Simultaneous determination of (fluoro)quinolone antibiotics in kidney, marine products, eggs, and muscle by enzyme-linked immunosorbent assay (ELISA)

A direct competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect a broad range of (fluoro)quinolones in various matrices. In the optimized generic test, anti-sarafloxacin antibodies in combination with norfloxacin conjugate showed 50% binding inhibition at 0.21 ng mL(-)(1) for sarafloxacin in buffer. Screening for this class of antibiotics is accomplished using a simple, rapid extraction carried out with a 1:1 mixture of methanol and phosphate-buffered saline adjusted to pH 7.4. This common extraction was able to detect 15 (fluoro)quinolone residues such as sarafloxacin, norfloxacin, difloxacin, ciprofloxacin, pefloxacin, ofloxacin, cinoxacin, danofloxacin, enrofloxacin, marbofloxacin, lomefloxacin, enoxacin, flumequine, oxolinic acid, and nalidixic acid in pig kidney, poultry muscle, egg, fish, and shrimp. The assay's detection capabilities (CCbeta) for most of these compounds were <10 microg kg(-)(1) except for the sarafloxacin-, oxolinic acid-, flumequine-, and cinoxacin-spiked matrices, the estimated CCbeta values of which were <4, <25, <100, and <200 microg kg(-)(1), respectively.     

Development of a monoclonal antibody-based cELISA for the analysis of sulfadimethoxine. 2. Evaluation of rapid extraction methods and implications for the analysis of incurred residues in chicken liver tissue

Several rapid extraction methods were evaluated for use with a monoclonal antibody-based competitive inhibition ELISA (cELISA) to detect sulfadimethoxine (SDM) in chicken liver tissue. These methods included extraction of the samples with (1) aqueous buffer with or without ultrafiltration, (2) acetonitrile/water, (3) methanol/water, or (4) acetone. The organic extraction methods were evaluated with or without solvent evaporation prior to dilution into assay buffer for the cELISA. The aqueous-based extraction methods were compatible with the cELISA. However, of the organic extraction methods, only the acetone liver extract with solvent evaporation prior to analysis was compatible with the cELISA. The cELISA method coupled to aqueous- or acetone-based sample extraction as well as an HPLC method was evaluated for the analysis of chicken liver tissues fortified with SDM at levels from 0.2 to 0.025 ppm. Mean SDM recoveries for the HPLC method and for the cELISA method using samples prepared by aqueous extraction, aqueous extraction and ultrafiltration, or acetone extraction, evaporation, and reconstitution were 68.9, 95.7, 60.1, and 52.5%, respectively. For the analysis of samples obtained from an SDM incurred residue study, HPLC and cELISA analysis of the same organic extract gave results that were highly correlated (R(2) = 0.976; p < 0.0001). However, results obtained from the analysis of aqueous extracts by cELISA did not correlate well with those obtained by HPLC (R(2) = 0.61, p > 0. 0006). This was attributed to the coextraction of cross-reactive SDM-related residues that were not quantified by the HPLC method. The presence of these residues should be considered during data interpretation when ELISA methods coupled with rapid aqueous extraction of samples are used in SDM residue monitoring programs.     

Evaluation and validation of a commercially available enzyme-linked immunosorbent assay for the neonicotinoid insecticide imidacloprid in agricultural samples

The performance of a commercially available microtiter plate ELISA kit for the determination of the neonicotinoid insecticide imidacloprid was evaluated for sensitivity, selectivity, influence of organic solvent used for extraction procedure, matrix interference originated from agricultural sample, accuracy, and method comparison with conventional HPLC analysis. The limit of detection for the kit (0.1 or 0.5 ng/mL) was determined. The working range (1-39 ng/mL) experimentally calculated on the basis of a criterion, which is determined as the range from I(20) to I(80), was comparable to that established by the manufacturer (1-50 ng/mL). The linearity of the standard curve based on the kit-assembled standard solutions agreed with the one based on the self-made standard solutions. Specificity studies indicate that the imidacloprid monoclonal antibody can readily distinguish the target compound from other structurally related neonicotinoid analogues and some metabolites, with the exception of clothianidin, the cross-reactivity of which was approximately 12%. To extract imidacloprid from an agricultural sample (apple) as simply and rapidly as possible, some extraction methods were examined. Consequently, the extraction method with hand-shaking for 5 min was the best among the examined methods. For the analysis of imidacloprid in apple samples, it was extracted directly with methanol and the extracts were diluted 10-fold (100-fold in the well) with water prior to ELISA analysis. No significant matrix interference was observed with the dilution factor. Recoveries of imidacloprid from fortified apple samples ranged from 87.7 to 112.0%. The results obtained with the ELISA kit correlated well with those by the reference method (conventional HPLC analysis) for apple samples (r > 0.998). These findings strongly indicate that the ELISA kit may be employed routinely for an on-site imidacloprid residue analysis of apple samples.     

