Rapid fluorescence screening assay for enrofloxacin and tetracyclines in chicken muscle

A simple, rapid fluorescence assay was developed for screening both enrofloxacin (ENRO) and tetracyclines in chicken muscle at the U.S. tolerance levels (300 ng/g and 2 microg/g, respectively). Screening for both classes of antibiotics is accomplished using one extraction, thus simplifying and expediting the process. The method requires an initial extraction of chicken muscle with 1% acetic acid in acetonitrile, centrifugation, and analysis of the supernatant for ENRO fluorescence. After addition of ammonium hydroxide, magnesium chloride, and methanol, followed by centrifugation and filtration, the supernatant can be measured for tetracycline fluorescence. Chlortetracycline (CTC) was chosen as a representative tetracycline to demonstrate the method, as it displays intermediate sensitivity among the three tetracyclines approved in the U.S. Comparison of the fluorescence of control and tolerance-level-fortified samples of both ENRO and CTC shows no overlap. Setting a threshold as the average fortified fluorescence minus 3sigma allows for successful screening, as illustrated with blind samples as controls or fortified with ENRO and/or CTC over a range of concentrations. This method can provide an alternative or supplemental approach to currently used microbial screening assays.     

Development of a chemiluminescent ELISA for determining chloramphenicol in chicken muscle

An indirect competitive enzyme-linked immunosorbent assay (ELISA) with chemiluminescent (CL) detection for chloramphenicol (CAP) in chicken muscle was developed. CAP-specific polyclonal antibody was raised in rabbit with a CAP-succinate derivative conjugated with bovine serum albumin. Luminol solution was used as the substrate of horseradish peroxidase. The detection limit was 6 ng/L. The CL-ELISA was 10 times more sensitive compared to the colorimetric-ELISA. When CAP was spiked in chicken muscle at levels of 0.05-5 microg/kg, recoveries ranged from 97 to 118% with coefficients of variation of 6-22%. In an actual residue study, the results obtained by CL-ELISA correlated well with those obtained by gas chromatography with microcell electron capture detector. The residue levels of CAP in treated chicken decreased with time and dropped rapidly after the first 6 h from around 50 to 10 microg/kg. After 3 days, CAP was not detected in chicken muscle. The developed method is therefore suitable for screening of CAP in chicken muscle samples.     

Rapid, low cost thin-layer chromatographic screening method for the detection of ochratoxin A in green coffee at a control level of 10 microg/kg.

A thin-layer chromatographic (TLC) screening method was developed for the detection of ochratoxin A (OTA) in green coffee at a control level of 10 microg/kg. The method is based on extraction of OTA with a mixture of phosphoric acid and dichloromethane, purification by liquid-liquid partition into sodium hydrogen carbonate, separation by normal-phase TLC, and detection by visual estimation of fluorescence intensity under a UV lamp at 366 nm. The method was validated by performing replicate analyses of uncontaminated green coffee material spiked at 3 different levels of OTA (5, 10, and 20 microg/kg), and also by comparing results obtained on a series of test trial green coffees naturally contaminated with OTA (range 0.2 to 136.7 microg/kg) with those measured by a quantitative immunoaffinity/HPLC method. The agreement between the two methods was excellent, and neither false positive nor false negative results were recorded. This screening method is rapid, simple, robust, and very cheap, which makes it particularly well adapted for implementation in coffee-producing countries.     

Time-resolved luminescence screening of antibiotics in tissue matrices without centrifugation and filtration: spiked recovery studies

Analyses of chemical residues in animal tissue matrices require multistep sample preparation. To simplify this process, a methodology was developed that combines sorbent extraction and solid-matrix time-resolved luminescence (TRL); it was applied to tetracycline screening in milk. Reported here is an effort to extend its application to tissue matrices, illustrated by oxytetracycline (OTC) screening in catfish muscle. Extraction and enrichment are accomplished by immersing small C18 sorbent strips into tissue homogenates for 20 min, followed by a 3 min rinse in water and a 2 min dip in a reagent solution. After desiccation, TRL is measured directly on the sorbent surface. Tissue particulates no longer interfere via attenuation or scattering, rendering centrifugation and filtration unnecessary. The integrated TRL intensity shows a linear dependence on OTC concentration in the 0-8 microg/g range (R2 = 0.9992) with a 0.026 microg/g limit of detection. To screen OTC at 2 microg/g, the U.S. regulatory tolerance level, a threshold is established at x2-3sigma2, where x2 and sigma2 are the mean and standard deviation, respectively, of the TRL signals from 15 samples fortified at 2 microg/g. Among 45 blind samples randomly fortified at 0-4 microg/g, 41 were screened correctly and 4 negative samples were presumed positive. This method has the potential to improve throughput and save assay costs by eliminating acids, organic solvents, centrifugation, and filtration.     

