Nitrite in feed: from animal health to human health.

Nitrite is widely consumed from the diet by animals and humans. However the largest contribution to exposure results from the in vivo conversion of exogenously derived nitrate to nitrite. Because of its potential to cause to methaemoglobin (MetHb) formation at excessive levels of intake, nitrite is regulated in feed and water as an undesirable substance. Forages and contaminated water have been shown to contain high levels of nitrate and represent the largest contributor to nitrite exposure for food-producing animals. Interspecies differences in sensitivity to nitrite intoxication principally result from physiological and anatomical differences in nitrite handling. In the case of livestock both pigs and cattle are relatively susceptible. With pigs this is due to a combination of low levels of bacterial nitrite reductase and hence potential to reduce nitrite to ammonia as well as reduced capacity to detoxify MetHb back to haemoglobin (Hb) due to intrinsically low levels of MetHb reductase. In cattle the sensitivity is due to the potential for high dietary intake and high levels of rumen conversion of nitrate to nitrite, and an adaptable gut flora which at normal loadings shunts nitrite to ammonia for biosynthesis. However when this escape mechanism gets overloaded, nitrite builds up and can enter the blood stream resulting in methemoglobinemia. Looking at livestock case histories reported in the literature no-observed-effect levels of 3.3mg/kg body weight (b.w.) per day for nitrite in pigs and cattle were estimated and related to the total daily nitrite intake that would result from complete feed at the EU maximum permissible level. This resulted in margins of safety of 9-fold and 5-fold for pigs and cattle, respectively. Recognising that the bulkiness of animal feed limits their consumption, these margins in conjunction with good agricultural practise were considered satisfactory for the protection of livestock health. A human health risk assessment was also carried out taking into account all direct and indirect sources of nitrite from the human diet, including carry-over of nitrite in animal-based products such as milk, eggs and meat products. Human exposure was then compared with the acceptable daily intake (ADI) for nitrite of 0-0.07 mg/kg b.w. per day. Overall, the low levels of nitrite in fresh animal products represented only 2.9% of the total daily dietary exposure and thus were not considered to raise concerns for human health. It is concluded that the potential health risk to animals from the consumption of feed or to man from eating fresh animal products containing nitrite, is very low.

Autoregulatory properties of (+)-thujopsene and influence of environmental conditions on its production by Penicillium decumbens.

A Penicillium decumbens strain was collected from a water-damaged building, and the production of microbial volatile organic compounds (MVOCs) was investigated by means of headspace solid-phase microextraction, followed by GC-MS analysis. The strain was characterized by a high production of (+)-thujopsene. The influence of various temperatures, relative humidity (RH) values, substrates, and inoculum concentrations on fungal growth and (+)-thujopsene production was studied. The optimal temperature and relative humidity for P. decumbens growth were 30°C and 100% RH, respectively. In general, the more favourable the incubation parameters were for growth, the faster maximum (+)-thujopsene production was reached. Moreover, the antifungal activity of thujopsene was tested against 16 fungal strains. The growth of five of these fungal strains was negatively affected both by thujopsene alone and when grown in contact with the MVOCs produced by P. decumbens. Following these results and since growth of P. decumbens itself was also inhibited by thujopsene, an autoregulatory function for this compound was proposed. Few data are present in the literature about chemical communication between fungi. The present research could, therefore, contribute to understanding fungal metabolism and behaviour in indoor environments.

The use of library identification and common fragments for the identification of corticosteroids in forensic samples.

The detection of corticosteroids and sex steroids in samples with no content indication, which are confiscated for forensic investigation, is a challenge in doping analysis. A screening method based on the identification of androgens, estrogens, gestagens, and their esters by means of a mass spectral library, along with a fast ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method, was recently developed in our lab for the analysis of dietary supplements. However, for forensic investigations, it is important to extend the scope of the method to corticosteroids in various matrices. Therefore, 36 corticosteroids were added to the mass spectral library, and the sample preparation step was modified so that androgens, gestagens, corticosteroids, and their esters could be analyzed with only one injection with the UPLC-MS method. A complementary tool to the existing library identification was found in the extraction of common fragment ions out of the full scan data obtained for the library search. The fragment ion with m/z 147 was found to be a good marker for the detection of steroids. Extra confirmation was obtained from the fragment ions with m/z 135 (for all steroids) and 237 (specific for corticosteroids) or from the fragment ions with m/z 77, 91, and 105. The effectiveness of this approach was evaluated on some samples previously screened for forensic investigation with thin-layer chromatography and confirmed with a targeted gas chromatography-mass spectrometry method. This study shows that the combination of the library identification and the common fragment ions approach can be a valuable tool in the detection of steroids without defining any target at the start of the analysis.