Sensitive streptavidin-biotin enzyme-linked immunosorbent assay for rapid screening of chloramphenicol residues in swine muscle tissue

A sensitive, competitive enzyme-linked immunosorbent assay (ELISA) for chloramphenicol (CAP) in swine muscle tissue has been developed. The ELISA is based on an earlier procedure. To improve sensitivity, different optimization procedures were investigated. The introduction of a streptavidin-biotin system and the use of a coating antigen with a lower CAP incorporation resulted in the most sensitive ELISA: the CAP concentration giving 50% inhibition decreased from 125 ng/mL to 3.0 ng/mL. This ELISA procedure was applied for a rapid screening of CAP residues in swine muscle tissue. The tissues were extracted with demineralized water. A concentrated phosphate-buffered saline solution was added to the filtered aqueous extract and this sample solution was directly submitted to the ELISA procedure. The results were compared to values obtained by analysis of a corresponding blank. This blank was prepared by treating a part of the aqueous sample solution with an immobilized monoclonal antibody preparation. This treatment was necessary because aqueous extracts of different swine muscle tissues showed a high variation in dose-response curves, probably caused by the complexity and variability of the matrix. In spiked tissues, the presence of CAP at concentrations of 10 micrograms/kg and higher can be easily demonstrated.     

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.     

Determination of spinosad and its metabolites in food and environmental matrices. 3. Immunoassay methods

Spinosad is an insect control agent that is derived from a naturally occurring soil bacterium and is effective on several classes of insects, especially Lepidopteran larvae. Spinosad is registered in many countries for use on a variety of crops, including cotton, corn, soybeans, fruits, and vegetables. Residue methods utilizing a magnetic particle-based immunoassay (IA) test kit have been developed and validated for determining spinosad in environmental and food matrices. These methods involve an extraction of the residues from the matrices with appropriate solvents. For some matrices, the sample extracts can be diluted and measured directly by IA without any cleanup. For other matrices, sample extracts are purified using liquid-liquid partitioning and/or solid phase extraction prior to measurement by IA. The methods determine the total residue of spinosad, which includes the active ingredients (spinosyns A and D) and several minor metabolites, including spinosyn B, spinosyn K, and N-demethylspinosyn D. The methods have validated limits of quantitation of 0.0001 microgram/mL in water, 0.05 microgram/g in sediment, and 0.010 microgram/g in crops, crop processed commodities, and animal tissues. This paper briefly summarizes the residue methodology and method validation data for spinosad in 34 food, feed, and environmental matrices.     

Monitoring of five sulfonamide antibacterial residues in milk by in-tube solid-phase microextraction coupled to high-performance liquid chromatography

A simple, rapid, and sensitive method for the quantitative monitoring of five sulfonamide antibacterial residues in milk was developed by coupling in-tube solid-phase microextraction (SPME) to high-performance liquid chromatography with an ultraviolet detector. A poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column was selected as the extraction medium for this on-line technique. To obtain optimum extraction efficiency, several parameters relating to in-tube SPME were investigated. By simple extraction with ethanol, dilution with phosphate buffer solution, and centrifugation, the sample solution then could be directly injected into the device for extraction. The calculated detection limits for sulfadiazine, sulfamethazine, sulfamethoxazole, sulfamonomethoxine sodium, and sulfacetamide sodium were 2.0, 2.8, 1.7, 2.5, and 22 ng/mL, respectively. The method was linear over the range of 20-5000 ng/mL (100-5000 ng/mL for sulfacetamide sodium) with a correlation coefficient R (2) value >0.9980. Excellent method reproducibility was found by intra- and interbatch precisions, yielding the relative standard deviations of <10.0 and <9.94%, respectively. The proposed method was proved to be robust in monitoring sulfadiazine, sulfamethazine, sulfamethoxazole, sulfamonomethoxine sodium, and sulfacetamide sodium residues in milk.     

