Determination of carbaryl, carbofuran and methiocarb in cucumbers and strawberries by monoclonal enzyme immunoassays and high-performance liquid chromatography with fluorescence detection. An analytical comparison

Carbaryl, carbofuran and methiocarb are three of the most important N-methylcarbamate pesticides. In the present work, the application of laboratory-developed monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) to the determination of these compounds in fruits and vegetables is described. Cucumbers and strawberries were spiked with the three carbamates at 10, 50 and 200 ppb. After extraction and clean-up, samples were analyzed by immunoassay and by HPLC with post-column derivatization and fluorescence detection (US Environmental Protection Agency Method 531.1). Results obtained by ELISA correlated well with those obtained by HPLC, both in terms of accuracy and precision. Recoveries were in the 60-90% range by ELISA and in the 50-90% range by HPLC, depending on the particular combination of commodity, pesticide, and fortification level under consideration. ELISAs were also applied to the analysis of non-purified sample extracts with excellent results: recoveries close to 100% were obtained, while maintaining similar precision values. This approach avoids the use of solid-phase extraction columns, saves time, and considerably increases the sample throughput. Results clearly indicate that the developed immunoassays may be suitable for the quantitative and reliable determination of carbaryl, carbofuran and methiocarb in fruits and vegetables even without including clean-up steps. These considerations make these ELISAs very useful analytical tools for monitoring and regulatory programs, without the need of complex and expensive instrumentation.     

Determination of pesticides in soy-based infant formula using liquid chromatography with electrospray ionization tandem mass spectrometry

A sensitive method using liquid chromatography with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was developed and validated to quantify and confirm 13 pesticides, including aldicarb sulfoxide, aldicarb sulfone, oxamyl, methomyl, formetanate, 3-hydroxycarbofuran, carbendazim, thiabendazole, aldicarb, propoxur, carbofuran, carbaryl, and methiocarb, in soy-based infant formula. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring of 2 fragment ion transitions to provide a high degree of sensitivity and selectivity for both quantitation and confirmation. Different approaches to constructing calibration curves were compared and discussed to address issues of the extraction efficiency or recovery, and matrix effects. Matrix-matched standard calibration curves with the use of isoprocarb as an internal standard were finally used to achieve the best accuracy of the method. Under most circumstances, recoveries of 13 pesticides, spiked at 5.0, 25.0, and 45.0 microg/kg, were close to 100%. The method detection limits (signal-to-noise ratio > or =3:1; microg/kg) of 13 pesticides were 0.2 for thiabendazole and methiocarb, 0.6 for aldicarb, and 0.1 for the others.     

Immobilization strategies of Brucella particles on optical fibers for use in chemiluminescence immunosensors

Immunosensors are powerful analytical tools in clinical and veterinary diagnostics. This has led us to design a chemiluminescent immunosensor aimed at identifying anti-Brucella antibodies using optical fibers as the transducer. In order to develop the optimal transducer, to achieve an optimal chemical modification thereby allowing an optimal covalent binding of the protein receptor, several cleaning strategies and silane coupling agents were investigated. Brucella killed organisms were used as a model receptor for quantifying anti-Brucella IgG antibodies in a suspension compared to conventional colorimetric and chemiluminescent ELISA. A silane-benzophenone derivative was selected as the best performing silane coupling agent: the optical fiber immunosensor (OFIS) has showed the lowest limit of detection at 0.207 μg/ml, compared to 0.828 μg/ml and 0.414 μg/ml achieved by colorimetric and chemiluminescent ELISAs, respectively. These results, together with the additional advantages of rapidity, lower reagent volumes and moderate operating conditions, have set the grounds for further study in order to adapt this platform for on-site diagnostics of brucellosis disease markers.     