Acid-induced phase separation of anionic surfactants for the extraction of 1,4-dichlorobenzene from honey prior to liquid chromatography

The acid-induced liquid-liquid phase separation of anionic surfactants in aqueous solutions and its applicability to cloud point extraction methodology were applied as a tool for the extraction of 1,4-dichlorobenzene (p-DCB) from aqueous samples. p-DCB is extracted into the micelles of sodium dodecane sulfonate (SDSA) in a 4.2 M HCl solution. The micellar phase is separated from the bulk aqueous solution after centrifugation and collected from the surface of the suspension. The micellar extracts are injected into a high-performance liquid chromatographic apparatus and quantified at 225 nm with a reference wavelength of 280 nm. Following the proposed methodology, a preconcentration factor of ca. 160 is achieved (starting from 50 mL solutions) allowing for detection limits at the low microg/L level. Application to honey samples produced detection limits of 2.5 microg/kg with quantification limits of 7.5 microg/kg, while the recoveries of the method ranged from 85% at high concentrations to 95% at lower concentrations of p-DCB. The combined uncertainty of the entire analytical procedure was 4.5% at the concentration level of 30 microg/kg allowing for reliable and reproducible results for the determination of p-DCB at the concentration levels considered as thresholds for EU and U.S. legislation (10 microg/kg).     

Validation of the tetrasensor honey test kit for the screening of tetracyclines in honey

Regarding anti-infectious agents, no maximum residue limits are fixed for honey in the European legislation. Discussions are being conducted in order to set working limits at the European level; for example, for tetracyclines, 20 microg/kg was proposed. The Tetrasensor Honey test kit is a receptor-based assay using dipsticks for a rapid screening (30 min) of honey on the presence of tetracyclines. The test was validated according to Commission Decision 2002/657/EC. The test detects tetracycline, oxytetracycline, chlortetracycline, and doxycycline in honey in a specific and sensitive way. Depending on the type of tetracycline, detection capabilities (CCbeta) between 6 and 12 microg/kg were obtained (4-7 microg/kg for dried dipsticks). The test is rugged and participation with the test in an international ring trial gave compliant results. It can be concluded that the Tetrasensor Honey test kit is a simple and reliable test that can even be used at the production site.     

Determination of florfenicol amine residues in animal edible tissues by an indirect competitive ELISA

Florfenicol (FF) is a broad-spectrum antibiotic used increasingly in aquaculture, livestock, and poultry to treat diseases. To avoid using labor-intensive instrumental methods to detect residues of FF in food and food products, a simple and convenient indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method for florfenicol's major metabolite, florfenicol amine (FFA), was developed using a polyclonal antibody prepared in this study. FFA was covalently attached to carrier protein as immunogen by using the glutaraldehyde method. The antibodies obtained were characterized by an ELISA method and showed excellent specificity and sensitivity with the 50% inhibition values (IC 50) of 3.34 microg/L for FFA in PBS buffer. In the ELISA, sample extractions were performed by ethyl acetate/ammonium hydroxide (90 + 10, v/v) following combined acid hydrolysis of FF and its known metabolites. The limits of detection (LOD) calculated from the analysis of 20 known negative swine muscle, chicken muscle, and fish samples were 3.08, 3.3, and 3.86 microg/kg (mean + 3 SD), respectively. Recoveries of FFA fortified at the levels of 5, 50, 100, and 300 microg/kg ranged from 64.6 to 124.7%, with coefficients of variation of 11.3-25.8% over the range of FFA concentrations studied. Validation of the ELISA method with FFA-fortified swine muscle at the levels of 10, 50, 100, and 200 microg/kg was carried out using GC, resulting in a similar correlation in swine muscle ( r = 0.97). The results suggest that this ELISA is a specific, accurate, and sensitive method, which is suitable for use as a screening method to detect residues of FFA in animal edible tissues.     

