Validation of an enzyme-linked immunosorbent assay for qualitative screening of neomycin in muscle, liver, kidney, eggs and milk.

A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) was used for the qualitative screening analysis of neomycin in food of animal origin (muscle, liver, kidney, eggs and milk) at levels corresponding to the European Union maximum residue limit (MRL) set for this substance. The method validation was performed according to the criteria of Commission Decision 2002/657/EC established for qualitative screening methods. In this regard, the following parameters were determined: detection capability (CCß), specificity, detection limit (LOD), quantification limit (LOQ), recovery, precision, linearity and ruggedness. LODs ranged from 5.7 microg kg(-1) in kidney to 29.3 microg kg(-1) in milk; LOQs ranged from 11.4 microg kg(-1) in kidney to 59.7 microkg(-1) in eggs. The recoveries from spiked samples at the MRL, half the MRL and double the MRL levels ranged from 65.8% to 122.8%, with a coefficient of variation (CV) between 5.9% and 28.6%. The CCß value was less than the MRL for all examined matrices. Moderate variations of some critical factors in the sample pretreatment for muscle, milk and eggs were deliberately introduced for ruggedness evaluation and had a slight but not statistically significant effect on method performance. The proposed method is suitable for qualitative screening analysis of neomycin in the above-mentioned food in conformity with current European Union performance requirements.

Determination of ten sulphonamides in egg by liquid chromatography-tandem mass spectrometry.

A precise and reliable method for the determination of 10 sulphonamide antibiotics (sulfadiazine, sulfathiazole, sulfamerazine, sulfamethazine, sulfamethoxypyridazine, sulfachloropyridazine, sulfamethoxazole, sulfamonomethoxine, sulfadimethoxine and sulfaquinoxaline) in egg by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. Drugs were extracted using a mixture of dichloromethane/acetone (50:50, v/v), acidified with acetic acid and then cleaned-up on a cation-exchange solid-phase extraction (SPE) cartridge. The chromatographic separation was performed by gradient on a C(18) column with a mobile phase of methanol-water containing 0.1% formic acid and 5mM ammonium acetate, then sulphonamides were detected in a triple-quadrupole mass spectrometer operated in positive electrospray ionization mode (ESI(+)). The method was validated at 15, 30 and 45 microgkg(-1). These levels were much lower than the corresponding maximum residue limit of 100 microgkg(-1) set for sulphonamides in several matrices but not in eggs, where the presence of such residues is not permitted. Results were quantitated against the selected internal standard (13)C(6)-sulphamethazine and also according to the matrix-matched approach. The within-laboratory reproducibility, expressed as a relative standard deviation, never exceeded 21%. All decision limit (CCalpha) values lied in the range between 16.1 and 20.5 microgkg(-1) and the corresponding results for detection capability (CCbeta) were 16.9 and 25.7 microgkg(-1). Ruggedness was estimated according to the Youden robustness test.

Modulation of vitellogenin synthesis through estrogen receptor beta-1 in goldfish (Carassius auratus) juveniles exposed to 17-beta estradiol and nonylphenol.

Many synthetic chemicals, termed xenoestrogens, have been shown to interact as agonists with the estrogen receptor (ER) to elicit biological responses similar to those of natural hormones. To date, the regulation of vitellogenesis in oviparous vertebrates has been widely used for evaluation of estrogenic effects. Therefore, Carassius auratus juveniles were chosen as a fish model for studying the effects of estradiol-17beta and different concentrations (10(-6) and 10(-7) M) of 4-nonylphenol (4-NP) on the expression of liver ERbeta-1 subtype; plasma vitellogenin and sex steroids (androgens and estradiol-17beta) were also evaluated together with the bioaccumulation process, through mass-spectrometry. C. auratus is a species widespread in the aquatic environment and, on the toxicological point of view, can be considered a good "sentinel" species. Juveniles of goldfish were maintained in tanks with only tap water or water with different concentrations (10(-6) and 10(-7) M) of 4-nonylphenol (4-NP), or 10(-7) M of estradiol-17beta. After 3 weeks of treatment, animals were anesthetized within 5 min after capture, and blood was immediately collected into heparinized syringes by cardiac puncture and stored at -70 degrees C; the gonads were fixed, then frozen and stored at -70 degrees C; the whole fish, liver, and muscle tissues were harvested and immediately stored at -70 degrees C for molecular biology experiments and bioaccumulation measurements. The estrogenic effects of 4-NP were evidenced by the presence of plasma vitellogenin in juveniles exposed both to estradiol-17beta and the two doses of 4-NP; moreover, exposure to 4-NP also increased aromatization of androgens, as suggested by decreasing androgens and increasing estradiol-17beta plasma levels. The changes of these parameters were in agreement with the increasing transcriptional rate of ERbeta-1 mRNA in the liver, demonstrating that both estradiol-17beta and 4-NP modulate the vitellogenin rate through interaction with the ERbeta-1 subtype. The present study also suggests that 4-NP at the concentration of 10(-6) M bioaccumulates in the liver.

