extraction procedure—were sufficient to allow the quantification of these levels as well in tissue samples. Validation results according to EC requirements concerning linearity, accuracy and precision were satisfactory. Limits of quantification of 0.375ngg⁻¹ were obtained for muscle and kidney tissues and of 1ngg⁻¹ for liver tissue, i.e. half the MRLs. Limits of detection were 0.09, 0.13 and 0.33ngg⁻¹ for muscle, kidney and liver tissues, respectively. Decision limits (CCα) were 1.2ngg⁻¹ for muscle and kidney tissues and 2.2ngg⁻¹ for liver tissue, while detection capabilities (CCβ) were 1.8, 1.9 and 2.5ngg⁻¹ for muscle, kidney and liver tissues, respectively. A simple deproteinization step with concentrated trichloroacetic acid followed by an extraction with ethyl acetate was sufficient to quantify DXM in plasma samples with a limit of quantification of 1ngml⁻¹. After intravenous injection of DXM to cattle, a distribution within 30min was observed followed by a phase of slow elimination characterized by a half-life of 9.2h. In muscle tissue, low levels of DXM were found and a quick elimination was observed, with the DXM level falling below the MRL within 4 days after administration. Higher DXM levels were found in liver tissue compared to kidney tissue up to 4 days after administration. Nevertheless, in liver tissue DXM was below the MRL after 8 days of withdrawal time, while it was still as high as 2.5ngg⁻¹ in kidney tissue.

Keywords: Dexamethasone; LC–APCI–MS/MS; Quantification; Validation; Intravenous administration; Residue depletion kinetics

Author Index (pp. 371-374).

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Analytica Chimica Acta (v.529, #1-2)

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Analytica Chimica Acta (v.529, #1-2)