Evaluation of fumonisin contamination in cornflakes on the Belgian market by "flow-through" assay screening and LC-MS/MS analyses

A total of 205 cornflake samples collected in Belgian retail stores during 2003-2004 were surveyed for the natural occurrence of fumonisin B1 (FB1), B2 (FB2), and B3 (FB3). These cornflake samples, originating from conventional as well as from organic production, were analyzed using an intralaboratory-validated LC-MS/MS method. Additionally, 90 cornflake samples were subjected to rapid screening using a flow-through enzyme immunoassay method to demonstrate the practicability of a screening test coupled to a validated confirmatory LC-MS/MS method for the management of food safety risks. FB(1) concentrations ranged from not detected (nd) [LOD (FB1) = 20 microg/kg] to 464 microg/kg with mean and median concentrations of respectively 104 +/- 113 and 54 microg/kg. For FB2 and FB3, the concentration ranges varied respectively from nd [LOD (FB2) = 7.5 microg/kg] to 43 microg/kg and from nd [LOD (FB3) = 12.5 microg/kg] to 90 microg/kg. Mean concentrations for FB2 and FB3 were respectively 12 +/- 8 and 21 +/- 15 microg/kg, while the median concentration was 11 microg/kg for FB2 and 19 microg/kg for FB3. From the statistical tests (chi2 and ANOVA model III), it could be concluded that the agricultural practice did not have any significant effect on the fumonisin concentrations but that the variation between different batches was significant (p < 0.0001).     

Development and validation of an optical SPR biosensor assay for tylosin residues in honey

In recent years there has been an increase in the use of tylosin in apiculture as bacterial brood diseases become resistant to oxytetracycline. Confirmatory mass spectrometry based methods have been developed but up until now there has been no complementary screening method available capable of sub 10 microg kg(-1) detection limits. In this paper the development and validation of a screening method using optical biosensor technology is presented. The honey was first dissolved in a phosphate buffer and following solid-phase extraction (SPE) cleanup was analyzed using a Biacore Q instrument. Using the criteria specified in European Commission Decision 2002/657/EC for qualitative screening methods, the detection capability (CCbeta) of the method was determined to be 2.5 microg kg(-)(1). Honey samples containing trace residue levels of tylosin were analyzed by both the biosensor screening method and a LC-MS/MS confirmatory procedure; the results were in good agreement.     

Quantification of imidacloprid uptake in maize crops

The systemic imidacloprid is one of the most used insecticides in the world for field and horticultural crops. This neurotoxicant is often used as seed-dressing, especially for maize, sunflower, and rape. Using a LC/MS/MS technique (LOQ = 1 microg/kg and LOD = 0.1 microg/kg), the presence of imidacloprid has been measured in maize from field samples at the time of pollen shed, from less than 0.1 microg/kg up to 33.6 microg/kg. Numerous random samples were collected throughout France from 2000 to 2003. The average levels of imidacloprid measured are 4.1 microg/kg in stems and leaves, 6.6 microg/kg in male flowers (panicles), and 2.1 microg/kg in pollen. These values are similar to those found previously in sunflower and rape. These results permit evaluation of the risk to honeybees by using the PEC/PNEC ratios (probable exposition concentrations/predicted no effect concentration). PEC/PNEC risk ratios were determined and ranged between 500 and 600 for honeybees foraging on maize treated with imidacloprid by seed dressing. Such a high risk factor can be related to one of the main causes of honeybee colony losses.     

