Improved methods for urinary atrazine mercapturate analysis--assessment of an enzyme-linked immunosorbent assay (ELISA) and a novel liquid chromatography-mass spectrometry (LC-MS) method utilizing online solid phase extraction (SPE)

Elimination of interfering substances in urine by solid phase extraction (SPE) prior to analysis resulted in 10-fold improvement in the sensitivity of atrazine mercapturate (AM) enzyme-linked immunosorbent assay (ELISA) compared to previous reports. Of the two tested SPE systems, Oasis^® HLB and MCX, the mixed-mode MCX gave good recoveries (82%) of AM in spiked samples measured by ELISA, whereas the reverse-phase HLB phase was not compatible with the immunochemical method. At relatively high concentrations of urinary AM (>20 ng mL⁻¹), sample dilution was effective enough for the elimination of interfering substances. The new liquid chromatography-mass spectrometry (LC-MS) method developed for AM utilizes online-SPE with Oasis^® HLB, column switching and a stable-isotope internal standard. The limit of quantification (0.05 ng mL⁻¹) indicates improved sensitivity compared with most previously published LC-MS methods for AM. Validation of all three methods, LC-MS, ELISA + SPE and ELISA + dilution with spiked urine samples showed good correlation between the known and measured concentrations with R² values of 0.996, 0.957 and 0.961, respectively. When a set (n = 70 plus 12 blind duplicates) of urine samples from farmers exposed to atrazine was analyzed, there was a good agreement (R² = 0.917) between the log normalized data obtained by ELISA + SPE and LC-MS. High correlation among the data obtained by the two tested methods and the LC-MS method by the Center of Disease Control and Prevention (CDC), together with low variability among the blind duplicates, suggests that both methods reported here would be suitable for the analysis of urinary AM as a biomarker for human exposure of atrazine.     

Evaluation and validation of commercially available enzyme-linked immunosorbent assays (ELISAs) specific for atrazine, chlorpyrifos, and diazinon in aqueous phase

The performance of commercially available microtiter plate enzyme-linked immunosorbent assays (ELISA) kits specific for the determination of triazines (atrazine), chlorpyrifos, and diazinon was evaluated for sensitivity, intra-assay repeatability, and accuracy using samples of known concentration in aqueous solution. Mean percent recovery values were not significantly different among concentrations for diazinon (One-way parametric ANOVA, P=0.46, n=72). However, mean percent recovery values were significantly different among concentrations for both atrazine and chlorpyrifos analyses (One-way parametric ANOVA, P<0.0001, n=36 for both analyses), and were highly dependent on concentrations for chlorpyrifos (% recovery=−0.155 (concentration)+238.448, r²=0.91, P<0.0001, n=36). All methods demonstrated a high degree of statistical separation between readings at various concentrations (One-way parametric ANOVA followed by Student-Neuman-Keuls (SNK) multiple range test, P<0.0001 for all analyses) and a close correlation between known concentrations and concentrations derived from ELISA for all three analytes (diazinon, r=0.985, P<0.0001, n=72; atrazine r=0.967, P<0.0001, n=36; chlorpyrifos r=0.947, P<0.0001, n=36). Statistical comparisons between known concentrations and concentrations derived from ELISAs showed that diazinon values were significantly (P<0.01, n=12 per concentration level) overestimated for all concentration levels. Chlorpyrifos concentrations were significantly (P<0.01, n=6 per concentration level) overestimated at lower concentrations and significantly (P<0.01, n=6 per concentration level) underestimated at higher concentrations. ELISA-derived atrazine concentrations were statistically similar to known concentrations for most concentration levels (P>0.05, n=6 per concentration level). Results indicate that ELISA kits are excellent for screening purposes, although consistent overestimation of ELISA for diazinon at all concentration levels and chlorpyrifos at lower concentrations levels must be resolved before the kits can be used routinely for regulatory compliance monitoring.     