Applicability of a yeast bioassay in the detection of steroid esters in hair.

The aim of the present study was to demonstrate the applicability of a yeast androgen and estrogen bioassay in the detection of steroid esters in hair samples of animals treated with a hormone ester cocktail. The outcome of the activity screenings was critically compared with the results previously obtained with LC-MS/MS analysis. Hair samples of one pour-on treated animal, 10 ml DMSO containing 25 mg estradiol benzoate (EB), 60 mg testosterone decanoate (TD) and 60 mg testosterone cypionate (TC), were selected and analyzed with the androgen and estrogen yeast bioassay. Results showed that by the introduction of a hydrolysis step, bioassays can be used to screen for the presence of hormone esters in hair samples. Based on the difference in fluorescence responses between the non-hydrolyzed and the hydrolyzed hair samples, it was possible to detect the presence of EB up to at least 56 days after a single pour-on treatment and to detect the presence of TC and TD up to at least 14 days after the treatment. Although the LC-MS/MS analysis could detect TC and TD up to 49 days after treatment, bioassays have the advantage that they can also detect any (un)known steroid ester.

Investigation of urinary steroid metabolites in calf urine after oral and intramuscular administration of DHEA.

DHEA (3beta-hydroxy-androst-5-en-17-one) is a natural steroid prohormone. Despite a lack of information on the effect, DHEA and other prohormones are frequently used as a food supplement by body-builders. DHEA is suspected for growth promoting abuse in cattle as well. Considering the latter, urine samples from a previous exposure study in which calves were exposed to 1 g DHEA per day for 7 days, were used. The calves were divided in three groups: one orally treated, one intramuscularly injected, and a control group. The effect of this treatment on the urinary profile of several precursors and metabolites of DHEA was investigated. Urine samples were collected several days before and during the 7 days of administration and were submitted to a clean-up procedure consisting of a separation of the different conjugates (free, glucuronidated, and sulfated forms) of each compound on a SAX column (Varian). An LC-MS/MS method was developed for the detection and quantification of several metabolites of the pathway of DHEA including 17alpha- and 17beta-testosterone, 4-androstenedione, 5-androstenediol, pregnenolone, and hydroxypregnenolone. Elevated levels of DHEA, 5-androstenediol, and 17alpha-testosterone were observed in the free and sulfated fraction of the urine of the treated calves, thus indicating that the administered DHEA is metabolized mainly by the Delta(5)-pathway with 5-androstenediol as the intermediate. Sulfoconjugates of DHEA and its metabolites were found to constitute the largest proportion of the urinary metabolites. The free form was also present, but in a lesser extent than the sulfated form, while glucuronides were negligible.

An immunochemical test for rapid screening of zearalenone and T-2 toxin.

An immunochemically based test for non-instrumental simultaneous detection of zearalenone (ZEA) and T-2 toxin (T2) in feed was developed. The method combines clean-up of sample extract, pre-concentration of analytes by immunoextraction and immunodetection through the enzymatic reaction of horseradish peroxidase (HRP). The test is housed inside a standard 1-mL solid-phase extraction column and consists of three layers: two test layers (one for ZEA and another for T2) with immobilised specific antibodies and one control layer with bound anti-HRP antibodies. Feed extract was passed through an additional column with clean-up layer, which was disconnected after extract application. Total assay time was about 15 min for six samples and detection time was 4 min after chromogenic substrate application. Under optimised conditions a cut-off level for ZEA and T2 of 100 microg/kg was established. Different feed types were analysed for ZEA and T2 contamination by the proposed method and results were confirmed by LC-MS/MS.