Occurrence of ochratoxin A in cocoa products and chocolate

In this work, the occurrence of ochratoxin A (OTA) in 170 samples of cocoa products of different geographical origins was studied. An immunoaffinity column with HPLC separation was developed to quantify low levels of OTA in cocoa bean, cocoa cake, cocoa mass, cocoa nib, cocoa powder, cocoa shell, cocoa butter, chocolate, and chocolate cream with >80% recoveries. The method was validated by performing replicate analyses of uncontaminated cocoa material spiked at three different levels of OTA (1, 2, and 5 microg/kg). The data obtained were related on the acceptable safe daily exposure for OTA. The highest levels of OTA were detected in roasted cocoa shell and cocoa cake (0.1-23.1 microg/kg) and only at minor levels in the other cocoa products. Twenty-six cocoa and chocolate samples were free from detectable OTA (<0.10 microg/kg). In roasted cocoa powder 38.7% of the samples analyzed contained OTA at levels ranging from 0.1 to 2 microg/kg, and 54.8% was contaminated at >2 microg/kg (and 12 samples at >3 microg/kg). Ochratoxin A was detected in cocoa bean at levels from 0.1 to 3.5 microg/kg, the mean concentration being 0.45 microg/kg; only one sample exceeded 2 microg/kg (4.7%). In contrast, 51.2% of cocoa cake samples contained OTA at levels > or =2 microg/kg, among which 16 exceeded 5 microg/kg (range of 5-9 microg/kg). These results indicate that roasted cocoa powder is not a major source of OTA in the diet.     

Residue analysis of tetracyclines and their metabolites in eggs and in the environment by HPLC coupled with a microbiological assay and tandem mass spectrometry

Tetracyclines are widely used in farm animals. This can cause drug residues in products of animal origin and, after excretion of these substances, in animal slurry and in soil fertilized with that slurry. In this paper, we present a method based on a microbiological assay coupled with HPLC for the detection of oxytetracycline, tetracycline, and chlortetracycline in eggs. After a simple liquid extraction of the samples and HPLC separation, fractions were collected on microtiter plates, and the tetracyclines were analyzed using the Staphylococcus aureus assay. This method was able to identify residues of tetracyclines in eggs at a level set by regulatory agencies (i.e., 200 microg/kg). In addition, it was shown that the described microbiological method can be used as a screening assay for the detection of tetracyclines and possible biologically active metabolites in animal slurry and soil samples. Employing the same extraction procedure, it was demonstrated that LC-MS-MS allowed the quantification of 20-400 microg/kg in eggs with recoveries ranging from 71 to 109% and RSDs of 3-15%.     

Liquid chromatographic determination of chloramphenicol residues in meat: interlaboratory study

A liquid chromatographic (LC) method for the determination of chloramphenicol (CAP) residues in meat at the 10 microgram/kg level was tested in an interlaboratory study. The method used, based on aqueous extraction and sample cleanup with a cartridge containing Extrelut, was published earlier. A prestudy to familiarize collaborators with the method was performed before the actual interlaboratory precision study. The meat samples used in the precision study were prepared by diluting dosed chicken and pig muscle tissues with blank tissues from other species. Fourteen laboratories received 20 meat samples; 13 laboratories actually participated in the study. Two blank samples and 2 positive samples each of pig, calf, chicken, lamb, and cow meat were tested. The chloramphenicol concentrations in the positive samples ranged from 6.5 to 21 micrograms/kg. The overall mean reproducibility coefficient of variation was 17.9% after the results per laboratory were corrected for the mean recovery obtained within each sample series. The overall mean recovery was 55.1% with a coefficient of variation of 18.0% at the 10 micrograms/kg level. The limit of detection, based on chromatograms of blank samples, was estimated to be 1.5 micrograms/kg of chloramphenicol. No false positives or false negatives were observed in the concentration range tested; only 2 false positive results above the detection limit (1.7 and 6 micrograms/kg) on a total number of 60 blank analyses (3.3%) were observed.     

Development of an indirect competitive ELISA for the detection of furazolidone marker residue in animal edible tissues

Due to its carcinogenicity and mutagenicity, furazolidone has been prohibited completely from being used in food animal production in the world since 1995. To monitor the illegal abuse of furazolidone, a polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the determination of tissue-bound furazolidone metabolite 3-amino-2-oxazolidone (AOZ). The highly specific antibody was targeted for PAOZ, the benzaldehyde derivative of AOZ. The 50% inhibition values (IC 50) of 0.91 microg/L for AOZ was achieved with the most sensitive antibody Ab-B1 by altering ELISA conditions. In the ELISA, sample extraction and cleanup were performed by an is MAX cartridge following combined hydrolysis of the tissue-bound AOZ and derivatization of the homogenized tissues with benzaldehyde. The limits of detection (LOD) calculated from the analysis of 20 known negative tissue samples (swine liver, swine muscle, chicken liver, chicken muscle,and fish muscle) were 0.3-0.4 microg/kg (mean+3 SD). Recoveries of AOZ fortified at the levels of 0.4, 1, and 5 microg/kg ranged from 55.8 to 96.6% in the tissues. The coefficients of variation were less than 20% over the range of AOZ concentrations studied. The linear detection range was between 0.1 and 25.6 microg/L. Validation of the ELISA method with swine muscle and liver from furazolidone-treated pigs was carried out using HPLC, resulting in a similar correlation in swine muscle (r=0.99) and in swine liver (r=0.98). The results suggest that this ELISA is a specific, accurate, and sensitive method of detecting AOZ residues in animal edible tissues.     

