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Nowadays, complaints about poor indoor air quality have become common. The variety of indoor air health problems include chronic fatigue, allergy, skin and eye irritation, and can be caused by several factors including fungi and their metabolites present in a building. The objective of this study was to establish a method for untargeted analysis of secondary fungal metabolites in indoor environments. As a detection technique, time-of-flight mass spectrometry was chosen, as it provided mass accuracy and higher sensitivity in full scan acquisition mode compared to tandem mass spectrometers. The method was first applied to fungal cultures, namely Penicillium brevicompactum and Chaetomium murorum, which were isolated from mouldy houses and grown on building materials under laboratory conditions for 7-21 days. Following the proposed strategy based on accurate mass measurement and post-acquisition data processing using principal component analysis, roquefortine C, brevianamide A and mycophenolic acid were identified in Penicillium sp., while chaetoglobosin A was found to be produced by Chaetomium sp. Subsequently, samples from mouldy inhabited buildings were analysed using the developed method. The actual presence of meleagrin was demonstrated in mouldy indoor environment. Applying the method to air and dust samples collected in these mouldy buildings, no metabolites were detected possibly due to generally low concentrations in these types of samples.

The design of imprinted polymers selective towards patulin (PAT) and their application in food analysis are reported for the first time. Different templates, functional monomers and molar ratios were evaluated related to binding capacity and specificity. Besides the toxin itself, the implementation of structural analogues (2-hydroxynicotinic acid, 5-indanol and 3-hydroxyphtalic anhydride) as templates was evaluated. A molecularly imprinted solid-phase extraction (MISPE) procedure was optimised for the selective clean-up of apple juice samples. Depending on the spiked concentration, recoveries after MISPE and non-imprinted solid-phase extraction varied respectively from 60% to 66% and from 40% to 41%. Limit of detection and limit of quantification were 10.0 µg/kg and 33.3 µg/kg, respectively. Equilibrium experiments and Scatchard analysis disclosed the presence of two classes of binding sites in the imprinted polymer. The dissociation constant (KD) of the higher affinity binding sites was 3.3 µmol/l, while the KD of the lower affinity binding sites was 260.7 µmol/l. The performance of the molecularly imprinted polymer throughout the clean-up was compared to liquid-liquid extraction and a C18 sorbent. Cross-reactivity experiments demonstrated that MISPE was substantially more selective than C18 clean-up. Moreover chromatograms, with less interfering peaks, were observed with MISPE resulting in a sensitive and reliable quantification of PAT.

The conditions of thin-layer chromatography separation of related aflatoxins and zearalenones in the presence of β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin were studied. Effects of the stationary phase and mobile phase composition were investigated. Analytical conditions for the separation and simultaneous semi-quantitative fluorescence detection of aflatoxins B1, B2, G1 and G2, zearalenone and α-zearalenol on normal-phase plates (silica gel, polyamide) and reversed-phase plates (C18) with cyclodextrin modified mobile phase were optimised. The limit of quantification was found 2 ng per spot for aflatoxin G1 and aflatoxin B2, 3.5 ng for aflatoxin B1 and aflatoxin G2 and 100 ng per spot for zearalenone and α-zearalenol. Addition of cyclodextrins to the mobile phase allowed a decrease in the amount of toxic solvents, and improved separation characteristics, but did not improve the limit of quantification.

2-Thio-thiozalidine-4-carboxylic-acid is synthesized and subsequently purified by high-pressure liquid chromatography. The spectral properties of the title compound are described and commented upon.

A molecularly imprinted polymer (MIP) for the recognition of fumonisin B analogues (FB) using 2-(diethylamino) ethyl methacrylate (DEAEM) as functional monomer and trimethylolpropane trimethacrylate (TRIM) as cross-linker was prepared by bulk polymerization in acetonitrile. Fumonisin B(1) (FB(1)) was used as a template molecule. A molecularly imprinted solid-phase extraction (MISPE) procedure was developed for further application in the analysis of FB. The performance of the MIP throughout the clean-up of spiked bell pepper, rice and corn flake sample extracts was compared with the results obtained when using non-imprinted polymer, C(18), strong anion exchange and immunoaffinity sorbents. Extracts were analysed for FB with liquid chromatography-tandem mass spectrometry (LC-MS/MS) after clean-up. Depending on the food matrix and the concentration range of the fumonisins, recoveries after MISPE varied from 62 to 86%, from 62 to 83%, and from 67 to 81% for fumonisin B(1) (FB(1)), fumonisin B(2) (FB(2)) and fumonisin B(3) (FB(3)), respectively. The selectivity of the synthesized MIP for mycotoxins belonging to the group of FB was confirmed by evaluating cross-reactivity from analogue structures and other mycotoxins. Analysis of 39 naturally contaminated samples (corn flakes) by liquid chromatography tandem mass spectrometry indicated that the synthesized MIP could be an excellent alternative for clean-up and pre-concentration of FB in food samples. Pearson correlations between immunoaffinity clean-up and MISPE were calculated and amounted to 0.923 for FB(1), 0.808 for FB(2), and 0.759 for FB(3). It was shown that the developed MIP could be reused more than 50 times. The synthesis of an FB(1) imprinted polymer and its application in food analysis is reported for the first time.

International audience; Different cheese samples, produced in Latvia (8) and Belgium (13), were analysed for their sterigmatocystin (STC) content. Only two (9.5 %) of the analysed samples were positive for STC with concentration levels of 1.23 and 0.52 µg kg-1 respectively. Five (24%) samples contained STC above the limit of detection (0.03 µg kg-1) but below the limit of quantification (0.1 µg kg-1), while five samples contained traces of STC. A sensitive liquid chromatography – electrospray positive ionization – tandem mass spectrometry (LC-MS/MS) method, which was developed before for the analysis of STC in grains, was modified and applied to the analysis of STC in cheese. This method consists of sample extraction with acetonitrile:water (90:10, v/v), defatting with n-hexane, solid phase extraction, separation on a reversed phase C18 column and STC detection by LC-MS/MS.