Determination of antidepressants in human postmortem blood, brain tissue, and hair using gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric (GC-MS) method in positive ion chemical ionization mode in combination with a solid phase extraction was optimized for new-generation antidepressants and their metabolites in postmortem blood, brain tissue, and hair. Twelve antidepressants and their active metabolites (i.e., mirtazapine, viloxazine, venlafaxine, citalopram, mianserin, reboxetine, fluoxetine, fluvoxamine, sertraline, maprotiline, melitracen, paroxetine, desmethylfluoxetine, desmethylmianserin, desmethylmirtazapine, desmethylsertraline, desmethylmaprotiline, desmethylcitalopram, and didesmethylcitalopram) could be quantified. In this article, in addition to the validation of the GC-MS method, four postmortem cases are discussed to demonstrate the usefulness of the described method in forensic toxicology. In these cases, sertraline, fluoxetine, citalopram, and trazodone in combination with their active metabolites were quantified. Blood concentrations ranged from subtherapeutic to toxic concentrations, while brain to plasma ratios ranged from 0.8 to 17. Hair concentrations ranged from 0.4 to 2.5 ng/mg depending on the compound and hair segment.

Application of a new anti-zearalenone monoclonal antibody in different immunoassay formats.

Monoclonal antibodies against zearalenone (ZEA) were raised in mice according to the hybridoma technology and applied in different immunochemical techniques. More specifically, three formats based on the competitive direct enzyme immunoassay principle were developed: an enzyme-linked immunosorbent assay (ELISA), a flow-through gel-based immunoassay column and a flow-through membrane-based immunoassay. In ELISA, the 50% inhibitory concentration (IC50) was 0.8 ng/mL, and the limit of detection for ZEA standard solutions was 0.1 ng/mL. The antibodies showed a high ZEA (100%) and alpha-zearalenol (alpha-ZOL) (69%) recognition, while cross-reactivities with alpha-zearalanol, zearalanone, beta-zearalenol and beta- zearalanol were 42%, 22%, <1% and <1%, respectively. For standard solutions, a cut-off level at 10 ng/mL could be established for the gel- and membrane-based enzyme immunoassays. Assay time of both non-instrumental tests was 25 min for 10 samples. By including a simple sample extraction procedure, the methods were applied to wheat with IC50s in ELISA of 80 and 120 microg/kg (dilution up to 5% and 15% (v/v) of wheat matrix, respectively). The cut-off level of the gel- and membrane-based immunoassays was established at 100 microg/kg. Potentials and limitations of the developed methods were compared. The possible application for multi-mycotoxin analysis of the ELISA method based on a single monoclonal antibody was investigated. Therefore, principal component analysis and partial least squares regression data modelling were used to separate the immunoassay responses of two cross-reactants (ZEA and alpha-ZOL).

JEM Spotlight: Fungi, mycotoxins and microbial volatile organic compounds in mouldy interiors from water-damaged buildings.

Concerns have been raised about exposure to mycotoxin producing fungi and the microbial volatile organic compounds (MVOCs) they produce in indoor environments. Therefore, the presence of fungi and mycotoxins was investigated in 99 samples (air, dust, wallpaper, mycelium or silicone) collected in the mouldy interiors of seven water-damaged buildings. In addition, volatile organic compounds (VOCs) were sampled. The mycotoxins were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (20 target mycotoxins) and quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). Morphological and molecular identifications of fungi were performed. Of the 99 samples analysed, the presence of one or more mycotoxins was shown in 62 samples by means of LC-MS/MS analysis. The mycotoxins found were mainly roquefortine C, chaetoglobosin A and sterigmatocystin but also roridin E, ochratoxin A, aflatoxin B(1) and aflatoxin B(2) were detected. Q-TOF-MS analysis elucidated the possible occurrence of another 42 different fungal metabolites. In general, the fungi identified matched well with the mycotoxins detected. The most common fungal species found were Penicillium chrysogenum, Aspergillus versicolor (group), Chaetomium spp. and Cladosporium spp. In addition, one hundred and seventeen (M)VOCs were identified, especially linear alkanes (C(9)-C(17)), aldehydes, aromatic compounds and monoterpenes.

