Development of an ELISA and Immunochromatographic Assay for Tetracycline,Oxytetracycline, and Chlortetracycline Residues in Milk and Honey Based on the Class-Specific Monoclonal Antibody

A sensitive class-specific monoclonal antibody against tetracyclines (TCs) was generated and used to develop an enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic assay for TC, oxytetracycline (OTC), and chlortetracycline (CTC) detection in milk and honey samples. The dynamic range of detection for TC in ELISA was 0.26–2.00 μg L^?1 with an IC₅₀ of 0.72 μg L^?1. The IC₅₀ value of OTC and CTC was 3.2 and 6.4 μg L^?1, respectively. The recovery of TC, OTC, and CTC in milk samples was 82–102, 91–105, and 90–101 %, respectively, and 88–101, 89–101, and 89–95 % in honey samples, respectively. In the immunochromatographic assay, the cutoff values for TC, OTC, and CTC were 15, 15, and 50 μg L^?1 in milk, respectively, and 40, 40, and 40 μg L^?1 in honey, respectively. The results revealed that ELISA and the immunochromatographic assay can be applied for the rapid and sensitive detection of TC, OTC, and CTC in milk and honey samples.     

Development and Validation of a Sensitive Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Screening of Florfenicol and Thiamphenicol in Edible Animal Tissue and Feed

To monitor the illegal use of florfenicol (FF) and thiamphenicol (TAP) in edible animal tissue and feed, a sensitive monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been developed with simple sample preparation and cleanup. The obtained monoclonal antibody (5F4) that has isotype IgG1 showed an IC₅₀ value of 0.21 μg L^?1 for FF and 0.35 μg L^?1 for TAP, respectively. It did not exhibit measurable cross-reactivity with other antibiotics. The limits of detection (LODs) for FF and TAP in a muscle matrix ranged from 0.07 to 0.14 μg kg^?1 and in a feed matrix ranged from 2.9 to 5.2 μg kg^?1. The recoveries were 72.8 to 113.4 % with a coefficient of variation of less than 15 %. Good correlation between the ELISA and HPLC-MS/MS results in the tissues tested demonstrated the reliability of our ic-ELISA. This ELISA is a useful tool for screening FF and TAP in edible animal tissue and feed.     

Rapid Screening of Quinoxaline Antimicrobial Growth Promoters and Their Metabolites in Swine Liver by Indirect Competitive Enzyme-Linked Immunosorbent Assay

Quinoxaline feed additives are antimicrobial growth promoters (AGPs); use three of them is permitted, and two of them are illegally used. It results in residue of quinoxaline AGPs and their metabolites in edible animal tissues, which are potentially harmful to human health. In order to effectively monitor the multiple residues of them in swine liver, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed based on polyclone antibody preparation. Protein conjugates were synthesized and immune to New Zealand white rabbit according to designed schemes. The effective antiserum with 50 % inhibiting concentration (IC₅₀) value of 1.34 μg?L^?1 was obtained. A new synthesized quinoxaline with similar chemical structure to olaquindox but longer spacer arm was used as coating antigen. Cross-reactivity of other four quinoxaline AGPs and their eight metabolites was tested; seven of them have cross-reactivity over 10 %, with IC₅₀ of 0.10–2.50 μg?L^?1. At the spike level of 1 to 100 μg?kg^?1 in swine liver, the recoveries of all compounds ranged from 80.14 % to 96.90 %, with the inter-day variation coefficient (CV) of 5.67–13.82 % and the intra-assay CV of 6.22–14.19 %. The limit of detection ranged from 0.03?±?0.002 to 0.79?±?0.05 μg?L^?1. Positive samples were determined by the ic-ELISA method and successfully confirmed by liquid chromatography-tandem mass spectrometry. The proposed ELISA is feasible for screening quinoxaline AGPs and their metabolites in swine liver.     