A high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) was developed and validated for the detection of zearalenone (ZON), alpha-zearalenon (alpha-ZOL) and beta-zearalenol (beta-ZOL) in in vitro biological samples. Furthermore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the detection of ZON, alpha-ZOL, beta-ZOL, alpha-zearalanol (alpha-ZAL) and beta-zearalanol (beta-ZAL) in in vitro biological samples. Zearalanone (ZAN) was used as internal standard in both methods. The limit of detection/limit of quantitation (LOD/LOQ) values of ZON, alpha-ZOL and beta-ZOL were 2/7, 2/7 and 4/13 mu g 1(-1), respectively, for the HPLC-FLD method. For the LC-MS/MS method LOD/LOQ values for ZON, alpha-ZOL, beta-ZOL, alpha-ZAL and beta-ZAL were 6/20, 5/17, 4/14, 9/30 and 6/19 mu g 1(-1), respectively. Within-day and between-day precision were less then 11 and 14%, respectively for the HPLC-FLD method, and both less then 20% for the LC-MS/MS method. The recovery of ZON and its metabolities ranged between 73 and 89% for the HPLC-FLD method and between 69 and 112% for the LC-MS/MS method. The methods were used for the detection of the compounds in in vitro biological samples, obtained with human intestinal Caco-2 cells culture experiments. The 8-days post-confluent Caco-2 cells were treated with ZON or a mixture of ZON and imazalil (IMA). After an incubation time of 24 h the samples were analyzed with the HPLC-FLD method. Neither ZON nor its derivatives were detected in the samples. The disappearance of ZON could possibly point out the formation of phase II metabolities like glucuronide conjugates. There, samples were pretreated with beta-glucuronidase before LC-MS/MS analysis. The LC-MS/MS results showed that ZON, alpha-ZOL and beta-ZOL could only be detected in the beta-glucuronidase pretreated samples. This confirmed the formation of glucuronide conjugates and the hydroxylation of ZON during the incubation with Caco-2 cells. Copyright (C) 2008 John Wiley & Sons, Ltd.

Liquid chromatographic methods were used for the detection of ochratoxin A (OTA) and its metabolites ochratoxin alpha (OT alpha), 10-hydroxy OTA (10-OHOTA), 4R-hydroxy OTA (4R-OHOTA) and the ethyl ester of OTA (OTC) in in vitro samples, obtained with Caco-2 cell culture experiments and in in vivo urine samples from sheep. A high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method were developed and validated for the detection of OTA and its metabolites OTo 10-OHOTA, 4R-OHOTA and OTC, which was used as internal standard. The LOD/LOQ values for OT alpha, 4R-OHOTA and OTA were 0.63/2.11, 0.99/3.31 and 0.84/2.81 mu g/L, respectively, for the HPLC-FLD method and 0.98/3.28, 1.11/3.72 and 0.88/2.96 mu g/L, respectively for the LC-MS/MS method. Within-day and between-day precision were both

A simplified clean-up procedure was developed in combination with gas chromatography-tandem mass spectrometry (GC-MS/MS) for the determination of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) in adipose tissue. Clean-up was performed by the successive application of a Mega Bond Elut silica column and a Bond Elut PCB column. Validation of the method was conducted according to European Union Commission Decision 2002/657/EC. In order to evaluate the applicability of the method, 44 horse fat samples were analysed. The total PCB concentration (sum of PCBs 28, 52, 101, 118, 138, 153 and 180) ranged from 5.35 to 140 ng g(-1) lipid weight. The total PBDE concentration (sum of BDEs 28, 47, 99, 100, 153, 154 and 183) ranged from below the decision limit to 6.34 ng g(-1) lipid weight.

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

The use of anabolic steroids has been banned in the European Union since 1981. In this study, the metabolism of the anabolic steroid methenolone acetate, was investigated in a male veal calf. After daily oral administration of methenolone acetate, three main metabolites were detected in both urine and faeces samples. Among these metabolites, alpha-methenolone was apparently the main one, but 1-methyl-5alpha-androstan-3,17-diol and 3alpha-hydroxy-1-methyl-5alpha-androstan-17-one were also observed. The parent compound was still detectable in faeces. As a consequence, abuse of methenolone acetate as growth promoter can be monitored by analysing urine and faeces samples. A few days after the last treatment, however, no metabolites were observed. Alpha-methenolone was detectable in urine until 5 days after the last treatment, but in faeces no metabolites were detectable after 3 days.

An approach for ochratoxin A (OTA) fast cost-effective screening based on clean-up tandem immunoassay columns was developed and optimized for OTA detection with a cut-off level of 10 microg kg(-1) in spices. Two procedures were tested and applied for OTA detection. Column with bottom detection immunolayer was optimized for OTA determination in Capsicum ssp. spices. A modified clean-up tandem immunoassay procedure with top detection immunolayer was successfully applied for all tested spices. Its main advantages were decreasing of the number of analysis steps and quantity of antibody and also minimizing of matrix effects. The total duration of the extraction and analysis was about 40 min for six samples. Chilli, red pepper, pili-pili, cayenne, paprika, nutmeg, ginger, white pepper and black pepper samples were analyzed for OTA contamination by the proposed clean-up tandem immunoassay procedures. Clean-up tandem immunoassay results were confirmed by HPLC-MS/MS with immunoaffinity column clean-up. Among 17 tested Capsicum ssp. spices, 6 samples (35%) contained OTA in a concentration exceeding the 10 microg kg(-1) limit discussed by the European Commission. All tested nutmeg (n=8), ginger (n=5), white pepper (n=7) and black pepper (n=6) samples did not contain OTA above this action level.