Enzyme-linked immunosorbent assay for microcystins in blue-green algal blooms

A direct competitive enzyme-linked immunosorbent assay (ELISA) for the freshwater blue-green algal toxin microcystin (MCYST) in algae and water was developed. The assay involves coating anti-MCYST-variant leucine-arginine (LR) antibody to the ELISA plate and the use of MCYST-LR-peroxidase as the enzyme marker. The linear portion of the standard curve for MCYST in phosphate buffer containing saline (PBS) was 0.5-10.0 ng/mL (25-500 pg/assay). The minimum detection level for MCYST-LR was 0.20 ng/mL (10 pg/assay). Contaminated water could be directly used in the ELISA. The overall analytical recoveries for MCYST-LR added to water at levels of 1-20 ng/mL was 83.4%. For analysis of cellular MCYST, the toxin was first extracted from the algae with 0.1M ammonium bicarbonate, diluted with PBS to less than 0.5 mg dried algae/mL (less than 5.0 mg wet weight/mL) and directly used in the ELISA. C-18 reverse-phase Sep-Pak cartridges effectively adsorbed MCYST from the toxin-containing solutions. The toxin could be recovered from the cartridge by eluting with 60% methanol. Using this approach, an algae extract that was relatively free of MCYST was prepared and was used in a recovery study. The overall analytical recovery of MCYST added to the algae extract in the range of 0.25-20 ppm was 83% with a coefficient of variation of 11.9%. The detection limit for MCYST in dried algae was about 0.25-0.5 microgram/g (0.25-0.5 ppm) lyophilized algae sample. This method was applied for the analysis of several naturally occurring algal blooms. Limited samples were also analyzed for MYCST by liquid chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Monoclonal antibody-based enzyme linked immunosorbent assay of aflatoxin B1, T-2 toxin, and ochratoxin A in barley

Aflatoxin B1 (B1), T-2 toxin (T2), and ochratoxin A (OA) were assayed in a single extract from barley grain by using competitive enzyme linked immunosorbent assays (ELISAs) with monoclonal antibodies. B1 and T2 monoclonal antibodies were conjugated to horseradish peroxidase for direct competitive ELISA while an indirect competitive ELISA was used for OA determination. The competitive ELISA detected 0.1 ng/mL of B1, 10 ng/mL of T2, or 1 ng/mL of OA. Acetonitrile-0.5% KCl-6% H2SO4 (89 + 10 + 1) extracts of barley grain either were diluted 1:10 for direct assay or were subjected to a simple liquid-liquid cleanup procedure to concentrate the extract 10:1 before assay. For cleanup, water was added to the acetonitrile extract to partition water-soluble interfering substances, and then the mycotoxins were re-extracted with chloroform. The chloroform extract was evaporated to dryness and redissolved in Tris HCl buffer for ELISA. The mean recoveries from barley spiked with 4-60 ng/g of B1, 50-5000 ng/g of T2, and 5-500 ng/g of OA were, respectively, 93.8, 80.6, and 95.8%. The mean within-assay, inter-assay, and subsample coefficients of variation by ELISA of barley grain colonized with toxigenic fungi were less than 12% for B1 and OA but as high as 17% for T2.     

吉林省畜禽粪便自然堆放条件下粪便/土壤体系中Cu、Zn的分布规律

本文通过对不同养殖场鸡、猪、牛粪便自然堆放条件下粪便和土壤样品的采集和实验室分析,研究了吉林省部分养殖场不同类型畜禽粪便Cu、Zn含量,以及粪便自然堆放对土壤Cu、Zn含量的影响。结果表明,鸡粪、猪粪和牛粪中Cu的含量范围和均值分别为7.12～26.04mg·kg-（1风干样,下同）（均值18.24mg·kg-1）、87.99～463.7mg·kg-（1均值243.6mg·kg-1）、3.95～27.63mg·kg-（1均值12.28mg·kg-1）;Zn的含量范围和均值分别为179.2～340.8mg·kg-（1均值276.9mg·kg-1）、140.5～455.8mg·kg-（1均值381.8mg·kg-1）、150.5～292.3mg·kg-（1均值188.1mg·kg-1）。不同类型粪便中Cu、Zn含量均是猪粪〉鸡粪〉牛粪,但不论是哪种类型的畜禽粪便重金属含量均是Zn〉Cu。在自然堆放条件下,鸡粪和猪粪中的Cu可从粪便向底土迁移,迁移量随粪便中Cu含量的增加而增加,并主要积累在土壤表层,而牛粪在自然堆放过程中对底土的Cu含量影响不显著,粪底土Cu含量分布规律为猪粪底土〉鸡粪底土〉牛粪底土;鸡粪、猪粪和牛粪中的Zn也可向底土迁移,使粪底土Zn含量有不同程度的增加,但主要积累在粪底土的表层,且不同类型粪便底土Zn含量差异不明显。     