Development of an Enzyme-Linked Immunosorbent Assay for the Detection of Difenoconazole Residues in Fruits and Vegetables

An indirect, competitive enzyme-linked immunosorbent assay (ic-ELISA) for the detection of difenoconazole was developed. Two haptens were designed and successfully synthesized. Hapten 1 had a particular moiety of difenoconazole, while hapten 2 had its full structure. The polyclonal antibodies against hapten-protein conjugates were prepared by immunizing rabbits. After optimization of the conditions, the detection limit (IC₁₅) and sensitivity (IC₅₀) were 4.58 and 29.10 μg L^?1, respectively. The cross-reactivities of the antibody with 11 triazole fungicides were all less than 0.1%, which showed that the antibody had excellent specificity. The recoveries of difenoconazole from the spiked samples ranged from 89.70 to 102.31% with good accuracy. The matrix effect was easily removed using a simple, rapid, and efficient extraction method on fruits and vegetables. The detection limit was all 229 μg kg^?1 in fruits and vegetables. To validate the ic-ELISA, samples were spiked with difenoconazole at three different concentrations and simultaneously analyzed using high-performance liquid chromatography (HPLC). The results showed a good correlation between the ic-ELISA and HPLC data (R ² = 0.9970). As a result, the developed immunosorbent assay is suitable for the quantitative determination of difenoconazole in fruits and vegetables.     

Fluorescence Polarization Immunoassay for Highly Efficient Detection of Imidaclothiz in Agricultural Samples

A homogeneous fluorescence polarization immunoassay (FPIA) based on a monoclonal antibody for the detection of imidaclothiz was developed. Two fluorescein-labeled imidaclothiz tracers containing two different bridge lengths were synthesized and purified. Under optimal conditions, the 4-aminofluorescein-labeled imidaclothiz conjugate (AMF-labeled imidaclothiz), which contains a shorter bridge length, showed a higher sensitivity in the FPIA for detecting imidaclothiz, and the full analysis was achieved in less than 11 min. The IC₅₀ and limit of detection (LOD, IC₁₀) were 87.94?±?10.18 and 0.57?±?0.16 μg/L, respectively. The spiked recoveries were 83 to 117 % measured in tomato, pear, rice, apple, cucumber, cabbage, and paddy water, with RSDs of 5 to 12 %. Furthermore, the results of FPIA for the authentic samples correlated well with those acquired by HPLC. Overall, the developed FPIA provided a simple, rapid, sensitive, and accurate method that was used for the quantitative detection of imidaclothiz in agricultural samples.     

Development of an Indirect Competitive ELISA for Analysis of Alternariol in Bread and Bran Samples

Alternariol (AOH) is one of the major mycotoxins produced by various species of Alternaria fungi. Natural occurrences of AOH have been reported in various foods, including fruits; processed fruit products such as apple juice, tomato products; wheat and other grains; oilseeds and products thereof, such as sunflower seeds, oilseed rape meal, and flax seed/linseed; and pecans. In this study, AOH-specific polyclonal antibodies were generated and developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for monitoring AOH in bread and bran samples. The assay was very sensitive with a limit of detection (LOD) of 2.4?±?0.6 ng/g and a half maximal inhibitory concentration (IC₅₀) of 15.2?±?2.6 ng/g in bread and a LOD of 8.4?±?1.2 ng/g and IC₅₀ of 52.8?±?10.8 ng/g in bran extract. The assay was very specific to AOH and showed no cross-reactivity to alternariol monomethyl ether, altertoxin, altenuene, tentoxin, or tenuazonic acid. The effect of organic solvents on the assay was tested. The ELISA system tolerated methanol and acetonitrile as co-solvents at up to 5% content without significant loss of IC₅₀ value. Recoveries in all cases were greater than 75%, and the results using this method were comparable to those obtained from mass spectrometry methods. We conclude that this method is suitable for rapid detection of AOH in bread and bran samples, without expensive analytical equipment or time-consuming sample preparation.     