Rapid antibody-based mycotoxin screening techniques are designed to be used outside a laboratory environment, at the place of sampling. Results are expected immediately, so that commodities can be further processed without delay. Because they are used for mycotoxin analysis, very low levels (ppb and ppt range) should be detected. A further requirement is that the obtained results are accurate with a false negative rate of5% at the level of interest.At first, plastic microtiter plates were used as solid phase materials for immobilizing antibodies (enzyme-linked immunosorbent assays). However, to increase speed and user-friendliness, plastics were replaced by microporous membranes. As an example a flow-through enzyme immunoassay for the detection of fumonisins in cornflakes with a cut-off value of 275 μg/kg is described. No false negative results were observed and the false positive rate was 18%. However, enzyme labels, used to enable visual evaluation of results, did not seem to be completely satisfactory in terms of stability and repeatability of the generated signal. Therefore microparticle labels such as colloidal gold particles are used more and more,e.g. in a lateral flow dipstick immunoassay. When applied to the detection of aflatoxin B1 in pig feed a cut-off value of 5 μg/kg could be reached with no false negative results and a false positive rate of only 10%. Sample pretreatment for screening techniques should be rapid and simple. Preferably a simple solvent extraction is used, followed by a filtration and dilution step. However, for strongly coloured or complex food matrices, this did not seem to work. The combination of clean-up and detection in one single test device is a new approach. When using this clean-up tandem assay column for the detection of ochratoxin A in roasted coffee, a cut-off value of 6 μg/kg was reached. No false positive results were obtained, however, the false negative rate was 8%.

The development of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of 16 mycotoxins possibly related to the 'Sick Building Syndrome' on filters and in fungal cultures is described. Fungi-surface sampling as regards the 'Sick Building Syndrome' preferably happens by scraping off fungal material and vacuuming onto cellulose filters. Therefore, these two media were used as samples. They were spiked with nivalenol, deoxynivalenol, zearalenone, diacetoxyscirpenol, T-2 toxin, verrucarol, verrucarin A, neosolaniol, sterigmatocystin, roridin A, ochratoxin A, aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2, which can be produced by isolates from fungi-damaged buildings. Deepoxy-deoxynivalenol was used as internal standard. Samples were extracted with organic solvents and the different mycotoxins were separated by high-performance liquid chromatography (HPLC) using a C18 reversed-phase SunFire analytical column and a mobile phase of variable mixtures of ammonium acetate (10 mM) and sodium acetate (20 microM) in water (solvent A) and in methanol (solvent B). The samples were run on-line with a Micromass Quattro Micro triple quadrupole mass spectrometer in positive electrospray ionisation mode using multiple reaction monitoring (MRM). The detection limits of the procedure varied from 50 to 0.009 pg/microL for filter samples and from 75 to 0.04 pg/microL for fungal culture samples. As the method includes few and non-labourious sample treatment steps, it should allow for a high throughput of samples.

As the mycotoxin ochratoxin A (OTA) can occur in roasted coffee, regulations and maximum limits are necessary. The need for simple, rapid and inexpensive detection methods which require a minimum of equipment led to the development of a clean-up tandem assay column for the detection of OTA in roasted coffee. In this study the column comprised two superposed layers: a layer capable of adsorbing at least part of the interfering fraction of the sample and a detection layer containing antibodies that were able to capture the analyte. No expensive instruments were needed as results were visually evaluated. The cut-off level, i.e. the smallest mycotoxin concentration resulting in no colour development, was 6 μg kg−1. Assay validation was performed using samples fortified with OTA. The method gave a low percentage of false negative results and no false positive results. Assay performance was evaluated by screening naturally contaminated samples using the clean-up tandem assay column. The described method offers a rapid, simple and cost-effective screening tool, contributing to more effective quality control procedures.

Polychlorinated biphenyls (PCBs) were monitored in Belgian human adipose tissue samples from deceased individuals (n=100). Their mean age was 52, ranging from 2 to 91 years. There were 57 men and 43 women. Other known variables were date of autopsy and place of residence. No information about diet or occupation was available. The seven marker congeners PCB 28, 52, 101, 118, 138, 153 and 180 were analysed in the samples with a GC-MS/MS method validated according to Commission Decision 2002/657/EC. Extracted fat was cleaned-up over a glass column filled with n-hexane, acid silica, deactivated alumina and anhydrous sodium sulfate. The whole procedure was subjected to a rigorous quality control programme with retention times, ion chromatograms and intensity ratios of the monitored product ions as identification criteria. The total PCB concentration ranged between 10 and 1640 ng g-1 fat, with a mean value of 658 ng g-1 fat. In the age groups of 0-9 (n=1), 10-19 (n=4), 20-29 (n=11), 30-39 (n=13), 40-49 (n=15), 50-59 (n=14), 60-69 (n=14), 70-79 (n=20), 80-89 (n=6) and 90-99 (n=2), the mean total PCB concentrations were 10, 134, 253, 445, 557, 687, 807, 962, 959, and 1191 ng g-1 fat, respectively. So, there was an increase of PCB body burden with age. For the male subjects (n=57; mean age of 53) the mean total PCB concentration was 633 ng g-1 fat. For the female subjects (n=43; mean age of 52) it was 690 ng g-1 fat. There was no significant sex-related difference in the concentrations of marker PCBs.

Tracheoesophageal vocal prostheses (TVP) in laryngectomized patients commonly deteriorate due to overgrowth by yeasts, particularlyCandidaspecies. We describe the first case of colonization of such devices by a member of theFusarium solanispecies complex in a patient with a history of glottal carcinoma. Three isolates, from three prostheses, were found morphologically consistent with the traditional picture ofF. solani.Ribosomal sequence analysis showed that the isolates belonged to a distinct, as yet apparently unnamed phylogenetic species within theF. solanispecies complex. This species, one of two distinct genetic types (genotype 2) traditionally considered part of the plant-pathogenic subtaxonFusarium solanif. sp.radicicola, has not previously been identified as an agent of human or animal disease, although it is closely related to a known etiologic agent of mycetoma, anAcremonium-like species recently renamedFusarium falciforme. Sequence and multisatellite M13 polymorphism analysis revealed no distinctions among the case isolates. Production of cyclosporine was detected for all three case isolates.

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 (a < 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.