Molecular and cellular effects of food contaminants and secondary plant components and their plausible interactions at the intestinal level.

The intestinal mucosa is not simply a barrier allowing entry of compounds such as nutrients or chemicals, and restricting that of others. Intestinal cells and activities perform selective absorption, biotransformations and efflux back to the lumen. Furthermore, food substances affect both bioavailability and intestinal function. Some are able to act as transcriptional regulators and enzyme modulators. This review points out plausible interactions between food contaminants and/or natural constituents at molecular and cellular levels and focuses on the effects of classical (pesticides and veterinary drugs), environmental (heavy metals, PCBs, dioxins, etc.) and food processing generated (PAHs, heterocyclic amines, etc.) contaminants on absorption, metabolism and efflux. Special attention is given to secondary metabolites of molds (mycotoxins) and plants (polyphenols). Molecular targets are briefly described as well as regulation mechanisms. Where possible, data referred to deal with human intestinal functions in vivo, and with in vitro studies on human intestinal Caco-2 cells; however, since data related to the intestine are rather scarce, effects on molecular targets in liver are also considered. This review also points out the urgent need for fully validated high throughput in vitro tools to screen combinations of substances, at realistic intestinal concentrations. A higher priority could then be given to combinations of nutrients, xenobiotics and food contaminants, with hazardous or beneficial impacts on human health.

Importance of a canteen lunch on the dietary intake of acrylamide.

A food and drink intake survey was carried out among university students and staff members. Consumption data were collected on days when the participants took hot lunch in a university canteen. The dietary acrylamide exposure was calculated through a probabilistic approach and revealed a median intake of 0.40 microg/kg bw/day [90% confidence interval: 0.36-0.44], which is in accordance with previous exposure calculations. Biscuits (35.4%), French fries (29.9%), bread (23.5%), and chocolate (11.2%) were identified to be the main sources of dietary acrylamide. Foodstuffs consumed in between the three main meals of the day (so called snack type foods) contributed the most to the intake (42.2%). The exposure was lower in an intervention group which received free portions of fruit and vegetables, indicating that a nutritionally balanced diet may contribute to a decreased acrylamide intake. French fries had a significant impact on the acrylamide intake, due to the frequent consumption in the canteen. This demonstrates the important responsibility of caterers and canteen kitchens in the mitigation of acrylamide exposure through reduction of acrylamide in their prepared products, in particular in French fries.

Analysis of meat samples for anabolic steroids residues by liquid chromatography/tandem mass spectrometry.

A rapid, specific and highly sensitive multi-residue method for the determination of anabolic steroid residues in bovine, pork and poultry muscle tissues was developed. The sample preparation involves enzymatic digestion followed by extraction with methanol. The crude extract was cleaned up by solid-phase extraction (SPE) combining C18 and NH2 columns. The detection was carried out by a highly sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method using both positive and negative ionization modes. Natural and synthetic steroids covering different polarities could be extracted, concentrated and purified using one single method. Mobile phase composition and additives were optimized to achieve the highest sensitivity. The linearity was not good enough for quantitative analysis but the method was well-suited for qualitative confirmation. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) and detection capabilities (CCbeta) were below 0.5 ng g(-1) for all the compounds in the three types of meat studied. The developed method is suitable for routine analysis in our laboratories.

Comparison of electron and chemical ionization modes by validation of a quantitative gas chromatographic-mass spectrometric assay of new generation antidepressants and their active metabolites in plasma.