Determination of organophosphate and carbamate pesticides based on enzyme inhibition using a pH-sensitive fluorescence probe

A flow injection system for the determination of organophosphate and carbamate pesticides is described. A sensitive fluorescence probe was synthesized and used as the pH indicator to detect the inhibition of the enzyme acetylcholinesterase (AChE). The percentage inhibition of enzyme activity is correlated to the pesticide concentration. Several parameters influencing the performance of the system are discussed. The detection limits of 3.5, 50, 12 and 25 μg/l for carbofuran, carbaryl, paraoxon and dichlorvos, in pure water, respectively were achieved with an incubation time of 10 min. A complete cycle of analysis, including incubation time, took 14 min. The detection system has been applied to the determination of carbofuran in spiked vegetable juices (Chinese cabbage and cole), achieving recovery values between 93.2 and 107% for Chinese cabbage juice and 108 and 118% for cole juice at the different concentration levels assayed.     

Analysis of carbamate pesticides in water samples using single-drop microextraction and gas chromatography–mass spectrometry

Single-drop microextraction (SDME) followed by gas chromatography–mass spectrometry detection was used for the determination of some carbamate pesticides in water samples. The studied pesticides were thiofanox, carbofuran, pirimicarb, methiocarb, carbaryl, propoxur, desmedipham and phenmedipham. Two alternative sample introduction methods have been examined and compared; SDME followed by cool on-column injection (without derivatization) and SDME followed by in-microvial derivatization and splitless injection. Acetic anhydride was used as derivatization reagent. Parameters that affect the derivatization reaction yield and the extraction efficiency of the SDME method were studied and optimized. The analytical performances and possible applications of both approaches were investigated. Relative standard deviations for the studied compounds ranged from 3.2 to 8.3%. The detection limits obtained by the derivatization method were found to be in the range 3–35 ng/L. Using cool on-column injection (without derivatization), the detection limits were between 30 and 80 ng/L.     

Room temperature imidazolium ionic liquid: a solvent for extraction of carbamates prior to liquid chromatographic analysis

A simple and rapid method for preconcentration of carbamate insecticides, including methomyl, propoxur, carbofuran, carbaryl, isoprocarb, methiocarb and promecarb, has been developed. It was based on a liquid-liquid microextraction using a [C₄MIM][PF₆] room temperature ionic liquid as an extraction solvent prior to analysis by high performance liquid chromatography with UV detection. Experimental parameters affecting the extraction performance, such as the volumes of sample, extractant and dissolving solvent, and extraction time, were studied. Under the selected conditions, the enrichment factors in the range between 10 and 25 could be achieved with the limit of detection in the range of 2-40 μg L⁻¹, and with the relative standard deviations of lower than 0.6 and 10.2% for retention time and peak area, respectively. The proposed method offers advantages in reduction of the exposure danger to toxic solvents used in the conventional liquid-liquid extraction, simplicity of the extraction processes, rapidity, and sensitivity enhancement. The method was demonstrated to apply to the analysis of fruit and natural surface water samples.     

Multivariate curve-resolution analysis of pesticides in water samples from liquid chromatographic-diode array data

Liquid chromatographic-diode array detection data recorded for aqueous mixtures of 11 pesticides show the combined presence of strongly coeluting peaks, distortions in the time dimension between experimental runs, and the presence of potential interferents not modeled by the calibration phase in certain test samples. Due to the complexity of these phenomena, data were processed by a second-order multivariate algorithm based on multivariate curve resolution and alternating least-squares, which allows one to successfully model both the spectral and retention time behavior for all sample constituents. This led to the accurate quantitation of all analytes in a set of validation samples: aldicarb sulfoxide, oxamyl, aldicarb sulfone, methomyl, 3-hydroxy-carbofuran, aldicarb, propoxur, carbofuran, carbaryl, 1-naphthol and methiocarb. Limits of detection in the range 0.1-2 μg mL⁻¹ were obtained. Additionally, the second-order advantage for several analytes was achieved in samples containing several uncalibrated interferences. The limits of detection for all analytes were decreased by solid phase pre-concentration to values compatible to those officially recommended, i.e., in the order of 5 ng mL⁻¹.     

