Validation of a monoclonal enzyme immunoassay for the determination of carbofuran in fruits and vegetables

The N-methylcarbamate pesticide carbofuran is a very important insecticide used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) to determine this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 740 ng/L and with a dynamic range between 200 and 3100 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbofuran at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop, mean recoveries in the 43.9--90.7% range were obtained for purified samples and in the 90.1--121.6% range for crude extracts. The carbofuran immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbofuran at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was very good (y = 0.90x + 2.66, r(2)() = 0.958, n = 25), with HPLC being more precise than ELISA (mean coefficients of variation of 4.1 and 11.5%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.02x + 10.44, r(2)() = 0.933, n = 29). Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbofuran in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases the sample throughput.     

Enzyme-linked immunosorbent assay (ELISA) and sol-gel-based immunoaffinity purification (IAP) of the pyrethroid bioallethrin in food and environmental samples

Enzyme-linked immunosorbent assay (ELISA) and sol-gel-based immunoaffinity purification (IAP) methods for the pyrethroid bioallethrin were developed and applied for monitoring bioallethrin in spiked food, soil, and dust samples. Attempts to determine bioallethrin content in fruit and vegetable extracts revealed high variability between sample preparations and marked interferences with the assay. Sol-gel IAP followed by solid-phase sample concentration was effective in removing the interfering components and resulted in high recovery of bioallethrin from spiked crude acetonic extracts of fruits and vegetables, even in the presence of high extract concentrations (28%). Solid-phase treatment alone failed to remove the interfering components from the spiked sample. Gas chromatography-mass spectrometry analysis of the IAP samples revealed bioallethrin as a doublet unsolved peak because of the cis and trans isomer present in the standard with confirmation of its mass. Unlike fruit and vegetable extracts, soil and dust samples did not interfere with the ELISA, and the bioallethrin content in those samples could be determined with high precision without the need of any further purification.     

Comparative study of three different methods for the determination of aflatoxins in tahini

Aflatoxins spiked at three different levels (6.5, 13.0, and 19.5 microg/kg) in tahini, a sesame butter, were analyzed by using three different methods: high-performance liquid chromatography (HPLC), fluorometry, and enzyme-linked immunosorbent assay (ELISA). An immunoaffinity column was used for cleanup and purification of extracts prior to detection by HPLC and fluorometry. All methods were statistically evaluated for accuracy, precision, and simple correlations. Additionally, 14 tahini samples randomly obtained from Turkish retail markets were analyzed using an immunoaffinity column cleanup procedure coupled with the HPLC detection method. The fluorometric determination method involving an immunoaffinity column cleanup step was found to be highly correlated with the HPLC method (r = 0.978). Both methods were found to be effective due to their high recoveries and low variance for the prediction of total aflatoxin contamination in tahini samples. The ELISA method, due to its high variation in replicates, was found to be applicable only as a screening method. The survey study demonstrated the need for control of aflatoxin contamination of foodstuffs involving sesame seeds as an ingredient.     

Determination of pyrethroid residues in agricultural products by an enzyme-linked immunosorbent assay

To determine cypermethrin and permethrin in agricultural products, a competitive enzyme-linked immunosorbent assay (ELISA) method was employed. The matrix interferences were minimized by direct dilution of the extracts. No further cleanup was needed. A minimum matrix effect with a 1:10 dilution of white wine for cypermethrin and a 1:200 dilution of red and white wines, fruits, and vegetables for permethrin was found when phosphate-buffered saline containing 40% methanol was employed as the diluent. Good recoveries of spiked levels were observed. The mean percentage recoveries of cypermethrin spiked in white wine and permethrin spiked in red and white wines were 99.7, 74, and 78%, respectively. The mean percentage recoveries of permethrin spiked in apple, banana, cucumber, lettuce, onion, and peach were 99.2, 105, 70.2, 97.5, 94.4, and 89.4%, respectively. Validation of the ELISA method with permethrin-spiked lettuce and peach was carried out using gas chromatography with mass spectrometry, resulting in a good recovery and correlation.     

Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for the determination of ractopamine in incurred samples from food animals

A monoclonal antibody-based ractopamine immunoassay has been applied to incurred samples from sheep and cattle. Results obtained by immunoassay were compared with those from high-performance liquid chromatography (HPLC). Three sets of sample extracts containing primarily unmetabolized ractopamine were analyzed. Correlation of HPLC with enzyme-linked immunosorbent assay (ELISA) for beef liver samples gave an r(2) = 0.98 despite rather low ractopamine concentrations (range 1.1-13.4 ng/mL, n = 6). Ractopamine concentrations in cow urine samples treated by solid phase extraction, to remove ractopamine metabolites, also showed a high correlation between the HPLC and the ELISA results (r(2) = 0.95, range 1.0-275 ng/mL, n = 61). In contrast, HPLC and ELISA analyses of ractopamine in sheep urine were not well-correlated (r(2) = 0.58, range 0.85-51 ng/mL, n = 34). When ractopamine conjugates in urine samples were hydrolyzed with hydrolytic enzymes, ELISA and HPLC methods were highly correlated [r(2) = 0.94 for sheep (range 123-10 554 ppb, n = 60) and an r(2) = 0.98 for cattle (range 14-8159 ppb, n = 62)]. Tissues contained only minute amounts of ractopamine, and after 7-day withdrawal periods, less than 1 ppb of free ractopamine was detected. Ractopamine was rapidly metabolized in both cattle and sheep. The difference in ractopamine concentration of urine samples before and after hydrolysis indicated that only 1-5% of ractopamine was excreted unmetabolized. Results from this study indicate that the monoclonal antibody-based ELISA could be useful for a sensitive, quantitative, or qualitative ractopamine screening assay.     

Development of a solid-phase cleanup and portable rapid flow-through enzyme immunoassay for the detection of ochratoxin a in roasted coffee

A membrane-based flow-through enzyme immunoassay (patent application pending) for the detection of ochratoxin A (OA) in roasted coffee was developed. First, an extraction and solid-phase cleanup method was developed. A high partition coefficient for OA in the mobile phase was achieved by using methanol/5% aqueous NaHCO(3) as the sample extraction and cleanup solvent. The solid-phase (aminopropyl) cleanup was developed to chromatographically elute OA but retain cross-reacting compounds. Without using aminopropyl cleanup, cross-reacting compounds resulted in 100% false positives for both flow-through enzyme immunoassay and HPLC methods. However, after cleanup with aminopropyl, no false positives were observed. The flow-through results were visually evaluated. The sensitivity achieved for the flow-through was 4 microg kg(-1) in spiked roasted coffee. The assay was used to screen roasted coffee samples. Results were confirmed with HPLC with a detection limit of 1 microg kg(-1).     

Development of two enzyme-linked immunosorbent assays for detection of endosulfan residues in agricultural products

Two competitive immunoassays, a laboratory assay based on microwell plates and a field test based on the use of polystyrene tubes, have been developed for the detection of endosulfan in agricultural products. The limit of detection for the microwell plate format was 0.8 +/- 0.1 microg/kg, and the limit of detection for the tube format was 1.6 +/- 0.2 microg/kg. A simple, rapid, and efficient extraction method was employed, and 76-112% recoveries of spiked samples were obtained. Methanol extracts of some agricultural product samples such as grape, carrot, spinach, and tobacco could be analyzed directly by immunoassay after dilution in 0.5% fish skin gelatin-phosphate buffered saline. In contrast, extracts of green tea caused significant interference in the assay, and a number of simple cleanup methods were ineffective in removing interference. However, use of the coagulating reagent polyvinyl pyrrolidone removed the matrix effect effectively. For the validation of the enzyme-linked immunosorbent assay (ELISA) tests, samples were analyzed by ELISA and gas chromatography (GC) after solid phase extraction. The relationship between data obtained using the tube assay and microwell assay was good (the lowest r(2) value was 0.94), and also, the immunoassay assay data correlated well with data obtained from GC analysis (the lowest r(2) value was 0.93). The developed immunoassay methods are the suitable methods for the rapid quantitative and reliable determination of endosulfan residues in agricultural products.     