A gas chromatographic-mass spectrometric method (GC-MS) for the simultaneous determination of the 'new' antidepressants (mirtazapine, viloxazine, venlafaxine, trazodone, citalopram, mianserin, reboxetine, fluoxetine, fluvoxamine, sertraline, maprotiline, melitracen, paroxetine) and their active metabolites (desmethylmirtazapine, O-desmethylvenlafaxine, m-chlorophenylpiperazine, desmethylcitalopram, didesmethylcitalopram, desmethylmianserin, desmethylfluoxetine, desmethylsertraline, desmethylmaprotiline) in plasma using different ionization modes was developed and validated. Sample preparation consisted of a strong cation exchange mechanism and derivatisation with heptafluorobutyrylimidazole. The GC separation was performed in 24.8 min. Identification and quantification were based on selected ion monitoring in electron (EI) and chemical ionization (CI) modes. Calibration by linear and quadratic regression for electron and chemical ionization, respectively, utilized deuterated internal standards and a weighing factor 1/x(2). Limits of quantitation were established between 5 and 12.5 ng/ml in EI and positive ionization CI (PICI), and 1 and 6.25 ng/ml in negative ionization CI (NICI). During validation stability, sensitivity, precision, accuracy, recovery, and selectivity were evaluated for each ionization mode and were demonstrated to be acceptable for most compounds. While it is clear that not all compounds can be quantitated either due to chromatographic (trazodone) or derivatisation problems (O-desmethylvenlafaxine), this method can quantitate most new antidepressants (ADs) in the therapeutic range using EI. PICI and NICI lead to higher selectivity. Moreover, NICI is of interest for small sample volumes and high sensitivity requirements. This paper draws the attention to the pros and cons of the different ionization modes in the GC-MS analysis of these antidepressants in plasma.

Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of zearalenone and deoxynivalenol.

A multianalyte lateral-flow technique using colloidal gold-labeled monoclonal antibodies was developed for the rapid simultaneous detection of deoxynivalenol (DON) and zearalenone (ZEA). The results of this qualitative one-step test were interpreted visually. A very simple and fast sample preparation was used, and the assay procedure could be accomplished within 10 min. When applied to spiked wheat samples, the technique gave accurate and reproducible results. Cut-off levels of 1500 and 100 microg kg(-1) for DON and ZEA, respectively, were observed. The described multianalyte format can be used as a reliable, rapid and cost-effective on-site screening technique for the simultaneous determination of mycotoxins in grain samples.

Role of water upon the formation of acrylamide in a potato model system.

The moisture sorption isotherms of a commercial potato powder were investigated at 20 degrees C for water activities ranging from 0.11 to 0.97. The sorption isotherms were typical type-II sigmoidal curves, with a steep increase in moisture content for water activities above 0.9 and exhibiting hysteresis over the whole water activity range. On the basis of the isotherms, the influence of the initial water activity and moisture content on both Maillard browning and acrylamide formation was determined by heating oil containing potato powder mixtures in a closed stainless-steel tubular reactor. The Maillard browning, as determined spectrophotometrically, showed an optimum at intermediate water activities. The yields of acrylamide, expressed relatively to the molar amount of asparagine, remained constant below 0.8 aw and below moisture contents of about 20% (on a dry basis). For the more intense heat treatments, an increased acrylamide yield was however observed at higher moisture contents, with an optimum at water contents of about 100% (on a dry basis). However, this increase and optimum was not observed at less intense heat treatments. At moisture contents above 100%, a significant decrease in acrylamide yields was assessed, although the water activity increased only marginally in this area of the sorption isotherms. It was thus observed that the acrylamide content was rather dependent upon the moisture content than upon the water activity in the high-moisture potato powder model system.

Selection criteria for potato tubers to minimize acrylamide formation during frying.

A number of parameters linked to the selection of potato tubers were evaluated with regard to their potential to influence acrylamide formation in French fries. The formation of acrylamide, which is a potential human carcinogen, can be minimized for a big extent by the selection of an appropriate tuber. This study focused on the following selection criteria: variety as influenced by storage time and soil type, underwater weight, and tuber size. A total of 16 varieties were compared, concerning their potential for acrylamide formation. From that survey, certain varieties, such as Tebina and Quincy, could be appointed as unsuitable for frying. The differences in the potential of acrylamide formation between the varieties could mainly be explained by the reducing sugar content of the potato (R2 = 0.82, n = 96). The investigated type of soil and storage time at 8 degrees C appeared to have a minor influence on the acrylamide formation during frying. On the other hand, the tuber size of the potato did contribute in a significant manner to the acrylamide formation. Smaller tubers were more susceptible to acrylamide formation and should be avoided in the frying process. The last selection parameter, the underwater weight, appeared to be of minor importance in the acrylamide formation. On the basis of these simple selection criteria, it is possible to make a first screening of potatoes to reduce the acrylamide formation during frying.