Human exposure to dioxins through diet in Italy.

We have measured the content of polychlorinated dibenzo-p-dioxins, and polychlorinated dibenzofurans (together defined as "dioxins") in 269 samples of food of animal origin collected through the regional veterinary services, covering the national territory. Quantification of the dioxins was accomplished by isotope dilution method, and toxic equivalents (TEQ) were calculated. The average daily food intake was obtained from two main sources: national data collected by the National Institute of Nutrition, and data from an ongoing cohort study on diet and cancer including 40,000 Italian subjects. The mean value of dioxins measured in food of animal origin was 0.144 +/- 0.266 pg-TEQ/g (range: 0.003-1.655 pg-TEQ/g). Fish was the item with the highest content. The estimated intake of dioxins with main food items of animal origin is presented. The major contribution to dioxins intake with food comes from cow milk and fish consumption. These results are in agreement with what observed in studies conducted in other countries, such as Germany, Finland, Japan, Spain, and are below the limits set by the European legislation.

Hygienic quality and freshness of shell eggs collected at retail outlets in Italy.

Shell eggs sampled at retail outlets in two large Italian cities were tested to assess their freshness, food safety and the presence of veterinary drug residues. Some samples were found to be irregular due to lack of compliance with freshness requirements or shells were tainted by micro-cracks and foreign material. The most severe case of non-compliance was due to the presence of veterinary drug residues that either exceeded either the maximum acceptable residue limits or drugs that were prohibited.

Use of histopathological indicators on chub (Leuciscus cephalus) and brown trout (Salmo trutta fario) in evaluating river environments.

A multidisciplinary study was conducted on water from two rivers in the Abruzzo region of Italy. The study highlighted the importance of histopathological investigations in the evaluation of the environmental impact on fish. Brown trout (Salmo trutta fario) from the Aterno river and chub (Leuciscus cephalus) from the Vomano river were sampled in winter and then again in spring. Histopathological investigations of gills, kidneys and livers revealed inflammatory and degenerative lesions, early warning signals of environmental stress. Lesions were evaluated semi-quantitatively and findings were ranked. The histopathological features were compared with results obtained from the analysis of water samples and macroinvertebrates collected in the two rivers (extended biotic index). All results confirmed alterations of the environment.

Dioxin contamination of food in Italy: an overview of the situation 1999-2000.

The Istituto Zooprofilattico dell'Abruzzo e del Molise 'G. Caporale' (IZS A&M) has been monitoring contamination of food by the polychlorinated dibenzodioxins (PCDD) and polychlorinated dibenzo-furans (PCDF) as part of the National Surveillance Plan (NSP) in Italy since 1999, on license from the Italian Ministry of Health. Between 1999 and 2000, 238 samples (including meat, fish, eggs, milk, fat, feedstuffs) were analysed. The results of the tests were expressed in terms of international toxic equivalents (I-TEQs from NATO/CCMS, 1988) and World Health Organization toxic equivalents (WHO-TEQs). These results showed contamination levels comparable to those detected in similar studies conducted in other European countries for products such as milk (mean: 0.81 pg I-TEQ/g fat), meat (mean: 0.73 pg I-TEQ/g fat) and fat (mean: 0.51 pg I-TEQ/g fat). The highest dioxin content was found in fish (mean: 5.28 pg I-TEQ/g fat) and fish feeds (mean 6.60 pg ITEQ/ g fat). These two matrices also showed complete duplication of contamination profiles. Other edible matrices (milk, meat, eggs) revealed the presence of HpCDD and OCDD. This could be due to the introduction into Italy of the animal feed additive choline chloride contaminated by these congeners.

