Enzyme-linked immunosorbent assay based on a polyclonal antibody for the detection of the insecticide fenitrothion. Evaluation of antiserum and application to the analysis of water samples.

For development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the organophosphorus insecticide fenitrothion, the specificity of the antiserum R-3 generated with the bifunctional hapten, LysMNPA (2-[[[(3-methyl-4-nitrophenyl)oxy]methylcarbonyl]amino]-6-(2,4-dinitrophenyl)aminohexanoic acid) and the application to the residual analysis of some water samples were evaluated. At optimized ELISA conditions, the quantitative working range was from 1 to 39 ng/mL with a limit of detection of 0.3 ng/mL and an IC(50) value of 6 ng/mL. Cross-reactivity to structurally similar organophosphorus compounds and related chemicals was determined. The antiserum R-3 showed significant cross-reactivity with fenitrooxon and 3-methyl-4-nitrophenol, which have a 3-methyl-4-nitrophenoxy group as common structures, but showed relatively low cross-reactivity with other compounds. Each water sample (river water, tap water, purified water, and bottled water) had a matrix effect and was investigated by adding Tween 20 in the assay buffer. These four kinds of water samples were fortified with fenitrothion at several concentration levels and were directly analyzed with only dilution with an equal volume of antiserum solution. The mean recovery was 105.9%, and the mean coefficient of variation was 10.9%. The results suggested that the developed ELISA would be very suitable for a preliminary screening for fenitrothion in water samples at such low levels.     

Immunoaffinity purification of chlorimuron-ethyl from soil extracts prior to quantitation by enzyme-linked immunosorbent assay

A competitive-indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed to quantify chlorimuron-ethyl in soil. The linear working range of the assay was from 1 to 1000 ng mL(-)(1). The assay had an I(50) value of 54 ng mL(-)(1), with a limit of detection of 2 ng mL(-)(1) and a limit of quantification of 27 ng mL(-)(1). Three soils were extracted using a carbonate buffer (pH 9.0) and the extracts spiked with chlorimuron-ethyl. Because of the effects of coextractants (matrix effects) from soil on the accuracy and precision of the ELISA, immunoaffinity chromatography (IAC) was used to purify chlorimuron-ethyl from soil extracts prior to analysis. The immunoaffinity columns, which had a total binding capacity of 1350 ng of chlorimuron-ethyl mL(-)(1) of immunosorbent, were prepared by binding anti-chlorimuron-ethyl antibodies to protein G Sepharose 4B. Although the matrix effects were largely removed using the affinity column, they could be completely removed by first passing the extract through a column containing epoxy-coupled 1,6-diaminohexane (EAH) Sepharose 4B to remove organic acids prior to IAC. Assay sensitivity was increased 100-fold using IAC to purify and simultaneously concentrate chlorimuron-ethyl from soil extracts. The purification strategy (EAH followed by IAC chromatography) removed matrix effects from all three soils and allowed for the accurate quantitation of chlorimuron-ethyl in soil extracts.     

Development of an enhanced chemiluminescence ELISA for the rapid detection of acrylamide in food products

In this work, a polyclonal antibody for acrylamide (AA) was obtained by immunization of rabbits with N-acryloxysuccinimide (NAS) and keyhole limpet hemocyanin (KLH) conjugate. A direct enzyme-linked immunosorbent assay (ELISA) based on this antibody was developed with enhanced chemiluminescent (ECL) detection of AA in food samples. Assay conditions, such as concentrations of antibody and enzyme conjugate and competition time, were optimized. The effects of ionic strength and pH value were investigated. The optimized ECL-ELISA system allowed AA determination in a linear working range of 26.3-221.1 ng mL(-1) with an IC(50) value of 60.6 ng mL(-1) and a limit of detection of 18.6 ng mL(-1). Good recoveries with spiked food samples were obtained with a recovery range from 74.4 to 98.1%, and these results correlated well with those obtained using an HPLC method. This indicates that ECL-ELISA is applicable to the specific detection and routine monitoring of AA in food samples.     