Influence of fertilization on acrylamide formation during frying of potatoes harvested in 2003.

The quality of the potato has been found to vary, when grown under different agricultural and environmental conditions, such as the level of fertilization. Consequently these factors may influence the acrylamide formation during the preparation of French fries. These assumptions were studied on three varieties: Bintje, Ramos, and Saturna from the harvest of 2003. Decreasing N fertilization caused increases in the reducing sugar concentration from 60% up to 100% on DM for all varieties studied. Due to a high correlation between the reducing sugar content and the generation of acrylamide during frying, this resulted in a parallel increase in the acrylamide concentration of the French fries. Thus by lowering the amount of N fertilizer, an increase of 30-65% of the acrylamide generation during frying could be observed. It seems of extreme importance to find an appropriate balance between the level of N fertilizer in order to diminish acrylamide formation but on the other hand to obtain an acceptable tuber and to consider the environmental impact. All results reported should be seen in the perspective of the warm growing season of 2003.

Combination of liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry for the detection of 21 anabolic steroid residues in bovine urine.

For the detection of anabolic steroid residues in bovine urine, a highly sensitive liquid chromatographic/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method was developed using both positive and negative ionization. For four compounds the ESI mode was not sensitive enough and gas chromatographic/mass spectrometric GC/MS detection was therefore still necessary as a complementary method. The sample clean-up consisted of solid-phase extraction (SPE) on a C(18) column followed by enzymatic hydrolysis and a second solid-phase extraction on a combination of a C(18) and a NH(2) column. After this last SPE clean-up, the eluate was split into two equal aliquots. One aliquot was further purified and after derivatization used for GC/MS analysis. The other aliquot was analyzed with LC/MS/MS in both ESI+ and ESI- modes. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) were between 0.16 and 1 ng ml(-1) for the compounds detected with the LC/MS/MS method. The developed method is used in routine analysis in our laboratory.

Development of a solid phase extraction for 13 'new' generation antidepressants and their active metabolites for gas chromatographic-mass spectrometric analysis.

A solid phase extraction procedure (SPE) for 13 'new' antidepressants (venlafaxine, fluoxetine, viloxazine, fluvoxamine, mianserin, mirtazapine, melitracen, reboxetine, citalopram, maprotiline, sertraline, paroxetine and trazodone) together with eight of their metabolites (O-desmethylvenlafaxine, norfluoxetine, desmethylmianserine, desmethylmirtazapine, desmethylcitalopram, didesmethylcitalopram, desmethylsertraline and m-chlorophenylpiperazine) from plasma is optimized using HPLC-DAD as monitoring system. Special attention has been paid to the choice of washing and eluting solvent, resulting in a highly concentrated, clean and moisture free extract, also suitable for GC-MS. A total number of 10 sorbents (apolar, polymeric, ion-exchange and mixed mode) was evaluated. Based on recovery, reproducibility and absence of interfering substances the strong cation exchanger gave the best results. Recoveries were determined at low and high therapeutic and toxic levels and ranged between 70 and 109% for all compounds, except for trazodone (39%).

Liquid chromatographic tandem mass spectrometric determination of five coccidiostats in poultry eggs and feed.

A method is described which permits the quantitative detection of the chemical coccidiostats halofuginone, robenidine, diclazuril, nicarbazin and dimetridazole and its main metabolite 2-hydroxydimetridazole in poultry eggs and feed. Sample preparations were kept very simple and are based upon extraction with an organic solvent. Sample extracts were injected into the liquid chromatography tandem mass spectrometry (LC-MS/MS) system on a C18 column and a gradient elution was performed. Dimetridazole-D3 and diclazuril-bis, a structural analogue of diclazuril, were used as internal standards. Detection was performed on a triple quadrupole mass spectrometer in the selected reaction monitoring mode after ionisation in the positive or negative electrospray ionisation mode. Argon was applied as collision gas for collision induced dissociation. Validation of the methods was performed based on Commission Decision 2002/657/EC [Official Journal of the European Communities L221 (2002) 8].