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) method for the measurement of 2,4-dichlorophenoxyacetic acid in human urine

An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoxyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline containing 0.05% Tween and 0.02% sodium azide, with analysis by a 96-microwell plate immunoassay format. No clean up was required as dilution step minimized sample interferences. Fifty urine samples were received without identifiers from a subset of pesticide applicators and their spouses in an EPA pesticide exposure study (PES) and analyzed by the ELISA method and a conventional gas chromatography/mass spectrometry (GC/MS) procedure. For the GC/MS analysis, urine samples were extracted with acidic dichloromethane (DCM); methylated by diazomethane and fractionated by a Florisil solid phase extraction (SPE) column prior to GC/MS detection. The percent relative standard deviation (%R.S.D.) of the 96-microwell plate triplicate assays ranged from 1.2 to 22% for the urine samples. Day-to-day variation of the assay results was within ±20%. Quantitative recoveries (>70%) of 2,4-D were obtained for the spiked urine samples by the ELISA method. Quantitative recoveries (>80%) of 2,4-D were also obtained for these samples by the GC/MS procedure. The overall method precision of these samples was within ±20% for both the ELISA and GC/MS methods. The estimated quantification limit for 2,4-D in urine was 30 ng/mL by ELISA and 0.2 ng/mL by GC/MS. A higher quantification limit for the ELISA method is partly due to the requirement of a 1:5 dilution to remove the urine sample matrix effect. The GC/MS method can accommodate a 10:1 concentration factor (10 mL of urine converted into 1 mL organic solvent for analysis) but requires extraction, methylation and clean up on a solid phase column. The immunoassay and GC/MS data were highly correlated, with a correlation coefficient of 0.94 and a slope of 1.00. Favorable results between the two methods were achieved despite the vast differences in sample preparation. Results indicated that the ELISA method could be used as a high throughput, quantitative monitoring tool for human urine samples to identify individuals with exposure to 2,4-D above the typical background levels.     

Optimization and performance evaluation of the analysis of glyphosate and AMPA in water by HPLC with fluorescence detection

Summary The object of this work was to optimize and validate an analytical method for the analysis of glyphosate and its main metabolite AMPA (aminomethylphosphonic acid) in natural and drinking water, for sanitary control. The method uses a derivatization step which transforms glyphosate and AMPA into fluorescent products by reaction with 9-fluorenylmethylchloroformate (FMOC-Cl). The kinetics and yield of this reaction were studied by use of different samples. The derivatization products were injected directly into the liquid chromatograph and separated on an amino-functionalized silica gel column. The performance of the method (detection limit, quantification limit, linearity, and relative standard deviation) was studied and the results obtained showed the method was suitable for routine analysis of glyphosate and AMPA. An interlaboratory test with five laboratories confirmed that good results were obtained by use of this method.     

Confirmation and identification of the impurities in metolachlor using gas chromatography interfaced with orthogonal acceleration time-of-flight mass spectrometry (GC-oaTOFMS)

The confirmation and identification of the impurities in metolachlor (a herbicde) by using gas chromatography-orthogonal acceleration time of flight mass spectrometry (GC-oaTOFMS) and gas chromatography-quadrupole mass spectrometry (GC-qMS) are described. For the accurate mass measurement can be carried by GC-oaTOFMS, the elemental compositions of molecular and fragment ions in the spectra are suggested. In the experiment the average of mass deviations between the measured and theoretical values was below 2.5 mDa. Finally ten impurities were confirmed and identified. They accounted for 94% of the total impurities by weight.     