Development of an enzyme-linked immunosorbent assay for the detection of glyphosate

A competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed to quantitate the herbicide glyphosate [N-(phosphonomethyl)glycine] in water. The ELISA has a detection limit of 7.6 microg mL(-1) and a linear working range of 10-1000 microg mL(-1) with an IC(50) value of 154 microg mL(-1). The glyphosate polyclonal antisera did not cross-react with a number of other herbicides tested but did cross-react with the glyphosate metabolite aminomethylphosphonic acid and a structurally related herbicide, glyphosine [(N,N-bis(phosphonomethyl)glycine]. The assay was used to estimate, quantitatively with accuracy and precision, glyphosate concentrations in water samples. Water samples were analyzed directly, and no sample preparation was required. To improve detection limits, water samples were concentrated prior to analysis, resulting in the increase of the detection limits by 100-fold. After the sample preconcentration step, the detection limit improved to 0.076 microg mL(-1) with an IC(50) value of 1.54 microg mL(-1), and a linear working range was 0.1-10 microg mL(-1). Glyphosate concentrations determined by ELISA correlated well with those determined by high-pressure liquid chromatography (r(2) = 0.99). This assay contributes to reducing the costs associated with conventional residue analysis techniques for the quantitation of glyphosate in water.     

Selective kinetic determination of paraquat using long-wavelength fluorescence detection.

The reaction between paraquat, ascorbic acid, and Cresyl Violet in alkaline medium and in the presence of sodium dodecyl sulfate has been applied for the first time to the development of a kinetic-fluorometric method for the determination of paraquat. The reaction rate of this system is measured by using the stopped-flow mixing technique, which makes the method applicable to automatic routine analysis. Analytical data are obtained in approximately 30 s. The calibration graph is linear over the range 6-500 ng mL(-)(1), and the detection limit is 1.8 ng mL(-)(1). The relative standard deviation is <3%. The use of dynamic measurements at long wavelength favors the high selectivity of the method. Diquat behaves in this system similarly to paraquat, but its interferent effect is easily avoided by using cysteine. The proposed method has been applied to the determination of paraquat in tap water, milk, and white wine samples with recoveries of 89-104%.     

Quantification of uronic acids in tea polysaccharide conjugates and their antioxidant properties

A technique of high-performance liquid chromatography (HPLC) was described for the measurement of total uronic acids in tea polysaccharide conjugates. This method was applied to polysaccharide conjugate extracts obtained from green tea after most of the components that produce interference were removed. The preliminary extraction process was according to the procedure of isolation of polysaccharide conjugates. The uronic acid content of different polysaccharide conjugate fractions was quantified by HPLC on a Sugar-Pak I column with a 1.0 x 10(-)(4) mol x L(-)(1) calcium disodium ethylenediaminetetraacetic acid solution as the mobile phase and refractive index detection. The validation study showed high recoveries (>97.0%) and low coefficients of variance (<3.0%). The minimum detectable limit concentration of uronic acid was 10 microg x mL(-)(1). The analysis of a standard range of galacturonic acid concentrations (100-4000 microg x mL(-)(1)) yielded linear results. The use of the method on different polysaccharide conjugate fraction samples confirmed its effectiveness. With the high content of uronic acids in polysaccharide conjugates, the stronger reactive oxygen species scavenging activities were found.     

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)     

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.     

Development of an enzyme-linked immunosorbent assay for the determination of maduramicin in broiler chicken tissues.

Maduramicin is one of the most widely used coccidiostats in the world. A rapid and accurate analytical method for this drug should provide producers and users with an effective management tool. The current chromatographic methods are sensitive but labor-intensive. This paper reports the development of an enzyme-linked immunosorbent assay (ELISA) based on an immunoaffinity chromatography cleanup procedure for the analysis of maduramicin in broiler chicken tissues (including muscle, liver, and fat). Recoveries from fortified tissue homogenates at levels of 30.0-120.0 microg kg(-)(1) ranged from 76.4 to 107.5% with coefficients of variation of 3.8-16.4%. The limits of detection were 1.0 ng g(-)(1) in muscle, 2.8 ng g(-)(1) in liver, and 1.5 ng g(-)(1) in fat. The ELISA results from the analysis of incurred residue in tissue samples showed the cleanup procedure is viable.     