Development of a Competitive Indirect Enzyme-Linked Immunosorbent Assay for Screening Phenylethanolamine A Residues in Pork Samples

Phenylethanolamine A (PEA), a new alternative β-agonist, has been illegally used in farming to promote the muscle growth in food-producing animals. In this study, a sensitive and convenient competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed for determination of PEA residues in pork samples. The produced antibody was highly specific to PEA and exhibited a negligible cross-reactivity toward some other β-agonists. The developed technique was characterized by the limit of detection below 0.08 μg kg^?1 and the IC₅₀ value of 0.93 pmol mL^?1 (0.32 ng mL^?1). Validation of the technique was done using artificially spiked and naturally contaminated pork samples. The recoveries ranged from 79.6 to 112.6 % for the samples spiked at levels of 0.1–5 μg kg^?1 with the variation coefficients below 15 %. The analysis of naturally contaminated samples showed that the obtained data corresponded with the data obtained by the LC-MS/MS. The developed ciELISA was shown to be a feasible highly sensitive and specific screening tool for PEA residue analysis.     

Preparation of a Broadly Specific Monoclonal Antibody-Based Indirect Competitive ELISA for the Detection of Benzodiazepines in Edible Animal Tissues and Feed

Incorrect use of benzodiazepines could result in serious health problems. To monitor the illegal use of benzodiazepine compounds in animals, a group-specific monoclonal antibody (mAb) was prepared in this study. The obtained 3D7 mAb, which is an IgG1 isotype mAb, displayed an IC₅₀ value of 8.9 ng mL^?1 for diazepam and exhibited cross-reaction for diazepam (100 %), nitrazepam (49 %), nordiazepam (140 %), temazepam (32 %), oxazepam (17 %), estazolam (7.5 %), and alprazolam (2.4 %). Based on this mAb, for the first time in this study, an optimized indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) protocol that did not require complicated sample preparation and clean-up was developed. The detection limits of this ic-ELISA for benzodiazepines ranged from 1.2 to 3.3 μg kg^?1 in muscle matrix and from 25.2 to 55.4 μg kg^?1 in feed matrix. The recoveries ranged from 70.9 to 111.3 % with coefficients of variation below 15.0 %. Good correlations (r?>?0.9494) between the results of the ic-ELISA and high-performance liquid chromatography were also observed. This simple method reduced the time required for sample preparation, ensured greater throughput, and met the requirements for benzodiazepine residue analyses. In conclusion, the proposed method is a sensitive and rapid multi-residue technique that offers a cost-effective alternative to current published procedures without any concession in the ability to detect benzodiazepine sedative misuse.     

Development of a sensitive polyclonal antibody-based competitive indirect ELISA for determination of citrinin in grain-based foods

ABSTRACT

A sensitive competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed for the detection and quantification of citrinin (CIT) in grain-based food samples. The limit of quantification (IC₂₀) of the established method was 0.10 ± 0.02 ng mL^?1, with the limit of detection (IC₁₀) being 0.04 ± 0.007 ng mL^?1 in wheat and corn flour matrices with a coefficient of variation (CV) less than 20%. The assay was very specific to CIT and showed no cross-reactivity with other mycotoxins (OTA, T-2 toxin, HT-2 toxin, DON, patulin and zearalenone). In spiked wheat and corn flours, the recoveries ranged from 86.6% to 115.6% with CVs of less than 20%. The effectiveness of this method was verified by participating in a proficiency test (PT) from the Food Analysis Performance Assessment Scheme (FAPAS) 17181 corn flour. A successful z-score (?0.6) for this PT sample showed that the present method is comparable to the instrumental methods used by other laboratories in the PT testing scheme. A small survey of grain-based foods was conducted using this method and CIT was detected in 43% of the samples up to a concentration of 17.7 ng g^?1. This method is suitable for sensitive and rapid quantitation of citrinin in wheat and corn matrices.     

Deoxynivalenol in wheat,maize, wheat flour and pasta: surveys in Hungary in 2008–2015

Among Fusarium mycotoxins, deoxynivalenol (DON) is the most common contaminant in case of cereals and cereal-based foods in Hungary. In this study, Hungarian wheat (n = 305), maize (n = 108), wheat flour (n = 179) and pasta (n = 226) samples were analysed (N = 818). The samples were collected during 2008–2015 in Hungary. Applied methods of analysis were enzyme-linked immunosorbent assay and liquid-chromatography coupled with a mass spectrometer. Results were compared and evaluated with Hungarian weather data. Among cereal samples, in 2011, wheat was contaminated with DON (overall average ± standard deviation was 2159 ± 2818 µg kg^?1), which was above the maximum limit (ML). In case of wheat flour and pasta, no average values above ML were found during 2008–2015, but higher DON contamination could be observed in 2011 as well (wheat flour: 537 ± 573 µg kg^?1; pasta: 511 ± 175 µg kg^?1).     

