Development and Validation of a Gas Chromatography-Mass Spectrometry Method for Determining Acaricides in Bee Pollen.

Pesticides can be found in beehives for several reasons, including contamination from surrounding crops or for their use by beekeepers, which poses a risk to bee ecosystems and consumers. Therefore, efficient and sensitive methods are needed for determining pesticide residues in bee products. In this study, a new analytical method has been developed and validated to determine seven acaricides (atrazine, chlorpyrifos, chlorfenvinphos, α-endosulfan, bromopropylate, coumaphos, and τ-fluvalinate) in bee pollen using gas chromatography coupled to mass spectrometry. After an optimization study, the best sample treatment was obtained when using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method employing an ethyl acetate and cyclohexane as the extractant mixture, and a mixture of salts for the clean-up step. A chromatographic analysis (<21 min) was performed in an Agilent DB-5MS column, and it was operated under programmed temperature conditions. The method was fully validated in terms of selectivity, limits of detection (0.2-3.1 µg kg-1) and quantification (0.6-9.7 µg kg-1), linearity, matrix effect (<20% in all cases), trueness (recoveries between 80% and 108%), and precision. Finally, the proposed method was applied to analyze commercial bee pollen samples, and some of the target pesticides (chlorfenvinphos, α-endosulfan, coumaphos, and τ-fluvalinate) were detected.

Facile synthesis of a core-shell structured magnetic covalent organic framework for enrichment of organophosphorus pesticides in fruits.

A facile strategy was developed for the fabrication of a magnetic covalent organic framework (COF) via grafting of the monomers, 2,5-dihydroxyterephthalaldehyde (Dt) and 1,3,5-tris(4-aminophenyl) benzene (Tb) onto surface-modified Fe3O4 nanoparticles. The magnetic COF, named as magnetic COF-DtTb, was readily fabricated without high temperature or harsh reaction conditions. The synthesized magnetic COF-DtTb nanoparticles were fully characterized, presenting a regular core-shell spherical structure, large specific surface area, superparamagnetism, and good thermal stability. Their potential as an enrichment adsorbent was investigated to establish an efficient magnetic solid-phase extraction method for the determination of organophosphorus pesticide residues in fruits. Systematic method validation revealed good linearity in the concentration range of 1-200 µg L-1 (correlation coefficient >0.9957). The method limits of detection were in the range of 0.002-0.063 µg kg-1, the method limit of quantification was 1.00 µg kg-1 and recoveries ranged from 72.8% to 111% with RSDs lower than 12.3%. The results indicated that magnetic COF-DtTb possesses superior trace enrichment properties for organophosphorus pesticides in fruits.

An assessment of pesticide exposures and land use of honey bees in Virginia.

Large-scale honey bee colony loss threatens pollination services throughout the United States. An increase in anthropogenic pressure may influence the exposure of hives to household and agricultural pesticides. The objective of this survey was to provide an assessment of the risk of exposure to commonly used pesticides to honey bee colonies in Virginia in relation to land use. Adult honey bee, pollen, and wax samples from colonies throughout Virginia were evaluated for pyrethroid, organophosphate, organochlorine, and triazine pesticides using gas chromatography-mass spectrometry analysis. Of the 11 pesticides analyzed, nine were detected in one or more hive matrices. The probability of detecting a pesticide in pollen was less in forests than in pasture, agriculture, or urban landscapes. Coumaphos and fluvalinate were significantly more likely to be detected across all matrices with concentrations in wax as high as 15500 and 6970 ng/g (dry weight), respectively, indicating the need for further research on the potential effects of miticide accumulation in wax to larval and adult bees.

Identification and measurement of veterinary drug residues in beehive products.

There is an increasing concern about the negative impacts of veterinary drugs in beehive compartments. This study evaluates the presence and distribution of chemical residues in beeswax, bee bread and honey and determinates in what extension honeybees are exposed to them. Samples were analyzed by LC-MS/MS and GC-MS/MS with a wide scope of 322 chemical residues. Samples were collected from apiaries located in rural and forest areas, showing no difference in contamination of phytosanitary applications. Residues of acaricides used for sanitary treatments, coumaphos and two transformation products of amitraz (DMF and DMPF), were quantified at higher levels in wax and bee bread than in honey in most cases. Coumaphos, DMF and DMPF were detected in honey in the range 6-36 µg.kg-1; 45-541 µg.kg-1; 15-107 µg.kg-1, respectively. All, except one sample, were below the EU MRLs, 396/2005 Regulation. Other pesticide residues were detected in beeswax and bee bread at various levels.