Identification of Carbofuran And Methiocarb and their Transformation Products in Estuarine Waters by On-line Solid Phase Extraction Liquid Chromatography—Mass Spectrometry

Abstract

The degradation of the carbamate insecticides carbofuran and methiocarb in distilled and natural waters was determined. Degradation studies were carried out both under a xenon arc irradiation and natural sunlight at pesticide concentrations of 50–100 μg/L. 50–100 mL water sample were preconcentrated using automated online solid phase extraction (SPE) followed by liquid chromatography (LC), UV detection or post column fluorescence detection (EPA method 531.1 for carbamate insecticides). Structure identification was carried out by on-line SPE-LC-MS either with thermospray and/or high flow pneumatically assisted electrospray interfaces. Half-lives varying between 4–12.5 days for carbofuran and methiocarb were determined under natural sunlight exposure, being chemical hydrolysis the major degradation pathway. When using xenon arc lamp irradiation both pesticides degraded very rapidly with half-lives varying from 0.3–1.7 hours. The various degradation products identified were: methiocarb sulfoxide, 4-methylthio-3, 5-dimethylphenol, 3-hydroxy-7-carbofuranphenol and 2-hydroxy-3-(2-methylprop-1-enyl)-phenyl-N-methylcarbamate.     

Use of high-performance liquid chromatography with diode array detection coupled to electrospray-Qq-time-of-flight mass spectrometry for the direct characterization of the phenolic fraction in organic commercial juices

We have developed a direct method for the qualitative analysis of polyphenols in commercial organic fruit juices. The juices were diluted with water (50/50), filtered and directly injected. The analysis of phenolic compounds was carried out by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to photodiode array detection (DAD) and electrospray ionisation-Qq-time-of-flight mass spectrometry (ESI-Qq-TOF-MS). A unique gradient program has been optimized for the separation of several phenolic classes and the analysis time was only 5 min. The fruit juice samples were successfully analysed in positive and negative ionisation modes. In positive mode the anthocyanins were identified whereas the vast majority of polyphenols were identified using the negative ionisation mode. The sensitivity, together with mass accuracy and true isotopic pattern of the Qq-TOF-MS, allowed the identification of the phenolic compounds. Moreover, the advantage of the proposed method is the combined search of MS and MS/MS spectra, which improves the identification of compounds considerably, reducing ambiguities and false positive hits. Therefore the total fragmentation of the compound ion leading to the aglycone ion or other fragments was corroborated by MS–MS. The method was successfully employed to characterize diverse phenolic families in commercially available organic juices from four different fruits and consequently could be used in the future for the quantification purposes to compare different content of polyphenols in juices.     

Monitoring some phenoxyl-type N-methylcarbamate pesticide residues in fruit juices using high-performance liquid chromatography with peroxyoxalate-chemiluminescence detection

A systematic investigation of optimal conditions for determining three of the most common phenoxyl-type N-methylcarbamate pesticides (carbaryl, carbofuran and propoxur) in fruit juices by HPLC with peroxyoxalate-chemiluminescence detection is described. The required pre-column hydrolysis of pesticides and derivatization of their hydrolytic metabolites with dansyl chloride was simultaneously carried out in a short time thanks to the micellar catalytic effect provided by cetyltrimethyl ammonium bromide micelles on the hydrolysis step. The liquid chromatographic separation of the dansylated phenols was performed on a reversed-phase C18 column with isocratic elution. The analytes were detected by using an integrated derivatization chemiluminescence detection unit based on the bis(2,4,6-trichlorophenyl)oxalate-hydrogen peroxide system. Fruit juice samples containing 4.0-1500 microg/l pesticides were analysed with a precision of ca. 6.5%. After contamination of the fruit juice samples, average recovery > 93% at fortification levels of 10-100 microg/l was obtained.     