Development of a polyclonal antibody-based indirect competitive ELISA for determination of sterigmatocystin in wheat and corn flours

Sterigmatocystin (STC) is a toxic secondary metabolite produced by more than 50 fungal species, including Aspergillus flavus, A. parasiticus, A. nidulans, and A. versicolor. The Joint FAO/WHO Expert Committee on Food Additives concluded that sterigmatocystin is genotoxic and carcinogenic with the critical effect determined to be carcinogenicity. The present study describes a simple method to prepare hapten and immunogens in order to generate polyclonal antibodies against this metabolite. We developed a sensitive and specific polyclonal antibody-based competitive indirect enzyme-linked immunosorbent assay (ciELISA) for monitoring STC in wheat and corn flours without the need for derivatisation of STC or clean-up of samples by immunoaffinity chromatography for quantification. The half inhibitory concentration (IC₅₀) of the established method was 4.52 ± 0.81 ng mL^?1, with the limit of detection (IC₁₀) being 0.19 ± 0.04 ng mL^?1 in wheat and corn flour matrices with the coefficient of variation of less than 22%.The assay was very specific to STC and showed no cross-reactivity with its analogue structures. The ELISA allowed for up to 5% methanol without significant influence on the IC₅₀ value. Validation of the assay was performed by spiking STC into a blank flour matrix and the recoveries were in the range of 75.3 % to 104.5 % with a coefficient of variation less than 15%. A small retail survey was conducted by purchasing wheat (n = 8) and corn flours (n = 2) from local grocery stores. All of these retail samples were negative for STC using the developed ELISA method and were confirmed by LC-MS/MS. We demonstrated a rapid, simple, and reliable method for screening STC in wheat and corn flours.     

Micellar Electrokinetic Chromatography Method for Determination of the Ten Water-Soluble Vitamins in Food Supplements

The separation and determination of the ten water-soluble vitamins by using capillary electrophoresis in the micellar electrokinetic chromatography in a single run are proposed. The method uses low toxicity and cost solvent (ethanol) as modifier of background electrolyte (BGE) attending to the Green Chemistry principles. The electrophoretic method uses 10.0 % (v/v) ethanol, 2.0 % (w/v) SDS, 0.02 mol?L^?1 borate at pH 8.70 as BGE. The standard and real sample solutions were injected in the eletrophoretic system by hydrodynamic injection under pressure of 0.80 psi for 8 s, and the separation was carried out in a fused silica capillary under a potential of 28 kV at 25 °C; the analytical signals were monitored at 214 nm. The analytical method is precise (r.s.d.?<?6 %), accurate (better than 9 %), selective, sensitive, robust, simple, and presents high analytical frequency as ten water-soluble vitamins were separated in only 18 min, with migration times of 5.75?±?0.02, 6.81?±?0.02, 8.13?±?0.04, 8.80?±?0.07, 8.98?±?0.06, 11.10?±?0.08, 11.34?±?0.05, 13.85?±?0.15, 14.82?±?0.04, and 17.85?±?0.30 min. Detection and quantification limits of 0.34, 0.32, 0.27, 0.20, 2.50, 4.98, 4.92, 0.30, 0.86 and 0.28 mg?L^?1 and 1.02, 0.97, 0.83, 0.62, 7.56, 15.09, 14.91, 0.90, 2.59 and 0.83 mg?L^?1, for vitamins PP (nicotinamide), B₁₂ (cyanocobalamin), B₂ (riboflavin), B₆ (pyridoxine), B₈ (biotin), C (ascorbic acid), B₅ (pantothenic acid), B₃ (nicotinic acid), B₁ (thiamine), and B₉ (folic acid), respectively. Excellent recoveries (intra and inter-day) were obtained and, when the method was applied to food supplement analyses the results were in agreement with the conventional HPLC methods.     