Development of multianalyte flow-through and lateral-flow assays using gold particles and horseradish peroxidase as tracers for the rapid determination of carbaryl and endosulfan in agricultural products

Membrane-based competitive immunoassays using gold particles and horseradish peroxidase (HRP) as tracers in flow-through and lateral-flow formats for multianalysis of carbaryl and endosulfan were developed. For gold-based immunoassay, membrane strips were coated with goat anti-rabbit IgG (control line) and carbaryl hapten-ovalbumin (OVA) and endosulfan hapten-OVA (test lines). The visual detection limits for carbaryl and endosulfan were 100 and 10 microg/L in gold-based assays, respectively. For immunoassay using HRP as tracer, anti-carbaryl and anti-endosulfan antibodies were separately coated on the membrane as test lines, and the visual detection limits were 10 microg/L for carbaryl and 1 microg/L for endosulfan. The developed assays used gold particles and HRP as labels, respectively; 10 times enhancement in the visual detection limit using HRP label was obtained in the study. Matrix interference was eliminated by appropriate dilution of sample extracts with buffer. For the validation of the multianalyte assay, the samples were screened by multianalyte gold-based assay and confirmed by HPLC for carbaryl determination and by GC for endosulfan determination. The results of multianalyte gold-based flow-through assay for the determination of carbaryl and endosulfan were in good agreement with the results of instrumental analysis (HPLC with ultraviolet detection and GC with electron capture detection). The developed multianalyte immunoassays for which the results were interpreted visually can be used as convenient qualitative tools for on-site rapid screening of carbaryl and endosulfan simultaneously in agricultural products.     

Fluorescence polarization immunoassay based on a monoclonal antibody for the detection of the organophosphorus pesticide parathion-methyl

A fluorescence polarization immunoassay (FPIA) based on a monoclonal antibody for the detection of parathion-methyl (PM) was developed and optimized. Fluorescein-labeled PM derivatives (tracers) with different structures were synthesized and purified by thin-layer chromatography. The influence of immunogen and tracer structures on the assay characteristics was investigated. PM concentration determinable by the FPIA ranged from 25 to 10000 ppb. The detection limit was 15 ppb. Methanol extracts of vegetable, fruit, and soil samples were diluted 1/10 for the analysis. Recovery in spiked samples averaged between 85 and 110%. The method developed is characterized by high specificity and reproducibility (CV ranged from 1.5 to 9.1% for interassay and from 1.8 to 14.1% for intra-assay). The FPIA method can be applied to the screening of food and environmental samples for PM residues without complicated cleanup.     

Simultaneous determination of neonicotinoid insecticides in agricultural samples by solid-phase extraction cleanup and liquid chromatography equipped with diode-array detection

Effective sample pretreatment procedures based on solid-phase extraction (SPE) for multiresidue determination of seven neonicotinoid insecticides in agricultural products were investigated. After extraction with acetone and concentration, the insecticides in aqueous sample extracts were transferred into organic solvent phases with a Chem Elut SPE cartridge. Finally, the eluate from the cartridge was cleaned up with a SPE cartridge packed with graphitized carbon black and aminopropyl silica gel, which showed a higher cleanup efficiency than the classical silica gel SPE cartridge. Seven insecticides were separated on a reversed-phase C18 column and a gradient system of methanol and phosphate solution based on high-performance liquid chromatography. The established multiresidue determination has been applied to several artificially spiked agricultural samples, with the result that the average recoveries were excellent, with the exception of nitenpyram. The limit of detection of the method ranged from 0.01 to 0.03 mg/kg for the insecticides.     

Sodium borohydride/chloranil-based assay for quantifying total flavonoids

A novel sodium borohydride/chloranil-based (SBC) assay for quantifying total flavonoids, including flavones, flavonols, flavonones, flavononols, isoflavonoids, flavanols, and anthocyanins, has been developed. Flavonoids with a 4-carbonyl group were reduced to flavanols using sodium borohydride catalyzed with aluminum chloride. Then the flavan-4-ols were oxidized to anthocyanins by chloranil in an acetic acid solution. The anthocyanins were reacted with vanillin in concentrated hydrochloric acid and then quantified spectrophotometrically at 490 nm. A representative of each common flavonoid class including flavones (baicalein), flavonols (quercetin), flavonones (hesperetin), flavononols (silibinin), isoflavonoids (biochanin A), and flavanols (catechin) showed excellent linear dose-responses in the general range of 0.1-10.0 mM. For most flavonoids, the detection limit was about 0.1 mM in this assay. The recoveries of quercetin from spiked samples of apples and red peppers were 96.5 +/- 1.4% (CV = 1.4%, n = 4) and 99.0 +/- 4.2% (CV = 4.2%, n = 4), respectively. The recovery of catechin from spiked samples of cranberry extracts was 97.9 +/- 2.0% (CV = 2.0%, n = 4). The total flavonoids of selected common fruits and vegetables were measured using this assay. Among the samples tested, blueberry had the highest total flavonoid content (689.5 +/- 10.7 mg of catechin equiv per 100 g of sample), followed by cranberry, apple, broccoli, and red pepper. This novel SBC total flavonoid assay can be widely used to measure the total flavonoid content of fruits, vegetables, whole grains, herbal products, dietary supplements, and nutraceutical products.     