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of fumonisin B 1 (FB 1 ), B 2 (FB 2 ) and B 3 (FB 3 ) in cornflakes is described. During method development, special attention was paidto the selection of a suitable internal standard (IS) in order to offer a good alternative for deuterated FB 1 . In this respect, the C 1 2 -sphinganine analogue (2S,3R)-2-amino-dodecane-1,3-diol was chosen because of its structural similarity to the fumonisin backbone and its chromatographic elution between the target analytes. For the extraction of the fumonisins from the cornflakes matrix, MeOH/H 2 O (adjusted to pH 4 with 0.1 M HCl; 70:30, v/v), ACN/MeOH/H 2 O (25:25:50, v/v/v) and acidified ACN/MeOH/H 2 O (25:25:50, v/v/v; pH 4) were evaluated. Preference was given to acidified MeOH/H 2 O (70:30, v/v) with mean recoveries (n = 12) for FB 1 , FB 2 and FB 3 of, respectively, 84 ′ 10,78 ′ 7 and 85 ′ 9%. Cleanup was performed using immunoaffinity columns (FumoniTest®, VICAM). The chromatography was performed under isocratic conditions at a flow of 0.3 mL min - 1 with a mobile phase consisting of ACN/H 2 O (60:40, v/v) containing 0.3% formic acid. The mass spectrometer was operated in the positive electrospray ionization (ESI+) mode using multiple reaction monitoring (MRM). An intralaboratory validation was conducted with fortified samples determining limits of detection (LOD), limits of quantification (LOQ), precision, trueness, specificity and measurement uncertainty. The LOD concentrations for FB 1 , FB 2 and FB 3 were 20, 7.5 and 12.5 μg/kg. The LOQs were 40 μg/kg for FB 1 , 15 μg/kg for FB 2 and 25 μg/kg for FB 3 . The coefficients of variation (CVs) under repeatability conditions varied from 11 to 13% for FB 1 , from 9 to 14% for FB 2 and from 7 to 10% for FB 3 . Under within-laboratory reproducibility conditions, the CVs ranged from 12 to 17% for FB 1 , from 9 to 16% for FB 2 and from 7 to 13% for FB 3 . The percent bias for FB 1 varied from -12 to -10%, while for FB 2 and FB 3 bias ranged, respectively, from -4 to -2% and from -12 to -5%. The expanded measurement uncertainties for FB 1 , FB 2 and FB 3 were, respectively, 19, 18 and 22%.

The format of an existing flow-through enzyme immunoassay has been optimised for the detection of fumonisin B-1 (FB1) and B-2 (FB2) in maize. The visual detection limit i.e. the smallest mycotoxin concentration resulting in no colour development, is 1000 mug/kg. Assay validation was performed using samples spiked with respectively only FBI and a FB1 + FB2 mixture (ratio 1:1). Results showed that within-day and between-day coefficients of variation ranged from respectively 0.4-10.2% and 1.1-9.0%. Naturally contaminated samples were screened with the developed flow-through method and results were compared with fumonisin contamination values obtained by a validated HPLC method. The assay was demonstrated to be accurate and reliable giving no false compliant and only a low percentage of false non-compliant results. The described method offers a simple, rapid and cost-effective screening tool, thus contributing to a better consumers' health protection. (C) 2004 Elsevier B.V. All rights reserved.

A new baking methodology to study acrylamide formation, based on a closed stainless steel tube reactor, was tested on its repeatability. The main advantage of this frying procedure includes the possibility to study the acrylamide formation mechanism in different artificial mixtures, eliminating some variable factors during frying, such as heat flux, degradation of the frying oil and water evaporation. As a first application of this optimized heating concept, the influence of fat oxidation and fat hydrolysis on acrylamide formation was tested during baking of French fries, as well as during heating in the tube reactor. In both cases, no differences in acrylamide formation could be found between fresh oil and oxidized or hydrolyzed heating oils.

Aims This study evaluates the suitability of gas chromatographic-mass spectrometric (GC-MS) analysis to follow-up the extent of benzodiazepine (mis)use in a Belgian prison population and compares it to other analytical strategies (e.g. screening followed by confirmation of the positive samples). Design and participants From February to August 1998, 598 persons were jailed of which 188 (31.4% of the incoming detainees) volunteered to be screened. Urine samples (530 in total) were collected on the day of arrival and after 14, 30 and 90 days of imprisonment. Measurements All samples were screened by EMIT ® for benzodiazepines and ® screening yielded 117 (22.1%) positive samples, a number which increased to 174 (32.8%) after GC-MS analysis. Of these 174 GC-MS positive samples, 119 (68.4%) contained one benzodiazepine while for the remaining samples multiple benzodiazepine (mis)use could be demonstrated. A significant increase in benzodiazepine (mis)use was indicated only from day 0 to day 14 based on the GC-MS results but not on the immunoassay results, even when the latter were complemented with GC-MS analysis of the positively screened samples. The GC-MS data also demonstrated that benzodiazepines are mainly (mis)used by subjects on benzodiazepine prescription as almost 50% of these subjects took additional non-prescribed benzodiazepines. During GC-MS analysis other drugs were co-extracted unintentionally and chromatographed and 23.9% of the volunteers were positive for illegal drugs on the day of arrival. Conclusion Immunoassay results yield an underestimation of the problem of benzodiazepine (mis)use in prison due to the high false negative rate. GC-MS analysis of all samples therefore is the recommended strategy for this type of longitudinal study as it yields more correct and detailed information than the immunoassay results.