Determination of chlorinated pesticide residues in foods. II. Simultaneous analysis of chlorinated pesticide and phthalate ester residues by using AgNO3-coated Florisil column chromatography for cleanup of various samples.

A simplified method suitable for simultaneous analysis of chlorinated pesticide and phthalate ester residues in various foods was developed. Chemical residues were quantitatively extracted from fatty and vegetable samples with acetonitrile as follows: Chemical standard in 0.5 mL ethanol solution was added to 10 g homogenized sample. After 3 hr, pork and beef were extracted 3 times with 20 mL portions of acetonitrile. The acetonitrile layers were diluted with water and extracted with n-hexane. Rice samples were combined with 10 mL water, 5 mL acetonitrile and 1 mL ethanol and extracted 3 times with 20 mL portions of n-hexane. The n-hexane concentrate from each sample was submitted to AgNO3-coated Florisil column chromatography. The AgNO3 coating adequately adsorbed interfering coextractives. Extracts of fish and vegetable samples were separated into 2 fractions by the above column chromatography. Supplemental cleanup procedures were also developed to accurately determine phthalate esters eluted in the second fraction. Satisfactory gas chromatograms were obtained for most samples.     

Immunochemical assays for direct sulfonamide antibiotic detection in milk and hair samples using antibody derivatized magnetic nanoparticles

Two direct enzyme-linked immunosorbent assays (ELISAs) have been developed for detection of sulfonamide antibiotic residues in milk samples. One of them is using magnetic nanoparticles (MNP) for target capture/enrichment (Ab-MNP-ELISA), and the second is performed using microtiter plates. Selective polyclonal antibodies, raised against 5-[6-(4-amino-benzenesulfonylamino)-pyridin-3-yl]-2-methyl-pentanoic acid (SA1), used in combination with an enzyme tracer prepared with the same hapten, has allowed us to reach a limit of detection (LOD) lower than 0.5 microg L(-1) for both ELISA formats. Sulfapyridine, sulfamethoxypyridazine, sulfathiazole, and sulfachloropyridazine are detected below the maximum residue limits established by the European Union for these antibiotics in milk (100 microg L(-1)). Matrix effects and accuracy studies performed with full-cream milk and hair extracts indicated a lack of interference from these sample matrices and very good recovery values, especially when using the Ab-MNP format. Milk samples and hair extracts can be measured without any previous treatment. The results demonstrate the high potential of these methods as screening tools for food safety and inspection controls.     

Rapid sample preparation method for LC-MS/MS or GC-MS analysis of acrylamide in various food matrices

A fast and easy sample preparation procedure for analysis of acrylamide in various food matrices was developed and optimized. In its first step, deuterated acrylamide internal standard is added to 1 g of homogenized sample together with 5 mL of hexane, 10 mL of water, 10 mL of acetonitrile, 4 g of MgSO4, and 0.5 g of NaCl. Water facilitates the extraction of acrylamide; hexane serves for sample defatting; and the salt combination induces separation of water and acetonitrile layers and forces the majority of acrylamide into the acetonitrile layer. After vigorous shaking of the extraction mixture for 1 min and centrifugation, the upper hexane layer is discarded and a 1 mL aliquot of the acetonitrile extract is cleaned up by dispersive solid-phase extraction using 50 mg of primary secondary amine sorbent and 150 mg of anhydrous MgSO4. The final extract is analyzed either by liquid chromatography-tandem mass spectrometry or by gas chromatography-mass spectrometry (in positive chemical ionization mode) using the direct sample introduction technique for rugged large-volume injection.     