A Cassava Starch–Chitosan Edible Coating Enriched with <Emphasis Type="Italic">Lippia sidoides</Emphasis> Cham. Essential Oil and Pomegranate Peel Extract for Preservation of Italian Tomatoes (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.) Stored at Room Temperature

The effect of edible cassava starch–chitosan coatings incorporated with rosemary pepper (Lippia sidoides Cham.) essential oil and pomegranate peel extract on the shelf-life of tomatoes during storage at 25 °C for 12 days was investigated. Sixteen formulations, containing 10 g L^?1 cassava starch and various concentrations of chitosan (5, 10, 20, 30 g L^?1), essential oil (0, 2.5, 5, 10 mL L^?1) and pomegranate peel extract (0, 5, 10, 20 mL L^?1) were prepared and applied to tomatoes. Physical–chemical and microbiological analyses were performed on days 1, 4, 8 and 12. Most of the coatings delayed the ripening of tomatoes, lowering the total soluble solids (38?44 g sucrose kg^?1) and weight loss (93?128 g kg^?1) and maintaining constant firmness compared to the uncoated tomatoes (45 g sucrose kg^?1, 175 g kg^?1) at 12 days of storage. Conversely, except red intensity (a*), which was higher for the uncoated samples, the colour parameters (L*, b*) of the coated and control tomatoes were similar at the end of storage. Uncoated and coated tomatoes showed no contamination during storage. The coatings showed potential to maintain the quality of tomatoes during storage at 25 °C for 12 days. In this context, tomatoes coated with the formulation comprising 10 g L^?1 cassava starch, 10 g L^?1 chitosan, 10 mL L^?1 essential oil and 20 mL L^?1 pomegranate peel extract showed the lowest weight loss and reduced total soluble solids content compared with uncoated ones.     

Physicochemical and sensory characteristics of red wines from the rediscovered autochthonous Tintilia grapevine grown in the Molise region (Italy)

Tintilia is an autochthonous grapevine of the Italian Molise region which risked to disappear. However, recently, the production of Tintilia red wines is resuming and in the year 2011 the protected designation origin ‘Tintilia del Molise’ was officially registered. In this work, an analytical characterization of representative red wines from Tintilia grape is reported. A total of 36 different physicochemical variables were determined and discussed, considering those with an estimated coefficient of variation <25 % as more characterizing. These were found to be (mean): density (0.9949); dry extract (34.4 g L^?1) and ashes (3.8 g L^?1); ethyl alcohol (14.2 mL 100 mL^?1), glycerol (9.2 g L^?1) and total higher alcohols (1.7 g L^?1); pH (3.65); the titratable (5.9 g L^?1), fixed (5.4 g L^?1), and salified (2.5 g L^?1) acidity; buffering capacity (52.6 mM/L/pH); total phenols (2,341 mg mL^?1); total flavonols (223 mg mL^?1) and epicathechin (75.0 mg mL^?1); %Red (49.1 %) and %Yellow (43.6 %). Sensory analysis was also performed by professional wine tasters. Finally, the Tintilia results were compared with those of Montepulciano wines. Findings of this analytical study describe the Tintilia red wine as a full-bodied wine; alcoholic; with feeble, but stable acidic profile; rich of phenols, especially flavonols; and finally, with a color balanced between red and yellow pigments.     

Novel Procedure for Lager Beer Clarification and Stabilization Using Sequential Enzymatic,Centrifugal, Regenerable PVPP and Crossflow Microfiltration Processing