Biosensitive element in the form of immobilized luminescent photobacteria for detecting ecotoxicants in aqueous flow-through systems.

We demonstrated the possibility of long-term and efficient application of a biosensitive element (BE) in the form of Photobacterium phosphoreum photobacteria immobilized in poly(vinyl alcohol) (PVA) cryogel for detecting various ecotoxicants (Zn(2) (+) , Cu(2) (+) , Hg(2) (+) , Pb(2) (+) , 2,4-dichlorophenoxyacetic acid, 2,6-dimethylphenol, pentachlorophenol, coumaphos, malathion, chlorpyrifos and methyl parathion) in flow-through media. The range of detectable concentrations of ecotoxicants was determined at 1 × 10(-8) to 1 × 10(-4) M for heavy metal ions and at 1 × 10(-8) to 1 × 10(-5) M for phenol derivatives and organophosphorus pesticides. Immobilized cells of photobacteria quantitatively reacted with these ecotoxicants; cell sensitivity exhibited no flow rate dependence in the range from 45 to 180 mL/h. At a constant concentration of ecotoxicant in the flow, the bioluminescence quenching profile of immobilized cells demonstrated an integral response. The BE could remain in a flow-through medium for at least 10 days while retaining 95% of luminescent activity in the absence of ecotoxicants. The BE tested in this work was demonstrated to have a long shelf life (> 60 weeks) at -80°C without changes in the baseline level of bioluminescence. Copyright © 2016 John Wiley & Sons, Ltd.

Attomolar determination of coumaphos by electrochemical displacement immunoassay coupled with oligonucleotide sensing.

Coumaphos, an organophosphorus pesticide (OP) used worldwide, has raised serious public concerns due to its positive association with major types of cancer. Herein, a novel method for attomolar coumaphos detection was developed on the basis of an electrochemical displacement immunoassay coupled with oligonucleotide sensing. An optimized displacement immunoassay was constructed to improve the binding efficiency of an antigen-antibody pair, and a guanine-rich single-strand DNA label, in combination with oligonucleotide sensing, was used to amplify the detection signal with "direct" relationship to the analyte. As a result, coumaphos was sensitively determined from the enhanced catalytic cycle of guanine-Ru(bpy)(3)(2+) by chronoamperometry. The limit of detection (LOD) was down to 0.18 ng L(-1) (S/N = 3), which is equal to 49.6 amol in a sample solution of 100 µL. In comparison with conventional methods, the proposed method has the lowest LOD and better accessibility to high-throughput sensing systems. Besides, it can complete the whole analysis process in under 50 min and exhibits good performance of excellent selectivity to the OPs. With regard to the advantages of rapidity, convenience, low cost, and ease of operation, the proposed method has provided a promising platform capable of fast and in-field OP detection, which may make the system promising for potential applications in the detection of other small molecules.

Determination of coumaphos, chlorpyrifos and ethion residues in propolis tinctures by matrix solid-phase dispersion and gas chromatography coupled to flame photometric and mass spectrometric detection.

A new analytical method has been developed and successfully evaluated in routine application for the quantitative analysis of a selected group of organophosphate pesticides (coumaphos, chlorpyrifos and ethion) which can be found at trace levels in propolis tinctures (ethanolic propolis extracts); a valuable commodity used as raw material in the food and pharmaceutical industries for which there have been few attempts for pesticide residue analysis reported in the literature. The proposed methodology is based on matrix solid phase dispersion (MSPD) using aluminum sulfate anh. a novel dispersant material and subsequent column chromatography clean-up in silica gel prior to gas chromatography (GC) with both flame photometric detector (FPD) and mass spectrometry (MS) detection used for the routine quantification and identification of the residues, respectively. The limits of detection, for coumaphos, chlorpyrifos and ethion were below 26.0 µg/kg in FPD and 1.43 µg/kg for MS detection. Mean recoveries were in the range of 85-123% with RSD values below 13%, which suggests that the proposed method is fit for the purpose of analyzing pesticides in propolis tinctures containing high concentration of polyphenolics. The method has been successfully applied in our laboratory for the last 2 year in the analysis of real propolis tinctures samples.