Determination of some sulfonamides and trimethoprim in chicken, fish muscle and eggs by liquid chromatography-tandem mass spectrometry.

Sulfonamides represent a wide range of synthetic compounds commonly used in veterinary therapy for the treatment of several bacterial and protozoan infections in cattle, swine and poultry. Trimethoprim is another antibacterial agent mainly used in fish culture and often combined with sulfonamides in commercial preparations. Residues of these drugs in foodstuffs are of concern because of their potential carcinogenic character. Consequently, the European Union (EU) and United States Food and Drug Administration set maximum residue limits for both sulfonamides (100 microg/kg either as a single molecule or as a sum of all detected compounds within the class) and trimethoprim (TMP) (50 microg and 100 microg/kg, according to the matrix) in chicken, fish muscle and eggs.On the other hand, these limits have made of concern the development of confirmatory methods for the analysis of these molecules. LC-MS/MS technique, in particular, resulted fit for the detection of these medium polarity compounds. An effective multi-residue method is presented for the simultaneous determination of certain sulfonamides (sulfadiazine, sulfathiazole, sulfamerazine, sulfamethazine, sulfamethoxypyridazine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline) and TMP in products of animal origin (chicken muscle, fish muscle and eggs) by liquid chromatographymass spectrometry/mass spectrometry (LC-MS/MS) at levels in compliance with the legislation in force. The drugs were extracted with a mixture of dichloromethane/acetone (1:1, v/v) and clean-up was carried out by solid phase extraction (SPE) on a sulfonic acid column after addition of acetic acid to the extract, so as to allow for ionexchange. Sulfonamides and TMP were then eluted from the SPE column using a solution of ammonia in methanol. The chromatographic separation was performed on a C18 column by using a mobile phase of methanol/5 mM aqueous ammonium acetate and 0.1% formic acid in gradient, and the LC-MS/MS analysis was performed in a triplequadrupole mass spectrometer equipped with a TurboIonSpray source and operated in positive ion mode. The multiple reaction monitoring (MRM) approach was adopted for the identification and quantification of the molecules of concern and was applied by selecting three specific diagnostic ions (one precursor ion and two product ions) for each analyte, so as to meet the criteria set by the EU both for the minimum required number of identification points and for the ion intensity ratio tolerances. Calculated detection limits for sulfonamides (signal/noise ratio 3:1) ranged from 0.1 microg to 1.7 microg/kg, whereas the recovery rates varied between 69.5% and 94.2% throughout the different compounds and matrices. The corresponding values for TMP ranged from 0.2 microg to 0.4 microg/kg and between 51.9% and 52.8%, according to the matrix. The easy sample preparation procedure and the specific and selective mass spectrometric detection make the present method reliable and suitable for the unambiguous identification and quantitation of the analytes taken into account in chicken, fish muscle and eggs. Moreover, method application has successfully extended to other sulfonamides, such as sulfaguanidine, sulfapyridine, sulfamoxole and sulfamethizole.

Determination of rotenone in honey by high performance liquid chromatography and ultraviolet detection.

A method to determine residues of the insecticide rotenone in honey using high-performance liquid chromatography (HPLC) is described. The sample was extracted with an acetone/water mixture, followed by a liquid/liquid partition with diethyl ether. A solid-phase extraction (SPE) clean-up step on alumina neutral cartridge was then performed. The chromatographic analysis was conducted on a C18 column (250 x 4 mm, 5 microm) using acetonitrile-water (65:35, v/v) as mobile phase. Rotenone was detected in the ultraviolet range at a wavelength of 295 nm. The specificity of the method was demonstrated through analyses of raw and commercial honey samples. The limit of detection was equal to 40 microg kg(-1). The precision and accuracy of the method were evaluated trueness honey samples spiked at three concentration levels (100-250-500 microg kg(-1)). The intra-laboratory coefficient of variation (from 9.2 to 10.6%) and mean recovery values (from 81.4 to 86.6%) were satisfactory. The calibration curve was linear in the range 0.125-2 microg ml(-1), with a determination coefficient R2 of 0.9999. Rotenone levels in honey samples from bees treated with this miticide were in the range 120-430 microg kg(-1).