In this work, the crossflow microfiltration (CFMF) performance of different lots of lager beer, produced in a pilot scale at the Italian Brewing Research Centre (CERB, Perugia, Italy), was assessed in a bench-top plant, equipped with a 0.8-μm ceramic tubular membrane module, under constant crossflow velocity of 6 m s^?1, transmembrane pressure difference of 3.74 bar, temperature of ~10 °C, and periodic CO₂ backflushing. By feeding different beer samples (i.e., as such, precentrifuged (C), or pretreated with a commercial enzyme preparation to degrade the original arabinoxylans and β-glucans and then centrifuged (EC) to minimize the fouling contribution of yeast cells, aggregates, and polysaccharides), it was possible to increase the average permeation flux (expressed as mean value?±?standard deviation) from 112?±?13 to 199?±?17 or 330?±?22 L m^?2 h^?1, respectively. Only when using the EC-pretreated beer specimens, the permeate turbidity at 20 °C approached the limiting one (<0.6 EBC unit) recommended by the European Brewery Convention standards. As expected, the permeate chill haze at 0 °C was generally higher than the above haze target. By submitting EC-pretreated beer seeded with 0.5 g L^?1 of regenerable polyvinylpolypyrrolidone (PVPP) to CFMF, it was possible to reduce the initial total polyphenol content by 30 % and permeate chill haze to 0.60?±?0.01 EBC unit, but the average permeation flux fell to 84?±?4 L m^?2 h^?1. By performing sequentially EC pretreatments, PVPP stabilization, cartridge filtration, and CFMF, it was possible not only to re-enhance the average permeation flux at about 230 L m^?2 h^?1 near to those achievable with DE filters, but also to obtain a chill haze-free permeate ready for aseptic packaging.     

Aflatoxin B1 and total fumonisin contamination and their producing fungi in fresh and stored sorghum grain in East Hararghe,Ethiopia

Natural contamination of sorghum grains by aflatoxin B₁ and total fumonisin and their producing toxigenic fungi has been studied. A total of 90 sorghum grain samples were collected from small-scale farmers’ threshing floors and 5–6 months later from underground pits during 2013 harvest from three districts of East Hararghe, Ethiopia. Mycotoxin analysis was done using enzyme-linked immunosorbent assay (ELISA). The limits of detection were in the range 0.01–0.03 μg kg^–1. The results revealed that all sorghum grain samples were contaminated with both Aspergillus and Fusarium species. Aflatoxin B₁ was detected at levels ranging from ?1 grain. There were marked variations in aflatoxin B₁ concentrations between fresh and stored samples, with much higher levels in the latter. Total fumonisin levels varied between 907 and 2041 µg kg^?1 grain across the samples. Lowest total fumonisin was recorded in freshly harvested sorghum grain samples. Sorghum is a main staple cereal in the studied districts and its consumption per day per person is high. Daily intake of low doses of mycotoxin-contaminated food stuff over a period of time could lead to chronic mycotoxicosis.     

Use of Multivariate Optimization to Develop Methods for Direct Copper and Lead Determination in Breast Milk by Graphite Furnace Atomic Absorption Spectrometry

A direct method for lead and copper determination in breast milk by graphite furnace atomic absorption spectrometry, using aqueous calibration, was proposed in this study. Samples were diluted with hydroximethylaminomethane 80 %?v/v, pH 8. The dilution determination for Pb and Cu was 1:1 and 1:9, respectively. Fractional factorial (2^4?1) and central composite designs were used to optimize experimental conditions (pyrolysis and atomization temperatures, pyrolysis time, and modifier) using 10 μL samples introduced into a graphite furnace. The methods allowed for copper and lead determination under optimized conditions with an aqueous calibration curve between 0 and 180 μg L^?1 for Cu and 0 and 48 μg L^?1 for Pb. The detection limits were 0.92 μg L^?1 and 6.4 μg L^?1 for Pb and Cu, respectively. Intra and inter-assay studies revealed coefficients of variation of 5.0 and 6.3 %, and 6.4 and 5.5 % for Pb and Cu, respectively. Recovery studies at three concentration levels (three consecutive days, n?=?7/day) presented results between 107 and 109 % for Pb and 102 and 103 % for Cu. Good accuracy was obtained for both metals through recoveries studies using certified reference material (infant formula NIST® 1846). The method determined lead and copper in six samples and the concentrations ranged from 2.90 to 27.9 μg L^?1 for Pb and 384 to 1,212 μg L^?1 for Cu. While copper is an essential element, lead has no nutritional function and is cumulative at low concentrations. Therefore, safe, efficient, and validated methods should be available to determine its concentration in breast milk.     