Simultaneous fluorimetric determination of glyphosate and its metabolite, aminomethylphosphonic acid, in water, previous derivatization with NBD-Cl and by partial least squares calibration (PLS)

The reactions of 4-chloro-7-nitrobenzofurazan (NBD-Cl) with glyphosate (GLY) and with its main metabolite, aminomethylphosphonic acid (AMPA), have been studied. The resolution of binary mixtures of glyphosate and aminomethylphosphonic acid has been accomplished by partial least squares (PLS) multivariate calibration. The method of determination is based on the fluorescence emission of the derivatives formed in presence of NBD-Cl at 90 °C, in methanol and in basic medium. The dynamic ranges of the methods were comprised between 10 and 150 μg l⁻¹ for GLY and between 10 and 200 μg l⁻¹ for AMPA, being the detection limits 2 and 5.4 μg l⁻¹ for GLY and AMPA, respectively. The total luminiscence information of the derivatives has been used to optimize the spectral data set to perform the calibration, by analysis of the three-dimensional excitation-emission matrices. A comparison between the predictive ability of the multivariate calibration method, partial least squares type 1 (PLS-1), on two spectral data sets, emission and synchronous spectra, has been performed. The PLS-1 method, applied to the emission spectra, has been selected as optimum. The proposed method has been applied to the simultaneous determination of GLY and AMPA in river water. For concentrations ranging from 100 to 600 μg l⁻¹ of each compound in the samples, analytical recoveries range from 83 to 94% for GLY and from 104 to 120% for AMPA.     

Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells

Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). The binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture.     

Stable competitive enzyme-linked immunosorbent assay kit for rapid measurement of 11 active beta-lactams in milk, tissue, urine, and serum

Beta-lactams are used as veterinary drugs for the treatment of food-producing animals. For consumer protection, legislation is in place to set limits for their residues. An enzyme-linked immunosorbent assay (ELISA) was developed which allowed, in a single reaction, the class-specific measurement of 11 beta-lactams, with limits of detection below European maximum residue limits. Determinations were feasible in milk, tissue, urine, and serum with simple and rapid sample preparation. In this format, the specific capture antibodies were precoated on the microtiter plate and horseradish peroxidase-labeled conjugate was used to compete with free beta-lactams. The stability of the precoated microtiter plate and conjugate was at least 1 year when stored at 2 to 8 degrees C; upon reconstitution, the conjugate was stable for 6 days at 2 to 8 degrees C. The stability of lyophilized ampicillin standards was at least 6 months when stored at 2 to 8 degrees C and at least 1 year when stored at -20 degrees C. A low cross-reactivity, 3.6%, was observed with ampicillin with open beta-lactam ring relative to 100% for intact ampicillin. Generic recognition was shown by relative cross-reactivity values ranging from 22 (penicillin V) to 144% (nafcillin). Cross-reactivity for cephalosporins was <0.1%. Intra- and interassay precisions expressed as coefficient of variation were typically 2-8%. The inhibitory concentration with 50% binding for ampicillin was typically 2 ppb. Recovery for different spiked levels was >70% with all the matrixes.     

Development of a solid-phase extraction—enzyme-linked immunosorbent assay method for the determination of estrone in water

Estrone has been identified as a potential endocrine-disrupting chemical (EDC). To facilitate its analysis, a highly selective and sensitive enzyme-linked immunosorbent assay (ELISA) method with a simple solid-phase extraction (SPE) for analysis of estrone in aquatic environments has been developed. The specific polyclonal antibody was produced against a conjugate of estrone-3-hemisuccinate and keyhole limpet hemocyanin (KLH). The obtained ELISA showed specific recognition of estrone, without cross-reactions for three other major estrogenic compounds (17β-estradiol, estriol, and 17α-ethynylestradiol) commonly found in water. The ELISA had a limit of detection of 0.14 μg/l estrone in water. Combining a SPE method to extract and pre-concentrate estrone from water samples and ELISA to specifically quantify estrone content, the SPE-ELISA can detect estrone down to 1.25 ng/l level in water. Good recovery with spiked river water was obtained with this SPE-ELISA method. The developed SPE-ELISA system was applied to analyze the real influent and effluent samples of sewage treatment plant in Penrith (Australia) and the results correlated well with those obtained using GC and HPLC methods. The developed SPE-ELISA method is capable of being applied for the specific detection and routine monitoring of estrone in environmental water samples.     