Reliable enzyme-linked immunosorbent assay for the determination of soybean proteins in processed foods

Among allergenic foods, soybean is known as a food causing adverse reactions in allergenic patients. To clarify the validity of labeling, the specific and sensitive detection method for the analysis of the soybean protein would be necessary. The p34 protein, originally characterized to be p34 as an oil-body associated protein in soybean, has been identified as one of the major allergenic proteins and named Gly m Bd 30K. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of the soybean protein in processed foods was developed using polyclonal antibodies raised against p34 as a soybean marker protein and the specific extraction buffer for extract. The developed sandwich ELISA method was highly specific for the soybean protein. The limit of detection (LOD) and the limit of quantification (LOQ) of the developed ELISA were 0.47 ng/mL (equivalent to 0.19 microg/g in foods) and 0.94 ng/mL (equivalent to 0.38 microg/g in foods), respectively. The recovery ranged from 87.7 to 98.7%, whereas the intra- and interassay coefficients of variation were less than 4.2 and 7.5%, respectively. This study showed that the developed ELISA method is a specific, precise, and reliable tool for the quantitative analysis of the soybean protein in processed foods.     

Development of monoclonal ELISAs for azinphos-methyl. 2. Assay optimization and water sample analysis.

Two enzyme-linked immunosorbent assays (ELISA) for the insecticide azinphos-methyl have been optimized and characterized. Both ELISAs are based on monoclonal antibodies produced from an immunogen with a hapten containing a phthalimido moiety and on protein conjugates of heterologous ligands containing a 1,2,3-benzotriazine group. Assay I was performed in the conjugate-coated ELISA format and assay II in the antibody-coated format. Several physicochemical factors (ionic strength, pH, incubation times, and Tween 20 and BSA concentrations) that influence assay performance were studied and optimized. Regarding specificity, both monoclonal immunoassays highly cross-reacted with azinphos-ethyl and phosmet. Finally, both assays were applied to the analysis of azinphos-methyl in spiked real water samples. For assay I the sensitivity, estimated as the I(50) value, was 0.40 nM, with a practical working range between 0.10 and 1.75 ng/mL and a limit of detection of 0.05 ng/mL. For assay II the sensitivity was 1.01 nM, with a practical working range between 0.32 and 2.54 ng/mL and a limit of detection of 0.08 ng/mL.     

New approach to immunochemical determinations for triclopyr and 3,5,6-trichloro-2-pyridinol by using a bifunctional hapten,and evaluation of polyclonal antiserum

The present work describes the design and synthesis of the structurally unique hapten, "bifunctional hapten", to produce a group-specific polyclonal antiserum to triclopyr and 3,5,6-trichloro-2-pyridinol. A bifunctional hapten was designed and synthesized by conjugating commercially available Nepsilon-2,4-dinitrophenyl (DNP)-L-lysine to triclopyr, and then coupling this to carrier proteins such as bovine serum albumin (BSA). The synthesized bifunctional hapten greatly raised the antiserum titer in comparison with that of the conventional hapten, triclopyr. Antiserum with a sufficiently high titer to provide the determinations of targeted compounds was obtained only 63 days after the primary immunization. The obtained antiserum showed the highest affinity to triclopyr (IC(50) = 3.5 nM) and 3,5,6-trichloro-2-pyridinol (IC(50) = 5.1 nM) in homologous ELISA. The cross-reactivities to various agrochemicals and some chlorinated phenolic compounds were determined. Significant cross-reactivity was found to the herbicide 2,4,5-T. The antiserum reacted to both triclopyr and its metabolite. Assay sensitivity was evaluated for effects of various assay conditions, including pH value and concentrations of organic solvents and detergents. Under optimized assay conditions, the quantitative working range of triclopyr ELISA was from 0.1 to 5.2 ng/mL with a limit of detection (LOD) of 0.037 ng/mL, and an IC(50) of 0.72 ng/mL. On the other hand, the quantitative working range of 3,5,6-trichloro-2-pyridinol ELISA was from 0.13 to 6.0 ng/mL with a LOD of 0.052 ng/mL, and an IC(50) of 0.95 ng/mL. Water samples fortified with triclopyr or its metabolite at 1, 5, and 10 ng/mL were directly analyzed without extraction and cleanup by the proposed ELISA. The mean recovery was 101.6%, and the mean coefficient of variation (CV) was 7.1% in the case of the triclopyr ELISA. In the case of the 3,5,6-trichloro-2-pyridinol ELISA, the mean recovery was 99.8%, and the mean CV was 9.5%. The proposed ELISA turned out to be a powerful tool for monitoring of residual triclopyr or 3,5,6-trichloro-2-pyridinol in water samples at trace level.     