Different extraction and clean-up techniques used before HPLC analysis were compared in order to obtain a reliable method for the quantitative determination of zearalenone (ZEA) and α-zearalenol (α-ZOL) in animal feed. Immunoaffinity clean-up was compared to C 18 and Florisil column clean-up. Extracted samples were analysed by reversed-phase HPLC with fluorescence detection ( λ ex =274 nm, λ em =440 nm). A mobile phase of acetonitrile:water (50:50 (v/v)) and a flow-rate of 1.0 ml min −1 resulted in a good separation between ZEA and α-ZOL. Using immunoaffinity clean-up the linear range was between 25 and 600 μg kg −1 for ZEA and α-ZOL in maize. Intra-laboratory coefficients of variation (CV) (under repeatability conditions) were 9.16% for ZEA and 2.18% for α-ZOL. Recoveries for spiked ZEA and α-ZOL samples ranged from 89 to 110% with CVs between 5.2 and 11.2% (under within-laboratory reproducibility conditions). Using C 18 and Florisil solid-phase clean-up, matrix interference was too high. Therefore, naturally contaminated animal feed samples were analysed using the developed HPLC method coupled to the immunoaffinity clean-up.

With Council Regulation 2821/98 zinc bacitracin, spiramycin, tylosin and virginiamycin, and with Commission Regulation 2788/98 olaquindox, were banned from animal feed as feed additives. To control the compliance of the ban a single multi-analyte liquid chromatography–mass spectrometry (LC–MS) method for their detection and quantification in animal feed was developed. A 2.5 g aliquot of feedstuff was extracted with 10 ml of methanol/water (70/30, v/v), and 3 ml of this extract was applied on an OASIS ® column after dilution with 27 ml of water. Elution was performed with 2 ml of acetonitrile/water (50/50, v/v) and the eluant diluted to acetonitrile/water (30/70, v/v). An amount of 20 μl was injected onto a Kromasil C 18 liquid chromatography column. A gradient program, consisting of a mixture of acetonitrile and water (both containing 0.3% formic acid) was used for the separation of the compounds. The method was validated according to the draft SANCO 1085/2000. The detection capability of the method for all compounds was −1 .

A few days after her admittance to a hospital for a suicide attempt with benzodiazepines, a 15-year-old girl was found dead in bed. At autopsy, no specific anatomo-pathologic cause of death was identified. Systematic toxicological analysis (HPLC-DAD, GC-NPD, and GC-MS) of postmortem blood and urine revealed the presence of high concentrations of flecainide and its two major metabolites. Flecainide is a class IC anti-arrhythmic drug causing a decreased intracardiac conduction velocity in all parts of the heart. To identify and quantitate flecainide together with its metabolites in blood, urine, and other toxicologically relevant matrices, a new method was developed using high-performance liquid chromatography with diode-array detection. All compounds were separated on a Hypersil BDS phenyl column using water, methanol, and 1.5M ammonium acetate in a gradient system. Chromatographic analysis was preceded by an optimized solid-phase extraction procedure on RP-C18 extraction columns. The flecainide concentrations in blood and urine were 18.73 and 28.3 mg/L, respectively, and the metabolites were detected only in urine at the following concentrations: 9.4 mg/L for meta-O-dealkylated flecainide and 8.59 mg/L for meta-O-dealkylated flecainide lactam. Based on these results, it was concluded that the suicide was consistent with an overdose of this anti-arrhythmic drug.

A liquid chromatography combined to mass spectrometry (LC–MS/MS) method to confirm the presence of 11 synthetic glucocorticoids in cattle liver is described. After an enzymatic deconjugation step, liver samples were treated with methanol to extract the glucocorticoids. A further cleanup of the extracts was carried out by SPE. LC separations were carried out as described previously using a column packed with porous graphitic carbon. For the detection positive (ESI+) and negative (ESI−) electrospray ionization were evaluated, but the preference was given to the negative mode for most of the analytes. The complete method was validated according to SANCO/1085/2000, giving decision limits and detection capabilities in 0.08–0.38 μg kg −1 range for the banned glucocorticoids and between 0.1 and 0.5 μg kg −1 above the established maximum residue limits (MRLs) values for betamethasone, dexamethasone, prednisolone and methylprednisolone. Several incurred liver samples of animals treated with different glucocorticoids were analyzed. In order to obtain a maximum amount of unconjugated parent drug, enzymatic hydrolysis conditions were optimized using liver samples of a calf treated simultaneously with beta- and dexamethasone. A central composite experimental design was applied and the estimated response surface plots were used to select optimal conditions of pH, temperature and duration of the preliminary deconjugation step.

Every other year scientists working in the field of residue analysis participate the "International Symposium on Hormone and Veterinary Drug Residue Analysis" and "Euroresidue" conferences. In each symposium a lot of innovative information is presented. In order to obtain a retrieval system for this growing amount of information, a database dedicated to these conferences was created. The main principles of the database were explained during the Fourth Euroresidue Conference (2000, Veldhoven), but it will be extended from subsequent conferences. It is not the aim to compete with other databases but only to have an easy-to-operate database containing information on hormone and veterinary drug residue analysis. In this second edition, the abstracts of all the proceedings have been added. Moreover, the database may be downloaded up-to-date from a website. © 2002 Elsevier Science B.V. All rights reserved.

In sports doping, as well in man as in horseracing, stanozolol (Stan) was abused and became the subject of metabolism research. Also in veterinary practice, stanozolol became an important misused anabolic steroid. Like most other anabolic steroids, stanozolol has poor gas chromatographic behavior. It is difficult to detect in urine, because of low urinary excretion and renal clearance. This is due to the rapid metabolization, leading to low concentration levels of the parent compound found in urine. Therefore, most research studies have focused on the detection of its urinary metabolites. For the identification of the metabolites, different methods of extraction and detection are described in the literature. These are reviewed in this article. Most authors use a hydrolysis to free the phase II metabolites. Extraction procedures vary from solid-phase extraction (SPE), liquid-liquid (L-L) extraction to immunoaffinity chromatography (IAC). For the final detection, the use of gas chromatography (GC)-mass spectrometry (MS) can be compared with liquid chromatography (LC)-MSn. Different metabolites are identified depending on the administration of stanozolol in the animal experiment (oral or intramuscular). Analyses for these analytes in other matrices are also briefly discussed. © 2002 Elsevier Science B.V. All rights reserved.