Screening for testosterone, methyltestosterone, 19-nortestosterone residues and their metabolites in bovine urine with enzyme-linked immunosorbent assay (ELISA)

This work reports on the development of rapid enzyme-linked immunosorbent assay for testosterone, methyltestosterone, 19-nortestosterone and their metabolites in real samples of bovine urine. The assays were based on the competition between an immobilised testosterone-BSA conjugate and the analyte for corresponding antibodies, followed by the use of secondary anti-species (α-IgG-HRP) to determine the degree of competition. Urine samples were analysed after 10-fold dilution with the buffer, omitting extraction and hydrolysis. The limits of detection calculated for the original sample were ca. 74, 266 and 131 pg ml⁻¹ (ppt) for testosterone, methyltestosterone and 19-nortestosterone, respectively. In particular, testosterone and methyltestosterone assays offer the advantage to pick up both parent compounds and their major metabolites due to the high cross-reactivity pattern with corresponding antibody used in each assay. The concentration in bovine urine detected by developed method does indicate 19-nortestosterone administration to heifers. The developed assays were applied to urine samples of heifers treated with above androgens.     

间接酶联免疫吸附分析法对环境水样中左旋18-甲基炔诺酮含量的测定

本文报道测定环境水样中的左旋18-甲基炔诺酮(NG)的间接酶联免疫吸附分析法(ELISA)。通过对左旋18-甲基炔诺酮的化学修饰,将左旋18-甲基炔诺酮与蛋白质载体结合,制成完全免疫抗原,经过多次动物免疫得到了兔抗左旋18-甲基炔诺酮抗体,在优化实验条件的基础上,建立了灵敏度高、特异性强、简便、稳定的测定水样中左旋18-甲基炔诺酮的酶联免疫吸附分析方法。IC50值(标准曲线中吸光度抑制至最大吸光度值的50%时所对应的待测物浓度)在1μg/L~5μg/L,最低检出限为0.05μg/L~0.1μg/L。水样的加标回收率在88%~140%之间。真实环境水样中,均发现含有左旋18-甲基炔诺酮,浓度在0.3μg/L-0.6μg/L之间。     

Development of an analytical protocol for a fast,sensitive and specific protein recognition in paintings by enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) analysis of proteins offers a particularly promising approach for investigations in cultural heritage on account of its appreciated properties of being highly specific, sensitive, relatively fast, and cost-affordable with respect to other conventional techniques. In spite of that, it has never been fully exploited for routine analyses of painting materials in consideration of several analytical issues that inhibited its diffusion in conservation science: limited sample dimensions, decrease of binder solubility and reduced availability of antibody bonding sites occurring with protein degradation. In this study, an ELISA analytical protocol suited for the identification of aged denatured proteins in ancient painting micro-samples has been developed. We focused on the detection of bovine β-casein and chicken ovalbumin as markers of bovine milk (or casein) and chicken albumen, respectively. A systematic experimentation of the ELISA protocol has been carried out on mock-ups of mural and easel painting prepared with 13 different pigments to assess limits and strengths of the method when applied for the identification of proteins in presence of a predominant inorganic matrix. The analytical procedure has been optimized with respect to protein extraction, antibodies’ concentrations, incubation time and temperature; it allows the detection of the investigated proteins with sensitivity down to nanograms. The optimized protocol was then tested on artificially aged painting models. Analytical results were very encouraging and demonstrated that ELISA allows for protein analysis also in degraded painting samples. To address the feasibility of the developed ELISA methodology, we positively investigated real painting samples and results have been cross-validated by gas chromatography–mass spectrometry.     