Detection of coumaphos in honey using a screening method based on an electrochemical acetylcholinesterase bioassay.

An analytical protocol based on an electrochemical assay for the detection of acetylcholinesterase (AChE) inhibitors has been optimised for the detection of coumaphos in honey. Coumaphos is a phosphotionate insecticide requiring transformation in the corresponding oxo-form to act as an effective AChE inhibitor. The inhibition assay was based on the electrochemical detection of the product of AChE, choline, via a choline oxidase biosensors obtained using prussian-blue modified screen printed electrodes. A simple procedure for the oxidation of coumaphos via N-bromosuccinimide (NBS) and AChE inhibition was optimised. A calibration curve for coumaphos (8-1000 ng/ml) was obtained in buffer; the intra electrode CV ranged between 8 and 12% whereas the inter electrode CV was comprised between 12 and 14%. A detection limit (LOD) of 8 ng/ml was achieved, with an I(50%) of 105 ng/ml. The assay was then applied to detect coumaphos in honey samples. Despite the solubility of the samples in buffer, the assay was affected by many electrochemical interferences present in this sample matrix A simple C18 based solid phase extraction procedure has been then optimised and used for the assay. This allowed to eliminate all the electrochemical interferences with a satisfactory coumaphos recovery (around 86%) for a final LOD of 33 ng/g. The developed assay applied to detect coumaphos in different honey samples gave data well correlated with LC-MS detection.

Evaluation of coumaphos exposure among tick eradication workers.

OBJECTIVE: To evaluate both the cholinesterase monitoring program and newer field methods of determining coumaphos exposure among tick eradication workers. METHODS: Measured blood cholinesterase by the Ellman and field testing methods and tested urine for chlorferon pre- and postshift; conducted personal air sampling, patch sampling of clothing, and wipe sampling of hands for coumaphos. RESULTS: Fifteen workers had normal plasma cholinesterase and acetylcholinesterase levels. No significant changes occurred pre- to postshift. High correlation was found between plasma cholinesterase and acetylcholinesterase levels by field testing and Ellman methods (r = 0.91, P < 0.01 and r = 0.63, P < 0.01, respectively). Chlorferon levels rose 4 to 6 hours after use (P < 0.01). Airborne coumaphos was detected in only one sample, in a trace amount. The majority of patch and hand wipe samples detected coumaphos. CONCLUSIONS: Dermal exposure to coumaphos resulted in significant increases in urinary metabolites of coumaphos.

Miticide residues in Virginia honeys.

Fifty honey samples from Virginia USA were analyzed for the presence of fluvalinate and coumaphos residues. Samples were collected from hives and from bottled honey provided by beekeepers. No coumaphos or fluvalinate residues above the limit of quantification (0.05 mg/kg) were detected in any of the samples, although trace levels (<0.05 mg/kg) of coumaphos were detected in three samples from hives and trace levels of fluvalinate were found in one hive sample. No residues were detected in any of the bottled honey samples and none of the samples exceeded the US EPA tolerance levels for either miticide.

Combined use of GC-TOF MS and UHPLC-(Q)TOF MS to investigate the presence of nontarget pollutants and their metabolites in a case of honeybee poisoning.

The combined use of gas chromatography (GC) and ultrahigh-pressure liquid chromatography (UHPLC), both coupled to time-of-flight mass spectrometry (TOF MS), has been explored in this work for the investigation of several cases of honeybee poisoning. The procedure applied involves a previous extraction with acetone followed by liquid-liquid extraction with dichloromethane. Both techniques, GC-TOF MS and UHPLC-(Q)TOF MS, have been applied to discover the presence of compounds that might be responsible of honeybee deaths. The application of a nontarget methodological approach to a first case of poisoning allowed the detection of the insecticide coumaphos at high concentration levels in the samples. The presence of possible metabolites of this organophosphorus insecticide was investigated by using both techniques. UHPLC-(Q)TOF MS showed its higher applicability in this case, as most of the metabolites were more polar than the parent compound. Four metabolites were identified by UHPLC-(Q)TOF MS, whereas only two of them were found by GC-TOF MS. The developed methodology was applied to other subsequent poisoning cases in which insecticides such as coumaphos, thiamethoxam, pyriproxyfen, and chlorfenvinphos were identified by both techniques, whereas GC-TOF MS also allowed the detection of fenitrothion and methiocarb. In all positive cases, the confirmation of the presence of the compound detected was feasible by means of accurate mass measurements of up to five ions together with their ion ratio evaluation.