Atmospheric pressure glow discharge desorption mass spectrometry for rapid screening of pesticides in food

Flowing afterglow atmospheric pressure glow discharge tandem mass spectrometry (APGD-MS/MS) is used for the analysis of trace amounts of pesticides in fruit juices and on fruit peel. The APGD source was rebuilt after Andrade et al. (Andrade et al., Anal. Chem. 2008; 80: 2646-2653; 2654-2663) and mounted onto a hybrid quadrupole time-of-flight mass spectrometer. Apple, cranberry, grape and orange juices as well as fruit peel and salad leafs were spiked with aqueous solutions containing trace amounts of the pesticides alachlor, atrazine, carbendazim, carbofuran, dinoseb, isoproturon, metolachlor, metolcarb, propoxur and simazine. Best limits of determination (LODs) of pesticides in the fruit juices were achieved for metolcarb (1 microg/L in apple juice), carbofuran and dinoseb (2 microg/L in apple juice); for the analysis of apple skin best LODs were 10 pg/cm(2) of atrazine, metolcarb and propoxur which corresponds to an estimated concentration of 0.01 microg/kg apple, taking into account the surface area and the weight of the apple. The measured LODs were within or below the allowed maximum residue levels (MRLs) decreed by the European Union (1-500 microg/kg for pesticides in fruit juice and 0.01-5 microg/kg for apple skin). No sample pretreatment (extraction, pre-concentration, chromatographic separation) was necessary to analyze these pesticides by direct desorption/ionization using APGD-MS and to identify them using MS/MS. This makes APGD-MS a powerful high-throughput tool for the investigation of very low amounts of pesticides in fruit juices and on fruit peel/vegetable skin. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Contactless conductivity detection of sodium monofluoroacetate in fruit juices on a CE microchip

Rapid and quantitative determination of sodium monofluoroacetate in diluted fruit juices (dilution 1:9 v/v in deionized water) and tap water was performed by microchip CE, using contactless conductivity detection. A separation buffer consisting of 20 mM citric acid and histidine at pH 3.5 enabled the detection of the monofluoroacetate (MFA) anion in diluted apple juice, cranberry juice, and orange juice without lengthy sample pretreatments. The analyte was very well separated from interfering anionic species present in juices and tap water. LODs in diluted juices and tap water were determined to be 125, 167, 138, and 173 microg/L for tap water, apple juice, cranberry juice, and orange juice, respectively, based upon an S/N of 3:1. Taking into account the dilution factor, the LODs for juice samples range from 1 to 2 mg/L, which is adequate for monitoring the toxicity of MFA in these juice beverages and tap water. The calibration curves for MFA in diluted fruit juices were linear over the range of 500 microg/L to 80 mg/L. The total analysis time for detecting the MFA anion in fruit juices was less than 5 min, which represents a considerable reduction in analysis time compared to other analytical methods currently used in food analysis.     

Determination of seventeen polar/thermolabile pesticides in apples and apricots by liquid chromatography/mass spectrometry

A simple liquid chromatography/mass spectrometry (LC/MS) approach for the determination of widely used representatives of polar/thermolabile pesticides in fruits was developed and validated. The group of pesticides comprised benzimidazoles and azoles (carbendazim, thiabendazole, imazalil, propiconazole, prochloraz, epoxiconazole, flusilazole, tebuconazole, bitertanol); N-methylcarbamates (carbaryl, carbofuran, methiocarb); and phenylureas and benzoylphenylureas (linuron, diflubenzuron, triflumuron, teflubenzuron, flufenoxuron). Matrixes (apple, apricot) were extracted with acetonitrile and crude extracts were cleaned up by solid-phase extraction (SPE) using either mixed cation exchange or hydrophilic lipophilic balance cartridges. LC separation of pesticides was performed on a reversed-phase column, Discovery C18. Electrospray ionization and ion trap MS/MS detection were applied. For most pesticides, overall recoveries ranged from 75 to 122%, and repeatability (as relative standard deviation) from 5 repetitive determinations of recovery ranged from 3 to 21%. Carbofuran was the only compound for which recovery was not satisfactory due to its loss in the SPE cleanup step. Limits of detection were 0.1-3 microg/kg for benzimidazole and azole fungicides and carbamate insecticides. For urea insecticides, detection limits were slightly higher (3-10 microg/kg).     