Application of bamboo charcoal as solid-phase extraction adsorbent for the determination of atrazine and simazine in environmental water samples by high-performance liquid chromatography-ultraviolet detector

In this article, a new method for the determination of triazine herbicides atrazine and simazine in environment aqueous samples was developed. It was based on solid-phase extraction (SPE) using bamboo charcoal as adsorbent and high-performance liquid chromatography-ultraviolet detector (HPLC-UV) for the enrichment and determination of atrazine and simazine at trace level. Related important factors influencing the extraction efficiency, such as the kind of eluent and its volume, flow rate of the sample, pH of the sample, and volume of the sample, were investigated and optimized in detail. Under the optimal conditions, the experimental results showed that excellent linearity was obtained over the range of 0.5–30 μg L⁻¹ with correlation coefficients 0.9991 and 0.9982, for atrazine and simazine, respectively; and the relative standard deviations of two analytes were 8.3, 8.7%, respectively. The proposed method was successfully applied to the analysis of tap water and well water samples. And satisfactory spiked recoveries were obtained in the range of 75.2–107.1%. The above results indicated that the developed method was an excellent alternative for the routine analysis in environmental field.     

A simple and rapid competitive enzyme-linked immunosorbent assay (cELISA) for high-throughput measurement of secretory immunoglobulin A (sIgA) in saliva

A simple competitive enzyme-linked immunosorbent assay (cELISA) was established for rapid measurement of secretory immunoglobulin A (sIgA) in saliva. The method was based on competitive reaction between the immobilized IgA and free IgA in the solution for the limited amount of horseradish peroxidase-conjugated rabbit anti-human IgA. In comparison with the conventionally used Sandwich ELISA, the cELISA is simpler, low-cost, and shows better reproducibility since it is not affected by the variation of capture antibodies from different batches. The assay time was also significantly reduced from more than 5 h to less than 3 h. Different curve-fitting models were compared, among which the fully specified logit-log model gave the best results. The linear working range and limit of detection were found to be 0.1-100 μg mL⁻¹ and 0.05 μg mL⁻¹, respectively. Matrix effects of saliva samples were investigated and a reasonable range of dilution factors were proposed. The developed method offers a very practical approach for high-throughput measurement of sIgA in saliva samples.     

A fast screening method for the presence of atrazine and other triazines in water using flow injection with chemiluminescent detection

Atrazine is a triazine herbicide which contains two secondary aliphatic amine groups. Previous studies have shown that aliphatic amines react with tris(2,2′-bipyridyl)ruthenium(III) to produce chemiluminescence. This paper describes the application of tris(2,2′-bipyridyl)ruthenium(III) to the detection of atrazine and related triazine herbicides in water by flow injection chemiluminescence analysis. The optimised experimental conditions were determined to be: sample and carrier flow rates of 4.6 mL min⁻¹, sample at pH 9 buffered with 50 mM borax, and reagent concentration of 1 mM tris(2,2′-bipyridyl)ruthenium(III) in 20 mM H₂SO₄ (pH 1). Under these conditions, the logarithm of the chemiluminescence intensity versus concentration was linear in the range of 2.15-2150 μg L⁻¹ for samples in MilliQ water, and the limit of detection of atrazine in water was determined to be 1.3 ± 0.1 μg L⁻¹. Validation of the method was performed using direct injection HPLC. The presence of natural organic matter (NOM) significantly increased the chemiluminescence, masking the signal generated by atrazine. Isolating the target analyte via solid phase extraction (SPE) prior to analysis removed this interference and concentrated the samples, resulting in a greatly improved sensitivity with a detection limit of 14 ± 2 ng L⁻¹.     

Development of a microparticle-based on-site immunoassay for the detection of atrazine in soil and water samples

Atrazine is widely used as a herbicide in agriculture and has been identified as a major groundwater contaminant in the US. Because of the possible hazard associated with its usage, there is a need for an efficient and economic screening method for on-site field testing of atrazine and other s-triazine herbicides in soil and water. We have developed a rapid, on-site test for the detection of atrazine based on the principle of microparticle agglutination inhibition immunoassay. The test detects 50 microg kg(-1) (0.050 ppm) atrazine in soil samples with direct extraction and 1.0 microg L(-1) atrazine in water samples when coupled with solid phase extraction.     