Monoclonal-based enzyme-linked immunosorbent assay for the detection of zearalenone in cereals

A monoclonal antibody against zearalenone (ZEA) was produced and used successfully to develop a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) for the analysis of ZEA in cereals. This DC-ELISA had a limit of detection of 0.15?±?0.02 µg l^?1 and an IC₅₀ value of 1.13?±?0.16 µg l^?1. Matrix interference was minimized by dilution of the sample extract before ELISA assays. Aqueous methanol (80%) gave good extraction efficiencies, and the recovery from spiked rice, barley, and corn samples averaged between 87 and 112%. Although ZEA was detected in seven (9%) of 80 rice samples and in eight (16%) of 50 barley samples, the concentration of ZEA in samples was around or below the limit of detection of DC-ELISA. Among 38 corn samples, ZEA was detected in nine (24%) samples in the range 41.0–909.8 µg kg^?1. Re-analysis of the ELISA-positive corn samples by high-performance liquid chromatography (HPLC) confirmed that seven (18%) corn samples were positive. The ZEA results for corn showed very good agreement between DC-ELISA and a commercial AgraQant® zearalenone kit (r ²?=?0.98). Thus, the monoclonal antibody-based DC-ELISA could be applied to the preliminary screening of ZEA contamination when analysis of a large sample number is needed.     

Aflatoxins in composite spices collected from local markets of Karachi,Pakistan

This survey was carried out to evaluate the occurrence of total aflatoxins (AFs; B₁+B₂+G₁+G₂) in unpacked composite spices. A total of 75 samples of composite spices such as biryani, karhai, tikka, nihari and korma masalas were collected from local markets of Karachi, Pakistan, and analysed using HPLC technique. The results indicated that AFs were detected in 77% (n = 58) samples ranging from 0.68 to 25.74 µg kg^?1 with a mean of 4.63 ± 0.95 µg kg^?1. In 88% (n = 66) samples, AFs level was below the maximum limits (ML = 10 µg kg^?1) as imposed by EU. Furthermore, 61% (n = 46) tested samples contained AFs level between 1 and 10 µg kg^?1, 9% (n = 7) exhibited AFs contamination ranged 10?20 µg kg^?1 and only 3% (n = 2) of the investigated samples contained AFs levels higher than the ML of 20 µg kg^?1 for total aflatoxins as set by the USA. It was concluded that there is need to establish a strict and continuous national monitoring plan to improve safety and quality of spices in Pakistan.     

Simple and fast spectrophotometric determination of low levels of thiabendazole residues in fruit and vegetables after pre-concentration with ionic liquid phase microextraction

There is a great importance of monitoring thiabendazole (TBZ) residues in fruits and vegetables to ensure food safety. Therefore, a new ionic liquid (IL) phase microextraction method using IL, 1-butyl-3-methylimidazoliumhexafluorophosphate [C₄mim][PF₆], as extracting solvent is proposed for simple and fast determination of low levels of TBZ in fruits and vegetables by spectrophotometry. The method is based on selective complex formation of TBZ with Cu(II) ions in presence of PF₆^– as counter ion at pH 5.5, and then microextraction of the complex into the fine micro-drops of IL phase. After optimisation of variables affecting microextraction efficiency, the analytical parameters of the method were determined by calibration curves. The method exhibits a linear relationship (0.3–280 μg L^?1), low detection limit (0.1 μg L^?1), good intra- and inter-day precision (2.4–4.5% as RSD_r%, 2.1–5.6% as RSD_R%), good recovery (≥95.1–98.2%) and high sensitivity enhancement factor (150) by solvent-based calibration curve. It allows a detection limit of 0.24 μg L^?1 and a range of 0.8–250 μg L^?1 by the matrix-matched calibration curve. After validation, the method was successfully applied to the determination of TBZ residues with method quantification limits in fruit and vegetables of 2.0 and 2.5 µg kg^?1 with and without adding polyvinylpyrrolidone (PVP-15) solution. Recoveries range from 85.5% to 98.2% after spiking (10, 50 and 100 µg kg^?1, n: 3).