HAI-RTP determination of carbaryl pesticide in different irrigation water samples of south Spain

In this work, a widely used pesticide named carbaryl, present in numerous water supplies, has been determined by the so-called heavy atom induced room-temperature phosphorescence (HAI-RTP) methodology. A detailed study of numerous instrumental variables such as sensitivity, slits, decay time, and gate time, and those of experimental type such as heavy atoms, oxygen scavenger, temperature, buffer solutions, and organic solvents, have been carried out. The detection limit was 2.8 ng mL(-)(1) with a relative standard deviation of 2.12% at the 150 ng mL(-)(1) level. Spiked irrigation water samples taken from different places near agricultural fields gave mean percentage recoveries of 95.7%. The results obtained in this study indicate that the proposed method is suitable for the determination of residues of carbaryl pesticide in water samples with good reproducibility and sensitivity for the analysis of this compound being rapid and very simple for routine analysis.     

Poly- and monoclonal antibody-based ELISAs for fipronil

An enzyme-linked immunosorbent assay (ELISA) for fipronil was developed by using polyclonal antibodies (pABs) or monoclonal antibodies (mABs), and its suitability of the determination of this analyte in spiked water samples was studied. The pABs-based assay showed I50 = 17.95 ppb, I90 = 203.40 ppb, and I10 = 0.066 ppb, whereas the mABs-based assay showed I50 = 5.99 ppb, I90 = 485.40 ppb, and I10 = 0.074 ppb. The recoveries of fipronil from tap water samples by pABs-based ELISA were 93.00-124.00% in the range of 0-500 ng/mL, and those obtained from the samples by mABs-based ELISA were 94.70-108.00%. Different types of water from pool, river, and sea were spiked at different levels (ranging form 0.1 to 10 microg/L) and were assayed by the indirect ELISA with mABs. The recoveries of fipronil by this ELISA were in the range of 80-120%. The results demonstrate that this assay is suitable for the quantitative detection of fipronil at trace levels in water samples.     

Development of an enzyme-linked immunosorbent assay for the detection of the organophosphorus insecticide acephate

A competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the organophosphorus insecticide acephate, O,S-dimethyl acetylphosphoramidothioate, was developed using a polyclonal antibody. Five different haptens mimicking the analyte were synthesized and conjugated with the carrier proteins bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) by the N-hydroxysuccinimide active ester and diazotization methods. Polyclonal antibodies raised against hapten-KLH conjugates in rabbits and hapten-BSA conjugates as coating antigens were screened and selected for the assay in the homologous and heterologous ELISA systems. The effects of various assay conditions such as detergent, organic solvents, pH, and preincubation of the mixture of the polyclonal antibody and the analyte on the sensitivity were evaluated. The IC(50) value for acephate was 25 ng/mL in an optimized heterologous system using hapten-4-BSA as a coating antigen and a polyclonal antibody no. 8377 against hapten-1-KLH, showing the detection range of 5-140 ng/mL and the lowest detection limit of 2 ng/mL. The cross-reactivities of the structurally related organophosphorus insecticides, including the major metabolite of the analyte, methamidophos, were less than 1%. Recoveries from the analyte-fortified tap water, mulberry leaves, and lettuce samples in the assay were in the range of 72-121% by simple extraction, concentration, and dilution. These results indicate that the ELISA could be a convenient and supplemental analytical tool for monitoring acephate residues in environmental and agricultural samples.     

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.     