Occurrence of zearalenone-4-beta-D-glucopyranoside in wheat

An LC-MS method was developed for the analysis of zearalenone-4-beta-D-glucopyranoside and zearalenone in wheat (Triticum aestivum). The limit of determination for zearalenone-4-beta-D-glucopyranoside and zearalenone was 10 microg/kg. The recovery rates were calculated to be 69% and 89% at a concentration of 100 microg/kg for zearalenone-4-beta-D-glucopyranoside and zearalenone, respectively. Twenty-four Bavarian wheat samples from a 1999 harvest were analyzed. Zearalenone was present in 22 of 24 field samples, the levels ranged from 11 to 860 microg/kg. Zearalenone-4-beta-D-glucopyranoside was found in 10 of the zearalenone positive samples (42%) at levels ranging from 17 to 104 microg/kg. The amounts of zearalenone-4-beta-D-glucopyranoside were correlated to those of zearalenone (r2 = 0.86, b = 0.10). After gastrointestinal hydrolyzation, zearalenone-4-beta-glucopyranoside might be implicated in the development of a zearalenone-syndrome. Therefore, more attention should be focused on conjugated mycotoxins in food and feed.     

Levels of benzo[a]pyrene (BaP) in "mozzarella di bufala campana" cheese smoked according to different procedures

The content of benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon, was determined by HPLC-FL in "mozzarella di bufala campana" cheese, a stretched cooked cheese, either experimentally smoked according to traditional procedures, using straw, cardboard, and wood shavings or aromatized with smoke flavoring. The BaP residues, researched also in cheese samples sold at retail, were detected in the rind, in the core, and in the slice (outer and inner parts). In the cheeses experimentally smoked with straw and cardboard the BaP levels, ranging from 0.38 to 2.12 microg kg(-1) and from 0.46 to 2.40 microg kg(-1), respectively, were statistically higher than those of the cheeses smoked with wood shavings and aromatized with liquid smoke (from 0.19 to 0.80 microg kg(-1) and from 0.18 to 0.50 microg kg(-1), respectively). However the cheeses treated with liquid smoke flavor showed a BaP content exceeding the level allowed by the European Union. In the samples sold at retail, smoked with straw, values were lower than those obtained from samples smoked experimentally with the same combustible. This is probably due to different smoking technologies among the several provinces of the Protected Designation of Origin (PDO) area. PDO is a term used to characterize foodstuffs produced and prepared in a given geographical region by the means of a recognized process. A standardization of the traditional smoking procedures and an improvement of liquid smoke purification treatments are recommended for mozzarella cheese.     

Improved Mass Screening of Tolerance to Iron Toxicity in Rice by Lowering Temperature of Culture Solution

Breeding for tolerance to iron (Fe) toxicity in rice (Oryza sativa L.) is hindered by lack of a suitable screening technique. In the culture solution methods used to date, a major difficulty has been maintaining an excess level of iron concentration in order to reveal toxicity symptoms. Experimental results showed that this problem was solved by lowering the solution temperature to around 20°C, at which leaf discoloration in susceptible cultivars became more pronounced. At 20°C Fe uptake was increased from a threshhold content of 300 mg/kg of dry weight of shoot for toxic symptom to more than 1000mg/kg in susceptible and in tolerant cultivars. Concentrations of other related minerals in the plant tissue, i.e., potassium (K) and phosphorus (P), were not affected by the low solution temperature itself but by Fe content, which can be inferred on the basis of their response curves to the excess Fe treatment. Using the proposed screening method makes it possible to obtain reproducible results in screening a large number of plants or breeding lines.     