Determination of N-methylcarbamate pesticides in water and vegetable samples by HPLC with post-column chemiluminescence detection using the luminol reaction

In this paper we proposed a reverse high performance liquid chromatography method for the simultaneous determination of three N-methylcarbamates (NMCs) named carbofuran, carbaryl and methiocarb, using the post-column chemiluminescence (CL) detection with the luminol reaction. This method is based on the enhancing effect of these analytes on the CL emission generated by the oxidation of luminol with potassium permanganate in alkaline medium. The separation was reached in less than 14 min using a C18 column and an isocratic binary mobile phase consisting of acetonitrile:water (50:50, v/v) pumped at a flow rate of 1 mL min⁻¹. CL reagents (luminol and KMnO₄) were incorporated by means of a peristaltic pump and were firstly mixed using a three-way connector. Then this stream was mixed with the eluate using another three-way connector just before reaching the detection cell. The optimization of variables affecting the CL reaction (reaction medium, concentration, flow rate of reagents and distance between both connectors) were optimized by means of experimental designs. Ethiofencarb, a NMC which has nowadays fallen into disuse was used as internal standard. For the analysis of theses pesticides in real water samples a pre-treatment step consisting of solid phase extraction (SPE) was conducted in order to reach sensitivity levels below the legal maximum concentration permitted. In the case of vegetable sample, SPE was used for matrix cleaning purpose.     

Stability studies of carbamate pesticides and analysis by gas chromatography with flame ionization and nitrogen-phosphorus detection

As a result of thermal stability studies of carbamate pesticides, a method has been proposed for their direct determination by gas chromatography in the ranges 1-20 and 0.1-1 mg l(-1), using flame ionization and nitrogen-phosphorus detection, respectively. The method allows the determination of propham, propoxur, carbofuran, carbaryl, methiocarb, isopropoxyphenol and naphthol in powdered potato samples. The analytes were previously extracted with a light petroleum-dichloromethane (1:1, v/v) mixture and preconcentred by solid-phase extraction through a C8 cartridge. The recoveries obtained from spiked potato samples (n=4 replicates) at two concentration levels, 10 and 0.5 mg of pesticide per kg of sample, were in the ranges 72-115 and 50-73%, with relative standard deviations of 2-7 and 5-8%, respectively. The detection limits were 50-210 and 41-53 microg kg(-1) with flame ionization and nitrogen-phosphorus detection, respectively, and reaching the maximum residue levels, 0.05 mg kg(-1) for methiocarb and propoxur, set by the Real Decreto 280/1994 (based on the European directive).     

超高效液相色谱-串联质谱法快速测定浓缩果汁中噻菌灵和多菌灵的残留量

建立了同时测定浓缩果汁中噻菌灵和多菌灵残留的超高效液相色谱-串联质谱快速检测法。样品用乙酸乙酯提取,以ACQUITY UPLC BEH C18色谱柱(50 mm×2.1 mm, 1.7 μm)进行超高效液相色谱分离,以电喷雾电离串联质谱正离子多反应监测(MRM)模式进行测定,以基质匹配标准溶液外标法定量。结果表明:在试验条件下,噻菌灵和多菌灵在0.5~10 μg/kg范围内线性关系良好,相关系数大于0.99,不同基质中的检出限(S/N=3)范围为0.12~0.23 μg/kg。在0.5、1.0和5.0 μg/kg 3个水平下噻菌灵和多菌灵的加标回收率为76.98%~108.7%,相对标准偏差(RSD)为2.95%~9.99%。同时,本研究对浓缩果汁中噻菌灵和多菌灵残留检测的基质效应进行了考察。本方法具有操作简便、快速、准确的特点,可用于浓缩果汁中噻菌灵和多菌灵残留量的日常检测。     

Sequential injection-bead injection-lab-on-valve coupled to high-performance liquid chromatography for online renewable micro-solid-phase extraction of carbamate residues in food and environmental samples