Separation and purification of sulforaphane from broccoli seeds by solid phase extraction and preparative high-performance liquid chromatography

A novel, rapid, and economical method to isolate and purify natural sulforaphane from broccoli seeds is described. The procedure involves solvent extraction of autolyzed seed meal, followed by separation by solid phase extraction (SPE) and purification by preparative high-performance liquid chromatography (HPLC). The SPE method provides higher yield of sulforaphane from crude extracts compared to conventional liquid-liquid extraction. High purity and recovery of sulforaphane product can be obtained by preparative HPLC with a C 18 column and 30% methanol in water as the mobile phase. The purified compound was characterized by MS and (1)H and (13)C NMR. The techniques described here are useful tools in the preparative-scale isolation of sulforaphane in a fast, cost-effective, and waste-conscious manner.     

Determination of oryzalin in water, citrus fruits, and stone fruits by liquid chromatography with tandem mass spectrometry

A new methodology is described for rapidly determining the herbicide oryzalin in water, citrus fruits, and stone fruits by liquid chromatography with negative ion electrospray ionization tandem mass spectrometry (LC/MS/MS). Oryzalin is extracted from water using a polymeric sorbent solid phase extraction (SPE) column and from fruit using methanol. The water samples require no further purification, but an aliquot of the fruit sample extracts is diluted with water and purified using a polymeric 96 well SPE plate. Purified extracts are concentrated prior to determination by LC/MS/MS at m/z 345 (Q1) and m/z 281 (Q3) using an external standard for calibration. The validated limits of quantitation were 0.05 microg/L in water (drinking water, surface water, and groundwater) and 0.01 microg/g in citrus fruits (oranges and lemons) and stone fruits (peaches and cherries). Recoveries averaged 102% for water samples and 85-89% for the various types of fruit samples. For all fortification levels combined, the relative standard deviations ranged from 4 to 6% for water and from 2 to 4% for fruit.     

High pressure liquid chromatographic determination of sugars in various food products.

A high pressure liquid chromatographic (HPLC) method has been developed which is fast, simple, specific, and reliable over a wide range of sugar concentrations in a variety of food matrices. With few exceptions, sample preparation is simple, requiring only a water-ethanol extraction, followed by a rapid mini-column cleanup before injection into the HPLC system. The majority of samples can be prepared for analysis within 1--1 1/2 hr, and the following sugars are separated in less than 45 min: fructose, glucose, sucrose, maltose, lactose, melibioals, chocolate products, chocolate sirups, cookies, health food products, molasses, preserves, processed fruits, and soy protein products.     

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.     

Thin layer chromatographic detection and indirect gas chromatographic determination of three carbamate pesticides.

Carbamate pesticide residues are extracted from vegetables and fruits with methylene chloride. The extracts are spotted on silica gel plates and the pesticides are detected by an enzymatic inhibition technique. For quantitative determination, aliquots of the methylene chloride extracts are evaporated to dryness in a rotary evaporator. After the residues are dissolved in ethanol, 0.5N NaOH is added in the hydrolysis step. To remove a number of possible interferences the hydrolyzed phenols are steam-distilled and treated with 1-fluoro-2,4-dinitrobenzene and/or 4-chloro-alpha,alpha,alpha-trifluoro-3,5-dinitrotoluene to form the ether derivatives. Efficiency in the conversion of the phenolic moieties to the phenyl ethers is about 100%. The resulting electron-capturing derivatives enable the carbamate pesticides to be detected in vegetables and fruits at the 0.05 ppm level. Recoveries of 90-94% were obtained from vegetables and fruits fortified with 0.5-2.0 ppm carbaryl, Mesurol, and propoxur.     