Production of polyclonal antibodies against ochratoxin A and its detection in chilies by ELISA

Polyclonal antibodies were produced for Ochratoxin A (OA) by injecting OA-bovine serum albumin (BSA) conjugate subcutaneously at multiple sites into a New Zealand White inbred rabbit. Antiserum could be used at a dilution exceeding 1:100 000 in an indirect competitive enzyme-linked immunosorbent assay (ELISA), and detected OA concentrations up to 0.1 ng/mL. The 50% inhibition binding (I(50)) of OA was 5 ng/mL. Antibodies did not react with ochratoxin B, coumarin, 4-hydroxycoumarin, L-phenylalanine, and aflatoxin B1. OA contamination in chilies (Capsicum annum L.) collected from commercial markets and cold storage units was determined. The mean recoveries from OA-free chilies spiked with 1 to100 microg of OA per kg of chili sample were 90-110% with a standard deviation of <10%. Of 100 chili samples tested, 26 were found to contain over 10 microg/kg of OA. In 12 samples the OA concentration varied from 10 to 30 microg/kg, in 10 samples from 30 to 50 microg/kg, in 3 samples from 50 to100 microg/kg, and in one sample it was 120 microg/kg. This is the first record in India of OA in chilies, a major component of cooked foods in this country, and it is noteworthy that OA contamination exceeded the permissible limit for human consumption of less than 20 microg/kg in over 26% of the market samples tested.     

Analysis of four 5-nitroimidazoles and their corresponding hydroxylated metabolites in egg, processed egg, and chicken meat by isotope dilution liquid chromatography tandem mass spectrometry

An isotope dilution liquid chromatography-electrospray ionization-tandem mass spectrometry method is presented for the simultaneous analysis of several 5-nitroimidazole-based veterinary drugs, which are dimetridazole (DMZ), ronidazole (RNZ), metronidazole (MNZ), ipronidazole (IPZ), and their hydroxylated metabolites (DMZOH, MNZOH, and IPZOH), in egg (fresh egg, whole egg powder, and egg yolk powder) and chicken meat. Data acquisition was achieved by applying multiple reaction monitoring, and quantitation was performed by means of five deuterated internal standards (ISs), namely, DMZ-d3, RNZ-d3, IPZ-d3, DMZOH-d3, and IPZOH-d3, whereas MNZ and MNZOH were quantitated using DMZOH-d3. At the lowest fortification levels (i.e., 0.5 microg/kg for fresh egg and chicken meat and 1.0 microg/kg for other egg-based matrices) and for compounds having their own corresponding deuterated analogue used as an IS, acceptable performance data were obtained (corrected recoveries, 88-111%; decision limits, 0.07-0.36 microg/kg; detection capabilities, 0.11-0.60 microg/kg; and within-lab precision, < or = 15%). The method failed to give acceptable quantitative results for MNZ and MNZOH due to the unavailability of the corresponding deuterated ISs. Nevertheless, a reliable identification of these two analytes at levels < or = 1 microg/kg was still feasible.     

Do fumonisin mycotoxins occur in wheat?

The fumonisin mycotoxins are mainly produced by the fungi Fusarium verticillioides and Fusarium proliferatum, which are both field pathogens of maize. The natural occurrence of fumonisins has been verified in maize and a large range of maize-based products in many countries of the world. However, occasional reports have emerged of fumonisins being detected in wheat, despite the main producing fungi not being pathogens of this cereal. An investigation was conducted into a recent report of the natural occurrence of fumonisins in the 2003/2004 South African wheat crop at levels up to 1.7 mg/kg, as determined by immunoaffinity column cleanup and direct fluorometric measurement. An AOAC International high-performance liquid chromatographic (HPLC) method for the determination of fumonisins in maize was modified and validated for the determination of fumonisins in spiked wheat samples. HPLC analysis of the wheat samples previously found to be positive for fumonisins revealed no detectable (<5 microg/kg) fumonisins in the 30 samples analyzed. These results, which lay doubt on previous reports of fumonisins in wheat, emphasize the fact that screening methods, especially if used outside their range or matrix of applicability, can produce false positive results despite the use of immunoaffinity cleanup. Such results should be validated and confirmed with a more definitive technique.