A gas chromatographic-mass spectrometric method was developed for the simultaneous analysis of 15 low-dosed benzodiazepines, both parent compounds and their corresponding metabolites, in human urine. The target compounds are alprazolam, alpha-hydroxyalprazolam, 4-hydroxyalprazolam, flunitrazepam, 7-aminoflunitrazepam, desmethylflunitrazepam, flurazepam, hydroxyethylflurazepam, nitrogen-desalkylflurazepam, ketazolam, oxazepam, lormetazepam, lorazepam, triazolam and alpha-hydroxytriazolam. Nitrogen-methylclonazepam is used as the internal standard. The urine sample preparation involves enzymatic hydrolysis of the conjugated metabolites with Helix pomatia beta-glucuronidase for 1 h at 56 degrees C followed by solid-phase extraction on a phenyl-type column. The extracted benzodiazepines are subsequently analyzed on a polydimethylsiloxane column using on-column injection to enhance sensitivity. The extraction efficiency exceeded 80% for all compounds except for oxazepam, lorazepam and 4-hydroxyalprazolam which had recoveries of about 60%. The LODs ranged from 13 to 30 ng/ml in the scan mode and from 1.0 to 1.7 ng/ml in the selected ion monitoring (SIM) mode. Linear calibration curves were obtained in the concentration ranges from 50 to 1000 ng/ml in the scan mode and from 5 to 100 ng/ml in the SIM mode. The within-day and day-to-day relative standard deviations at three different concentrations never exceeded 15%.

GC-MS screening conditions were developed for 15 low-dosed benzodiazepines, covering alprazolam, flunitrazepam, flurazepam, ketazolam, lorazepam and triazolam, and the corresponding metabolites alpha-hydroxyalprazolam, 4-hydroxyalprazolam; 7-aminoflunitrazepam, desmethylflunitrazepam, 7-aminodesmethylflunitrazepam; hydroxyethylflurazepam, N-desalkylflurazepam; oxazepam and alpha-hydroxytriazolam, respectively. Benzodiazepines are analyzed on a polydimethylsiloxane column in both the scan and the multiple ion monitoring modes using on-column injection to attain maximal sensitivity. The reactive compounds are acetylated with pyridine and acetic anhydride for 20 min. The derivatives are stable for at least 4 days. The relative standard deviation observed with standard compounds at the low nanogram-level ranged from 1.13 to 4.87% within-day and from 1.12 to 4.94% between-day. Unequivocal identification potential, high chromatographic resolution and sensitivity are combined with minimal thermal degradation. The presented screening conditions provide the basis for a unique routine screening method for low-dosed benzodiazepines with a broad polarity range.

In this study a new method for the simultaneous confirmation of betamethasone and dexamethasone residues in bovine liver is presented. A Quattro LCZ triple quadrupole mass spectrometer, equipped with an atmospheric pressure ionization (API) source, was coupled to a high performance liquid chromatograph (HPLC) system. Spiked liver samples were first extracted with acetonitrile, and the extracts were purified on C-18 columns. LC separations were performed on a Hypercarb column, with acetonitrile/water (90:10, v/v, +0.3% formic acid) as the mobile phase. Retention times for dexa- and betamethasone were 6.60 and 8.50 min, respectively. Fluorometholone had a retention time of 6.70 min and was used as the internal standard. The detection of the analytes was performed in the multiple reaction monitoring (MRM) mode. The assay was linear over the range of 0.5 to 8 µg/kg for both analytes. The estimated determination limits were 0.2 µg/kg for both beta- and dexamethasone and the quantification limits were 0.4 µg/kg for dexamethasone and 0.3 µg/kg for betamethasone. Analysis precision at 1, 2 and 4 µg/kg was lower than 6.1% (relative standard deviation, RSD) and accuracy was at least 97.5%. Recoveries at 1, 2 and 4 µg/kg ranged between 56 and 69%. Copyright © 2001 John Wiley & Sons, Ltd.

An immunoaffinity chromatographic (IAC) method for isolating sulfamethazine (SMZ) from incurred urine samples was developed. This was achieved by (i) generating polyclonal antibodies that recognize equally well SMZ and its major urinary metabolites, (ii) evaluating in an ELISA procedure the influence of methanol, salt and pH on the antigen-antibody interaction in order to determine the optimum conditions for IAC and (iii) covalent coupling of the IgG fractions of anti-SMZ to CNBr activated Sepharose for the preparation of re-usable immunoaffinity columns, having a high capacity for SMZ (1900 ng SMZ mL-1 gel). For desorbing SMZ from the immunoaffinity column, different elution modes were evaluated, with 40% MeOH-0.1 mol L-1 HOAc-0.5 mol L-1 NaCl being the most efficient combination. Using the IAC column for processing SMZ spiked urine samples resulted in high recoveries, ranging from 92 to 100%. Because of the high cross-reactivity with the major metabolites of SMZ present in urine of treated animals, the antibodies show excellent properties for use in both IAC and ELISA. For the isolation and concentration of the parent drug and its major metabolites, the urine could be applied directly to the IAC column, without the time-consuming step of deconjugation. Moreover, the use of IAC prior to ELISA for the analysis of incurred urine samples showed good efficiency for the elimination of matrix interferences. Owing to the urine-tissue relationship, the urine concentrations can be used to predict the presence of the parent drug in tissues and so possible violations of the maximum residue limit (MRL) can be controlled.

The major objective of this study was to determine the fatty acid composition of the Belgian diet. Food consumption data from a large representative sample (n = 11,302) of the Belgian population between 25 and 74 years of age (BIRNH study) were analyzed with regard to the intake of fatty acids. The fatty acid composition of the major fat sources in the Belgian diet was determined and used to calculate average intakes for fatty acids from C4 to C22. In addition, results are compared to other studies and to guidelines for n–3 and n–6 fatty acids. Saturated fatty acids provide 17% of the energy intake in the Belgian diet, polyunsaturated fatty acids 7%, and monounsaturated fatty acids 14%. The intake of total n–6 fatty acids is very high (6 en%), particularly of linoleic acid. The intake of n–3 fatty acids is low, only 0.8 en%, which results in a low ratio of n–3 to n–6 (0.15). The most important sources of n–6 and n–3 fatty acids are margarine and meat, respectively.