Determination of multiresidue of avermectins in bovine liver by an indirect competitive ELISA

An ELISA was developed to detect multiresidues of avermectins (AVMs) including abamectin (ABM), ivermectin (IVM), and eprinomectin (EPR) in bovine liver. The modified ABM, 4'-O-succinoyl-ABM was conjugated to bovine serum albumin as the immunogen for the preparation of polyclonal antibodies to AVMs and conjugated to ovalbumin as the coating antigen for the ELISA. Serum with the highest antibody titers to AVMs, which had a cross-reactivity of 100% with ABM, 145.4% with EPR, and 25% for IVM, was selected for the development of an indirect competitive ELISA. The ELISA could detect ABM, IVM, and EPR residues in bovine liver tissues, with a limit of quantitation of 1.06 ng/mL for all three AVMs. Optimal pH, ion strength, organic solvent, and duration of incubations were investigated to increase the sensitivity of the ELISA. Recoveries of these drugs ranged from 53.8% to 80.6% with inter-assay coefficients of variation (CV) of 3.4-17.9% and intra-assay CV of 5.5-14.7%. Analysis results of field samples by the ELISA were consistent with those by a previously developed HPLC method. The ELISA can be used as a rapid method for screening of AVMs residues in bovine liver.     

Spectrophotometric and adsorptive stripping square wave voltammetric determination of iron in olive oils,as complex with 5,5-dimethylcyclohexane-1,2,3-trione 1,2-dioxime 3-thiosemicarbazone (DCDT)

Two methods for the determination of iron in olive oil by spectrophotometry and by adsorptive stripping square wave voltammetry (Ad-SSWV) have been developed. These two methods are based on the formation of a 5,5-dimethylcyclohexane-1,2,3-trione 1,2-dioxime 3-thiosemicarbazone (DCDT)-iron(II) complex in strongly acid media. In both, iron is extracted from the olive oil by using HCl. Spectrophotometric determination of iron with DCDT is based on the feature that the DCDT-Fe complex shows an absorbance maximum at 550 nm. A calibration graph has been constructed from 0 to 4000 ng mL(-)(1), and the detection limit was 115 ng mL(-)(1) (57 ng g(-)(1) in olive oil). On the other hand, the voltammetric determination of the metal is based on the appearance of a peak due to an adsorptive reductive process of the complex that it is observed when the Ad-SSWV technique is used. A calibration graph has been constructed from 0 to 30 ng mL(-)(1), and the detection limit was 0.55 ng mL(-)(1) (13.75 ng g(-)(1) in olive oil according to the proposed procedure).     

Quantification of beta casein in milk and cheese using an optical immunosensor

beta-Casein was quantified in milk and cheese, using an optical immunosensor, based on surface plasmon resonance (SPR) measurement. The assay consists of a two-step sandwich strategy, with two anti-beta-casein antibodies directed against each extremity of the casein. This strategy permits only native beta-casein to be quantified and not its degradation products. The calibration curve was obtained with a reference milk powder of known beta-casein concentration. The analysis time per sample was less than 10 minutes. The antibody-coated surface could be used for more than 250 determinations. The detection limit was established at 85 ng x mL(-)(1) and the intra- and inter-assay variation coefficients were 2.6 and 6.2% respectively. The method was applied to raw milk to quantify intact beta-casein, with no pretreatment of the sample. A second application was realized with cheese, to follow the proteolysis of beta-casein during ripening.     

Development of an immunoassay (ELISA) for the quantification of thiram in lettuce

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 quantification of thiram in lettuces. Concerning the laboratory assay, the calibration curve for thiram had a linear range of 11 to 90 ng/mL and a detection limit of 5 ng/mL. Precision of the assay presented coefficient of variation values <9% and the recovery of thiram from lettuce averaged 89% across the range of the immunoassay method using 30 min extraction with water/acetone (50:50, v/v). The tube-based method was developed in order that an extract of lettuce, containing thiram at the MRL (8 ppm), would be found on the linear part of the standard curve. The calibration curve for thiram has a linear range of 100 to 800 ng/mL (1.39 to 11.1 ppm in lettuce) and a detection limit of 40 ng/mL.