Development of a sequential injection chromatography (SIC) method for determination of simazine,atrazine, and propazine

This paper describes the development of a sequential injection chromatography (SIC) procedure for separation and quantification of the herbicides simazine, atrazine, and propazine exploring the low backpressure of a 2.5 cm long monolithic C₁₈ column. The separation of the three compounds was achieved in less than 90 s with resolution >1.5 using a mobile phase composed by ACN/1.25 mmol/L acetate buffer (pH 4.5) at the volumetric ratio of 35:65 and flow rate of 40 μL/s. Detection was made at 223 nm using a flow cell with 40 mm of optical path length. The LOD was 10 μg/L for the three triazines and the quantification limits were of 30 μg/L for simazine and propazine and 40 μg/L for atrazine. The sampling frequency is 27 samples per hour, consuming 1.1 mL of ACN per analysis. The proposed methodology was applied to spiked water samples and no statistically significant differences were observed in comparison to a conventional HPLC–UV method. The major metabolites of atrazine and other herbicides did not interfere in the analysis, being eluted from the column either together with the unretained peak, or at retention times well‐resolved from the studied compounds.     

Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sudan I in food samples

The use of Sudan I as an additive in food products has been prohibited in the European Union and many other countries. In this study, a highly sensitive and specific monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of Sudan I in food samples was developed. The hapten derivative with a three-carbon-atom length of carboxylic spacer at the azobound para-position was synthesized and coupled to carrier proteins. The hapten-bovine serum albumin (BSA) conjugate was used as an immunogen, while the hapten-ovalbumin (OVA) conjugate was applied as a coating antigen. The mAb against Sudan I was produced by hybridoma technique and the corresponding ELISA was characterized in terms of sensitivity, specificity, precision and accuracy. At optimal experimental conditions, the standard curve was constructed in concentrations of 0.1-100 ng mL⁻¹. The values of IC₅₀ for nine standard curves were in the range of 1.1-2.0 ng mL⁻¹ and the LOD at a signal-to-noise ratio of 3 (S/N = 3) was 0.07-0.14 ng mL⁻¹. The cross-reactivity values of the mAb with Sudan II, III and IV were 9.5%, 33.9% and 0.95%; no cross-reactivity was found with other six edible colorants: Lemon yellow, Bright blue, Indigotin, Kermes, Amarant and Sunset yellow, indicating the assay displays not only high sensitivity but also high specificity as well. The organic solvent effect on the assay was tested. It was observed that the ELISA was tolerated to 30% of methanol and 10% of acetonitrile without significant loss of IC₅₀ value. Six food samples were spiked with Sudan I and the methanolic extracts after appropriate dilution were analyzed by ELISA. Acceptable recovery rates of 88.2-110.5% and coefficients of variation of 2.5-17.4% were obtained. The ELISA for nine spiked samples was confirmed by high-performance liquid chromatography (HPLC) with a high correlation coefficient of 0.9840 (n = 9). The mAb-based ELISA proven to be a feasible quantitative/screening method for Sudan I analysis in food samples with the properties of high sensitivity, specificity, simplicity of sample pretreatment, high sample throughput and low expense.     

Validation of an enzyme-linked immunosorbent assay (ELISA) for the determination of 4-nonylphenol and octylphenol in surface water samples by LC-ESI-MS

4-Nonylphenol (NP) and octylphenol (OP) were measured by direct ELISA in both laboratory-fortified and surface water samples collected monthly from 10 rivers. In this procedure, samples were concentrated by solid phase extraction (SPE) using Lichrolut RP-18 sorbent with good recoveries obtained for both LC-MS and ELISA, giving a low level of detection (LOD) at the range of low μg L⁻¹ and good reproducibility. Analysis of 40 surface water samples demonstrated that the ELISA may be a useful screening tool for the determination of the alkylphenols in surface water matrices. The concentration of NP and OP in surface waters ranged from 0.11 to 6.58 μg L⁻¹. A good correlation of results obtained by ELISA and LC-MS within the concentration range of 0.08-6.86 μg L⁻¹ was found in the river samples [R² = 0.924, n = 39]. The influence of various factors on assay determination was also discussed.