Simultaneous determination of ethidimuron, methabenzthiazuron, and their two major degradation products in soil

An analytical method has been developed for the quantification of two herbicides (ethidimuron and methabenzthiazuron) and their two main soil derivatives. This method involves fluidized-bed extraction (FBE) prior to cleanup and analysis by reverse-phase liquid chromatography with UV detection at 282 nm. FBE conditions were established to provide efficient extraction without degradation of the four analytes. (14)C-labeled compounds were used for the optimization of extraction and purification steps and for the determination of related efficiencies. Extraction was optimal using a fexIKA extractor operating at 110 degrees C for three cycles (total time = 95 min) with 75 g of soil and 150 mL of a 60:40 v/v acetone/water mixture. Extracts were further purified on a 500 mg silica SPE cartridge. Separation was performed on a C18 Purosphere column (250 mm x 4 mm i.d.), at 0.8 mL min(-1) and 30 degrees C with an elution gradient made up of phosphoric acid aqueous solution (pH 2.2) and acetonitrile. Calibration curves were found to be linear in the 0.5-50 mg L(-1) concentration range. Besides freshly spiked soil samples, method validation included the analysis of samples with aged residues. Recovery values, determined from spiked samples, were close to 100%. Limits of detection ranged between 2 and 3 microg kg(-1) of dry soil and limits of quantification between 8 and 10 microg kg(-1) of dry soil. An attempt to improve these performances by using fluorescence detection following postcolumn derivatization by orthophthalaldehyde-mercaptoethanol reagent was unsuccessful.     

Analysis of fat-soluble vitamins. XXVIII. High performance liquid chromatographic determination of vitamin D in pet foods and feeds: collaborative study

A high performance liquid chromatographic (HPLC) method for vitamin D in pet foods and feeds at low concentrations (2-8 IU/g = 50-200 ppb) was studied collaboratively. The procedure consists of the following purification steps: saponification, extraction of the unsaponifiable fraction, chromatography on alumina, cleanup on reverse phase HPLC, and quantitation with straight phase HPLC. The original method, developed by Knapstein, was simplified by deleting the quantitative TLC step. Six coded samples were distributed to 31 laboratories, along with a known sample containing 15 IU/g to allow practice of the rather complicated procedure. Eighteen collaborators returned their results. Results for the spiked samples show good recovery. The estimates of repeatability and reproducibility are 0.96 and 2.2 IU/g for spiked samples and 1.5 and 3.1 IU/g for commercial samples, respectively, which are considered acceptable for these low concentrations. The method has been adopted official first action.     

Determination of acequinocyl and hydroxyacequinocyl on fruits and vegetables by HPLC-DAD

A method for determining residues of the new reduced-risk pesticide acequinocyl and its deacetylated derivative hydroxyacequinocyl on fruits and vegetables (grapes, lemons, pears, and tomatoes) by HPLC is described. The pesticides were extracted from the fruits and vegetables with hexane and ethyl acetate solution (1:1, v/v), determined by HPLC-DAD at 250 nm and confirmed by LC/MS. No cleanup was necessary. This method is characterized by recoveries (0.01-4 mg/kg) > 77%, while the coefficient of variation was determined to be less than 11%. The limit of quantitation for both acequinocyl and hydroxyacequinocyl was 0.01 mg/kg for all matrixes.     

Development of an enzyme-linked immunosorbent assay for the fungicide imazalil in citrus fruits

Imazalil has been widely used in citrus fruits such as lemons, oranges, and grapefruits. A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the detection of residual imazalil in citrus fruits. A monoclonal antibody (MoAb) generated to the synthetic imazalil hapten (EIT-0073)-protein conjugate was used. This assay was applied to lemon, orange, and grapefruit matrices for an imazalil analysis. The acceptable residue level for lemons, oranges, and grapefruits in Japan is 5 ppm. The matrix interference was minimized by direct dilution of the sample homogenate. No further cleanup was needed. The detection limit for imazalil in these citrus fruits was 0.1 ng/mL. The recovery of each fortified citrus fruit sample was >81.0%. The imazalil recovery measured by the proposed ELISA was compared to the recovery determined by a conventional HPLC. A good correlation was observed between the proposed ELISA and the HPLC. This proposed ELISA would be useful for monitoring for residual imazalil.