A representative sample of the Belgian population, aged 25-74 years, was interviewed between 1980 and 1985. Dietary habits were assessed using a 24 h food record method. Age-, sex- and district-specific energy-adjusted averages of macronutrient intakes were compared with mortality rates from 1988-90, with special emphasis on the association between fat intake and cancer mortality. Univariate analyses were followed by multiple linear regression analyses, controlling for possible confounders such as fibre intake, smoking and educational level. In multivariate analyses, significant positive associations were found between all-causes mortality and saturated fat intake in men, and between all-causes mortality and the ratio of n-3 to n-6 fatty acids in men; colorectal cancer mortality was associated with polyunsaturated fat intake and with the ratio of unsaturated to saturated fat in men. Significant negative associations were found between all-causes mortality and polyunsaturated fat intake in men, and between all-causes mortality and the ratio of unsaturated to saturated fat in men; colorectal cancer mortality was associated with saturated fat intake in men. In women, only breast cancer mortality was associated with saturated and monounsaturated fat intake. Prostate cancer mortality was not related to any of the studied dietary fat components. For total cancer mortality, only weak non-significant associations with fat intake were found.

Samples of bovine muscle, liver and urine, zeranol-free (RM 508, RM 509 and RM 510, respectively) and zeranol-containing (RM 511, RM 512 and RM 513, respectively) were prepared and tested as candidate reference materials. Preliminary studies on achievement of target zeranol content, intercomparison of analytical methods (HPLC-RIA and GC-MS) and effects of lyophilisation and irradiation on zeranol content are described. The preparation of the materials and testing for homogeneity and stability of zeranol in the materials are discussed. The coefficients of variation for zeranol determinations for between-vial homogeneity (4.0%, 4.4% and 4.6% for muscle, liver and urine, respectively) are similar to those for the analytical method (4.0%, 7.3% and 6.8% for muscle, liver and urine, respectively) indicating that the materials are homogeneous. Stability data over a 12-month storage period at temperatures ranging from −18 °C to +37 °C indicate that the materials are sufficiently stable for use as reference materials. This paper is dedicated to the memory of Dr Hilary Stevenson (The Queen’s University of Belfast/DANI) who, together with Mr. W. Graham, facilitated the irradiation of the lyophilised materials.-->

A dipstick enzyme immunoassay for the rapid detection of Fusarium T-2 toxin in wheat was developed. An Immunodyne ABC membrane was precoated with rabbit anti-mouse immunoglobulins. After the strips were immersed in a solution of monoclonal anti-T-2 toxin antibodies, a direct competitive enzyme immunoassay was performed. This assay included the incubation of the antibody-coated dipsticks in a mixture of sample and T-2 toxin-horseradish peroxidase conjugate. Afterwards, the strips were placed in a chromogen-containing substrate solution (H202-3,3',5,5'-tetramethylbenzidine) for color reaction. The dot color intensity of toxin-positive dipsticks was visually distinguishable from that of the negative control. A portable colorimeter was used to confirm and quantify the visual observations. With coated strips, the tests could be performed in 45 min. The visual detection limit for T-2 toxin in buffer solution was 0.25 ng/ml. Artificially infected wheat samples were extracted with 80% methanol-water. A dilution of the raw extract of 1:8 was sufficient to avoid matrix effects. It was possible to make visually a clear distinction between the negative control and a wheat extract spiked with 12 ng/g.

A novel highly sensitive liquid Chromatographic method for the determination of some tetracycline antibiotics has been developed. It is based upon the measurement of fluorescence induced by complexation of tetracyclines with the zirconium cation, which is added post-column to the liquid Chromatographie eluate. The zirconium reagent concentration and post-column configuration were optimized. The effect of time upon the stability of the chelates was studied. The fluorescence intensity was linear in a concentration range of 0.01–10 μg ml −1 tetracycline. The proposed method has already been applied to the residue analysis for tetracycline, oxytetracycline and chlortetracycline in bovine milk. The most striking advantages of the method over the reported methods in the literature are the higher sensitivity and specificity achieved. The detection limits of the assay ( signal - to - noise ratio = 4:1) were 1 ng ml −1 2 ng ml −1 and 4 ng ml −1 for oxytetracycline, tetracycline and chlortetracycline, respectively.

Animal feed is often contaminated with different types of mycotoxins and plant toxins. These toxic substances can cause adverse effects on animal health, and carry-over of some toxins and/or their metabolites into edible tissues, milk and eggs may contribute to human exposure. Therefore risk assessments and official controls of feed samples have to be performed. In order to carry out these assignments, different tools such as rapid screening tests and chromatographic confirmatory tests have been developed to analyse and detect the toxic substances in different commodities. An overview of the detection and determination of mycotoxins and plant toxins in feed is presented.

An LC-MS/MS method was developed and validated for the simultaneous determination of deoxynivalenol, zearalenone, T-2-toxin, HT-2-toxin and metabolites, including 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, deoxynivalenol-3-glucoside, α-zearalenol, β-zearalenol, zearalenone-4-glucoside, α-zearalenol-4-glucoside, β-zearalenol-4-glucoside and zearalenone-4-sulfate in maize, wheat, oats, cornflakes and bread. Extraction was performed with acetonitrile/water/acetic acid (79/20/1, v/v/v) followed by a hexane defatting step. After filtration, the extract was evaporated and the residue was redissolved in mobile phase for injection. The mobile phase, which consisted of a mixture of methanol and water with 10 mM ammonium acetate, was adjusted to pH 3 with glacial acetic acid. A sample clean-up procedure was not included because of the low recoveries of free and masked mycotoxins and their differences in polarity. The method allowed the simultaneous determination of 13 Fusarium mycotoxins in a one-step chromatographic run using a Waters Acquity UPLC system coupled to a Quattro Premier XE mass spectrometer. The method was validated for several parameters such as linearity, apparent recovery, limit of detection, limit of quantification, precision, expanded measurement uncertainty and specificity. The limits of detection varied from 5 to 13 ng g−1; those for the limit of quantification from 10 to 26 ng g−1. The results of the performance characteristics of the developed LC-MS/MS method were in good agreement with the criteria mentioned in Commission Regulation (EC) No. 401/2006. Thirty samples of a variety of food and feed matrices were sampled and analysed between July 2010 and January 2011.