A sequential injection‐bead injection‐lab‐on‐valve system was hyphenated to HPLC for online renewable micro‐solid‐phase extraction of carbamate insecticides. The carbamates studied were isoprocarb, methomyl, carbaryl, carbofuran, methiocarb, promecarb, and propoxur. LiChroprep^® RP‐18 beads (25–40 μm) were employed as renewable sorbent packing in a microcolumn situated inside the LOV platform mounted above the multiposition valve of the sequential injection system. The analytes sorbed by the microcolumn were eluted using 80% acetonitrile in 0.1% acetic acid before online introduction to the HPLC system. Separation was performed on an Atlantis C‐18 column (4.6×150 mm, 5 μm) utilizing gradient elution with a flow rate of 1.0 mL/min and a detection wavelength at 270 nm. The sequential injection system offers the means of performing automated handling of sample preconcentration and matrix removal. The enrichment factors ranged between 20 and 125, leading to limits of detection (LODs) in the range of 1–20 μg/L. Good reproducibility was obtained with relative standard deviations of <0.7 and 5.4% for retention time and peak area, respectively. The developed method has been successfully applied to the determination of carbamate residues in fruit, vegetable, and water samples.     

Electrospun zeolitic imidazolate framework-8/poly(lactic acid) nanofibers for pipette-tip micro-solid phase extraction of carbamate insecticides from environmental samples

In the present study, electrospun zeolitic imidazolate framework-8/poly(lactic acid) nanofibers were successfully synthesized and characterized as a potential nanosorbent for the pipette-tip micro-solid phase extraction of chlorpropham, pirimicarb, carbaryl, and methiocarb carbamate insecticides from environmental water samples. The extraction procedure was followed by gas chromatography/mass spectrometry separation and determination of the target analytes. All the effective parameters of the extraction procedure were optimized through the one variable at-a-time method. Thanks to the very simple extraction procedure as well as the application of electrospun nanofibers with high surface area, the four analytes were efficiently extracted with as lowest extraction times as practicable. Under the optimal conditions, the calibration plots of the analytes were obtained within broad linear dynamic ranges of 0.5 – 150 ng mL^?1 for chlorpropham and pirimicarb plus 1.0 – 175 ng mL^?1 for carbaryl and methiocarb, respectively. Besides, limits of detection as low as 0.2 and 0.15 ng mL^?1 for chlorpropham and pirimicarb, respectively, as well as 0.5 ng mL^?1 for carbaryl and methiocarb indicate the favorable sensitivity of the analytical procedure. The applicability of the developed method was evaluated by quantitative determination of the target analytes in four different environmental water samples. Relative recoveries higher than 88.0% shows the acceptable accuracy of the method in the quantitative determination of the four carbamate insecticides.     

Validation of a method for the determination of multiclass pesticide residues in fruit juices by liquid chromatography/tandem mass spectrometry after extraction by matrix solid-phase dispersion

A multiresidue method was developed and validated for the determination of pesticide residues (omethoate, dimethoate, carbendazim, propoxur, thiabendazole, carbaryl, pirimicarb, azinphos-methyl, methidathion, and iprodione) in fruit juices. The samples were extracted by matrix solid-phase dispersion with diatomaceous earth and analyzed by liquid chromatography/tandem mass spectrometry. The method detection limits were <0.2 ppb for all pesticides; the relative standard deviations for analyses of samples fortified over the range of 2-50 ng/g were <9%, and the recoveries for each pesticide were all between 77 and 102%. The proposed method was used to analyze 21 commercial fruit juices; pesticide residues were found in 71% of the samples.     

Derivatization reactions of carbamate pesticides in supercritical carbon dioxide

Supercritical fluid carbon dioxide (SC-CO(2)) has been used to dissolve derivatizing agents (e.g. heptafluorobutyric anhydride, HFBA, and pyridine), which also act as a modifier in the fluid phase, for simultaneous extraction and derivatization of carbamates from the sample matrix. The derivatized carbamate pesticides (carbaryl, 3-hydroxycarbofuran, carbofuran, aldicarb, methiocarb) were then analyzed by GC-ECD or GC-MS with excellent sensitivity. Extraction and conversion of the carbamates was complete, as indicated by HPLC with post-column hydrolysis and o-phthalaldehyde derivatization then fluorescence detection. GC-MS (ion trap) was also used to confirm the formation of the carbamate derivatives. Compared with the same HFBA reaction in an organic solvent the derivatization reaction time was considerably shorter in SC-CO(2.) The described approach, combining both extraction and derivatization, simplifies the analysis of carbamate pesticides and eliminates the use of organic solvents associated with the derivatization step.