广州市兽用抗生素的环境残留研究

畜禽废物中的抗生素可造成土壤及水体的抗生素污染,利用高效液相色谱及高效液相色谱-串联质谱分析了广州市代表性养殖场畜禽废物、施用畜禽粪土壤和鱼塘水中尼卡巴嗪、喹乙醇、四环素类、磺胺类、喹诺酮类、氯霉素类抗生素的含量。结果发现：猪粪、鸡粪中抗生素含量最高的均为四环素类,分别为123.76、14.59 mg.kg-1;含量最低的均为氯霉素类,分别为2.35μg.kg-1、0.08 mg.kg-1;小猪猪粪中抗生素的含量明显高于大猪。鱼塘水中抗生素含量较高的是四环素（5.16μg.L-1）与磺胺对甲氧嘧啶（4.78μg.L-1）,土霉素在所有样点中均未被检出。施用禽畜粪土壤中四环素类、喹诺酮类含量较高,分别为70.40、49.77μg.kg-1;磺胺甲基嘧啶、磺胺对甲氧嘧啶、磺胺甲噁唑、甲砜霉素、氯霉素、喹乙醇均未被检出。粪样中的抗生素可导致土壤和水体的抗生素污染,TCs、QNs和NCZ可能对生态环境和人类健康安全的威胁更大。     

Establishment of a highly sensitive sandwich enzyme-linked immunosorbent assay specific for ovomucoid from hen's egg white

A highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) kit was established for quantifying ovomucoid from hen's egg white, which has been considered as one of the major allergen in egg white. The detection limit reached 0.041 ng/mL, and linearity ranged from 0.1 to 6.25 ng/mL. Intra- and interassay coefficient variations were all lower than 5% at three concentrations (0.5, 2.5, and 5 ng/mL). No cross-reactivity was observed with bovine serum, horse serum, goat serum, human serum, duck egg white, goose egg white, quail egg white, and pigeon egg white, but a low level of cross-reactivity was found with chicken serum. The ELISA kit was established on the basis of two monoclonal antibodies (mAbs) recognizing different epitopes of ovomucoid. However, these mAbs were generated using commercially purified ovalbumin as immunogen. Studies on the relative allergenicity and antigenicity of egg white protein have been performed by many researchers, but there were controversial opinions reported previously because of the impurity of each egg white protein used in various studies. In the present work we measured the degree of ovomucoid contamination in commercially purified ovalbumin sample, and the value was about 11%. We also determined the ovomucoid residue in influenza vaccine samples for the first time. These data showed that the ELISA kit we established could serve as an effective method for precisely quantifying concentrations of ovomucoid in the egg industry and as a useful tool for the research of allergenicity and antigenicity of hen's egg proteins.     

Characterization of the key aroma compounds in cooked grey mullet (Mugil cephalus) by application of aroma extract dilution analysis

Aroma and aroma-active compounds of wild grey mullet ( Mugil cephalus ) were analyzed by gas chromatography-mass spectrometry-olfactometry (GC-MS-O). According to sensory analysis, the aromatic extract obtained by simultaneous distillation and extraction (SDE) was representative of grey mullet odor. A total of 50 aroma compounds were identified and quantified in grey mullet. Aldehydes were qualitatively and quantitatively the most dominant volatiles in grey mullet. Aroma extract dilution analysis (AEDA) was used for the determination of aroma-active compounds of fish sample. A total of 29 aroma-active compounds were detected in aromatic extract of grey mullet, of which 24 were identified. On the basis of the flavor dilution (FD) factor, the most powerful aroma active compounds identified in the extract were (Z)-4-heptenal and nonanal, which were described as the strong cooked fish and green-fruity odor, respectively.     

Determination of ethalfluralin in canola seed, meal, and refined oil by capillary gas chromatography with mass selective detection

Ethalfluralin is a herbicide that is effective for weed control on a wide variety of crops, including canola. A method is described for the determination of ethalfluralin residues in canola seed, meal, and refined oil. Residues are extracted from canola sample matrixes with acetonitrile. An aliquot of the extract is diluted with water and purified by C(18) solid-phase extraction prior to analysis by capillary gas chromatography with mass selective detection. For all three sample matrixes, the method has a validated limit of quantitation of 0.02 microg/g and a limit of detection of 0.006 microg/g. Recoveries averaged 96 +/- 7% for canola seed, 87 +/- 6% for canola meal, and 89 +/- 5% for refined oil. In a magnitude-of-residue study, canola seed from field plots that had been treated with ethalfluralin at one to three times the maximum label rate for weed control were found to contain no detectable residue of the herbicide.     