Determination of synthetic acaricides residues in beeswax by high-performance liquid chromatography with photodiode array detector.

A multiresidue HPLC method for identification and quantification of the synthetic acaricides fluvalinate, coumaphos, bromopropylate and its metabolite 4,4'-dibromobenzophenone in beeswax has been developed. Different techniques were tested and modified. The method consists of a sample preparation with isooctane followed by solid phase extraction using Florisil columns. Determination of the synthetic acaricides is achieved by HPLC with a photodiode array detector. Analytical performance of the proposed method, including sensitivity, accuracy and precision was satisfactory. The LOD for the analytes varied between 0.1 and 0.2 microg g(-1) wax and the recoveries between 70 and 110%. Relative standard deviation of the repeatability of the method is <15% and reproducibility is <31%.

[Resolution of synchronous fluorimetric spectrum for carbaryl and coumaphos and its application study].

The synchronous fluorimetric method for the simultaneous determination of carbaryl and coumaphos is described. In the Britton-Robinson buffer at pH 3.0, synchronous scanning with deltalambda= 60 nm was carried out, and the overlapped fluorimetric spectra were better separated. The two synchronous fluorimetric peaks are located at 280 and 322 nm respectively, and can be used for the determination of these two pesticides. No preliminary separation is needed. The method is simple, rapid and successfully applied to the analysis of food samples. The linear ranges of the calibration curves for carbaryl and coumaphos are 0.016-0.384 microg x mL(-1) and 0.016-0.320 microg x mL(-1), respectively. The limits of detection are 0.015 microg x mL(-1) for carbaryl, and 0.010 microg x mL(-1) for coumaphos.

Analysis of organophosphorus pesticides in whole milk by solid phase microextraction gas chromatography method.

Solid phase microextraction (SPME) was used for the extraction of residual coumaphos and dichlorvos in whole milk. The residues were analyzed by capillary gas chromatography equipped with nitrogen phosphorus detector (GC-NPD). A manual SPME holder with a 100-microm polyacrylate fiber was used. The optimized conditions for extraction by SPME method were: sample agitation, absorption temperature of 30 degrees C, absorption time of 40 min, desorption time of 10 min, and sample volume was 16.0 mL in the vial. Under these conditions, the calibration graphs were linear in the range of 0.17 microgL-1 to 1.75 microgL-1 for coumaphos and 0.69 microgL-1 to 6.90 microgL-1 for dichlorvos. Precision was good with RSD values of 13% for coumaphos and 14% for dichlorvos. The detection limits (LOD) were 0.060 microgL-1 for dichlorvos and 0.052 for coumaphos. The quantification limits (LOQ) were 0.086 microgL-1 for dichlorvos and 0.066 microgL-1 for coumaphos. The results obtained in this study suggest that SPME is a suitable technique for residual pesticide analysis of milk. The data demonstrate that particular OP pesticides used in dairy farming in the region of Minas Gerais were found to contaminate cow whole milk, and the residues are not removed by treating the milk by boiling.

Determination of acaricide residues in saudi arabian honey and beeswax using solid phase extraction and gas chromatography.

Determination of acaricide residues of flumethrin, tau-fluvalinate, coumaphos, and amitraz in honey and beeswax was carried out using a rapid extraction method utilizing C-18 SPE cartridges and an analytical method utilizing GC with ECD, NPD, and MSD detectors for the four acaricides. Recovery percentages from the extraction method ranged from 90-102%, while the minimum detection levels ranged from 0.01-0.05 mg/kg for the acaricides. Nine of the 21 analyzed samples were found to be contaminated with the acaricides tau-fluvalinate and coumaphos. Neither flumethrin nor amitraz was detected in any of the honey or wax samples. Coumaphos was found only in honey samples in which two samples exceeded the tolerance levels set by EPA and EC regulations. It has not been detected in beeswax. Five honey samples and eight beeswax samples were found to be contaminated with tau-fluvalinate. One of the wax samples was contaminated with a relatively high residue of tau-fluvalinate and contained above 10 mg/kg.