Anabolic steroids are used as doping agents in sports. Notwithstanding the total EC ban, they are also used as growth promoters in animal production. The detection of anabolic steroids in the urine of untreated persons due to the consumption of contaminated meat has been described earlier. This paper describes further confirmation of a urine sample which was clostebol positive as a result of the above described interference phenomenon. This was done by gas chromatography—mass spectrometry. Both low resolution mass spectrometry (LRMS) and high resolution mass spectrometry (HRMS) were used after gas chromatographic separation. LRMS was used to determine the relative abundances of the ions and for calculating similarity indices. With HRMS, selectivity was increased by recording exact masses instead of nominal masses.

The detection of illegal growth promoters in cattle urine by conventional immunoassays, such as radioimmunoassay (RIA) and enzyme‐linked immunosorbent assay (ELISA) has been extensively described. ...

Two multi-analyte flow-through immunoassay formats for rapid detection of mycotoxins in a variety of food matrices (peanut cake, maize, and cassava flour) were developed and evaluated. The selected food matrices are typical staple foods and export products for most low-income communities around the world. The assay formats included gel-based and membrane-based flow-through assays and were based on the principle of indirect enzyme-linked immunosorbent assay. Using the same immunoreagents, the performance characteristics of both assays were compared. To the best of our knowledge, this is the first report on such a comparison. The gel-based format was developed to screen for ochratoxin A, fumonisin B(1), deoxynivalenol, and zearalenone detection at cut-off values of 3, 1,250, 1,000, and 200 μg kg(-1), respectively, while the membrane-based format can be used to screen ochratoxin A, aflatoxin B(1,) deoxynivalenol, and zearalenone at the following cut-offs: 3, 5, 700, and 175 μg kg(-1), respectively. The applicability of these assay formats was demonstrated by evaluating the performance characteristics of both tests through performing multiple experiments on different days. Both assays were further evaluated by analyzing naturally contaminated samples in the laboratory and also in the field under tropical conditions (Cameroon, West Africa). The false-negative rate with both formats was less than 5%, which is in good agreement with Commission Decision 2002/657/EC regarding the performance of analytical methods intended for screening purposes.

In poultry farming, anticoccidial drugs are widely used as feed additives for the prevention and treatment of coccidiosis. Because coccidiostats and veterinary medicines, in general, are often poorly absorbed, manure from treated animals may contain high concentrations of these compounds. Experimental studies have shown that the uptake of veterinary medicines by plants from soil containing contaminated manure may occur. This leads to several questions regarding the impact on the environment, resistance problems, and public health and allergy issues. This work describes the development of a quantification method for coccidiostats in vegetables. Vegetables were spiked at 100 μg kg(-1) (dry weight) with coccidiostats (monensin, narasin, lasalocid A, salinomycin, diclazuril, and nicarbazin) in order to optimize the extraction and clean-up. Possible critical factors (e.g., extraction solvent) were statistically examined by linear regression with the use of Plackett-Burman and full factorial designs. Final extracts were analyzed with ultra-performance liquid chromatography tandem mass spectrometry operating in multiple-reaction monitoring mode. Both the synthetic and ionophoric coccidiostats could be determined in a single run with an analysis time of 5 min. The developed method was validated taking into account the requirements of the Commission Decision 2002/657/EC as a guideline. The method is regarded as applicable for its intended purposes with quantification limits between 0.30 and 2.98 μg kg(-1). This method could be used to establish possible maximum residue limits for coccidiostats in vegetables, as already exist for eggs, meat, and milk.

The glucosinolate profile of black radish (Raphanus sativus L. niger) based dietary supplements has been investigated by HPLC-PDA, LC-ESI-MS/MS and LC-APCI-MS/MS systems. Optimization of the MS/MS parameters and LC conditions was performed using sinigrin reference standard and rapeseed certified reference material (BC190) respectively. An LC-ESI-MS/MS system was used to detect (screen) and identify the naturally occurring intact glucosinolates (GLs). The intact GLs identified were then desulfated and quantified on an HPLC-PDA system as desulfo-glucosinolates (DS-GLs). Prior to quantification, the DS-GLs were identified using an APCI-MS/MS. The HPLC-PDA method performance criteria were evaluated using glucotropaeolin potassium salt. The validated method was applied for the analysis of six dietary supplements. In total, six glucosinolates were identified and quantified in the dietary supplements; glucoraphasatin (0.20.48 mg/g), glucosisaustricin (0.370.91 mg/g), glucoraphenin (0.841.27 mg/g), glucoputrajivin (0.140.28 mg/g), glucosisymbrin (0.700.99 mg/g) and gluconasturtiin (0.060.12 mg/g). Glucoraphenin was the most abundant glucosinolate in all samples.

A sensitive and rapid screening method for the estimation of aflatoxin M 1 (AFM 1 ) in milk has been developed. Milk samples were first purified by immunoaffinity chromatography (IAC) using polyclonal antibodies raised in rabbits against aflatoxin M 1 -bovine serum albumin (AFM 1 BSA) and coupled to an activated sepharose matrix. The eluate was analyzed in an indirect competitive streptavidin-biotin modified enzyme linked immunoassay (EIA). Microtitre plates were coated with AFM 1 BSA that competes with the analyte in the sample for binding with the biotinylated antibody. Bound biotinylated antibody was detected using a streptavidin biotinylated horseradish peroxidase complex. The limit of detection of the EIA is 2 ng kg −1 . By combination of IAC and EIA, spiked milk samples were analyzed. In conclusion, IACEIA is a sensitive and rapid method for the estimation of AFM 1 in milk.