Membrane Dialysis Extraction (MDE): A Novel Approach for Extracting Toxicologically Relevant Hydrophobic Organic Compounds from Soils and Sediments for Assessment in Biotests

Goal, Scope and Background   Organic solvents are routinely used to extract toxicants from polluted soils and sediments prior to chemical analysis or bioassay. Conventional extraction methods often require the use of heated organic solvents, in some cases under high pressure. These conditions can result in loss of volatile compounds from the sample and the degradation of thermally labile target analytes. Moreover, extracts of soils and sediments also frequently contain substantial quantities of organic macromolecules which can act as sorbing phases for target analytes and in doing so interfere with both chemical analysis and bioassays. Membrane dialysis extraction (MDE) is described as a simple, passive extraction method for selectively extracting toxicologically relevant hydrophobic organic compounds (HOCs) from polluted soils and sediments and anaylzed for its applicability in ecotoxicological investigations. Methods   Toxicologically relevant hydrophobic organic compounds were extracted from wet and dry sediments by sealing replicate samples in individual lengths of pre-cleaned low-density polyethylene (LD-PE) tubing and then dialysing in n-hexane. The efficacy of the MDE method for use in ecotoxicological investigations was assessed by testing the concentrated extracts in the neutral red assay for acute cytotoxicity, in the EROD assay for the presence of dioxin-like compounds and in the Danio rerio fish egg assay for embryotoxic and teratogenic effects. Conditions of the sediment sample (with or without water content), dialysis membrane length and duration of dialysis were analyzed with respect to their impact on three endpoints. Results of the MDE investigations were compared to data obtained in samples prepared using conventional Soxhlet extraction. Results and Discussion   The membrane dialysis extraction was found to be at least as efficient as Soxhlet methodology to extract toxicologically relevant HOCs from sediment samples. In most cases, MDE-derived extracts showed a higher toxicological potential than the Soxhlet extracts. Lack of any significant effects in any MDE controls indicated these differences were not caused by contamination of the LD-PE membrane used. The elevated toxicological potential of MDE extracts is most likely the result of enhanced bioavailability of toxic compounds in consequence of lower amounts of organic macromolecules (i.e. sorbing phases) in the MDE extracts. This effect is probably the result of a size-selective restriction by the LD-PE membrane. Conclusion   Membrane dialysis extraction was found to be a simple, efficient and cost-effective method for the extraction of sediment samples. MDE can be used to extract toxicologically relevant hydrophobic organic compounds from both wet and dry sediments without the risk of loosing volatile and thermally labile target analytes. The size-selectivity of the LD-PE membrane also appears to have the capacity to increase the bioavailablity of potential target analytes in the resulting extracts by retaining much of the organic macromolecules present in the sample. Thus, results suggest that MDE may be particularly useful for the extraction of toxicologically relevant hydrophobic organic compounds from soils and sediments for bioassays and other ecotoxicological investigations. Recommendation and Perspective   Further validation of MDE has been initiated and the applicability of the methodology to other sample types will be investigated. Of particular interest is the potential application of MDE to recover hydrophobic target analytes from biological samples such as muscle, other soft tissues and blood.     

Development and comparison of three diagnostic immunoassay formats for the detection of azoxystrobin

The currently accepted method of detection for azoxystrobin, a strobilurin fungicide, involves a labor-intensive organic solvent extraction and gas chromatography analysis. Three diagnostic assay formats, i.e., enzyme-linked immunosorbent assay (ELISA), fluorescence polarization (FP), and time-resolved fluorescence (TR-FIA), were developed and compared with regard to detection and quantification of azoxystrobin in grape extract and river, lake, and well water samples. These three assay formats require no initial sample extraction and were not affected by any of the environmental matrices tested, and each had a linear working range of 0-400 pg/mL. The polyclonal antibodies used for each of the immunoassays were specific to azoxystrobin; that is, the highest cross-reactivity to other pesticides observed was 5.7%. The limits of detection of the immunoassays were similar at 3 (ELISA), 46 (FP), and 28 (TR-FIA) pg/mL, as were the respective IC50 values of 306, 252, and 244 pg/mL. Each of the three immunoassays developed was less labor-intensive and approximately 100-fold more sensitive than the gas chromatographic method. While the three formats were comparable in terms of performance, the fluorescence polarization assay was the least labor-intensive and required the least time to perform.