Cholinesterase sensors based on screen-printed electrodes for detection of organophosphorus and carbamic pesticides.

Cholinesterase sensors based on screen-printed electrodes modified with polyaniline, 7,7',8,8'-tetracyanoquinodimethane (TCNQ), and Prussian blue have been developed and tested for detection of anticholinesterase pesticides in aqueous solution and in spiked grape juice. The influence of enzyme source and detection mode on biosensor performance was explored. It was shown that modification of the electrodes results in significant improvement of their analytical characteristics for pesticide determination. Thus, the slopes of the calibration curves obtained with modified electrodes were increased twofold and the detection limits of the pesticides were reduced by factors of 1.6 to 1.8 in comparison with the use of unmodified transducers. The biosensors developed make it possible to detect down to 2 x 10(-8) mol L(-1) chloropyrifos-methyl, 5 x 10(-8) mol L(-1) coumaphos, and 8 x 10(-9) mol L(-1) carbofuran in aqueous solution and grape juice. The optimal conditions for grape juice pretreatment were determined to diminish interference from the sample matrix.

Determination of whole blood cholinesterase in different animal species using specific substrates.

Whole blood cholinesterase was measured using acetyl-, butyryl- and propionylthiocholine as substrates in 10 healthy adult dogs, cats, horses, pigs, goats, sheep and cows, in order to determine and characterise the cholinesterase activity in whole blood of the main domestic animals. An in vitro exposure test with two anticholinesterase compounds, the organophosphate insecticide coumaphos and the carbamate insecticide imidocarb, was also performed. In whole blood of ruminants and pigs, acetylthiocholine yielded the highest cholinesterase activity and other substrates were poorly hydrolysed; in dogs and cats, although acetylthiocholine showed the highest cholinesterase activity, butyryl- and propionylthiocholine also produced high cholinesterase values; in horses, propionylthiocholine was the substrate that yielded the highest cholinesterase activity, closely followed by butyrylthiocholine. All within- and between-run coefficients of variation observed in whole blood samples were less than 5 and 7 per cent, respectively, except when butyrylthiocholine was used as substrate in ruminant blood samples. Butyryl- and propionylthiocholine were the substrates that yielded higher inhibitions after coumaphos exposure, whereas the use of acetylthiocholine showed the highest cholinesterase inhibition after imidocarb exposure. The use of at least two substrates (acetyl and butyrylthiocholine) is recommended for whole blood cholinesterase analyses in domestic animals since it will allow monitoring of both acetyl- and butyrylcholinesterase activities, respectively, and a more accurate detection of exposure to anticholinesterase compounds. However, acetylthiocholine could be used as a unique substrate for whole blood cholinesterase determination in porcine and ruminant samples since butyrylcholinesterase activity is very low in these species. Additionally, propionylthiocholine could be used as an alternative substrate to butyrylthiocholine in horse whole blood samples.

Determination of malathion, coumaphos, and fluvalinate residues in honey by gas chromatography with nitrogen-phosphorus or electron capture detectors.

A rapid, reliable, and inexpensive extraction method was developed to determine acaricide residues in honey by gas chromatography (GC) with nitrogen-phosphorus (NP) or electron capture (EC) detectors. Because of the high selectivity of the NP detector, no interfering peaks were present and no cleanup was necessary. A simple cleanup step is proposed for the GC-ECD analysis. Recoveries from spiked honey samples ranged from 79 to 94.4%, with coefficients of variation of 0.3-18.5%. The quantitation limit obtained was 0.015 mg/kg for malathion, 0.020 mg/kg for coumaphos, and 0.005 mg/kg for fluvalinate. The method was used to determine the disappearance of malathion and coumaphos residues from honey samples collected from beehives treated with these acaricides. The disappearance of both acaricides was rapid and followed a first-order model for the duration of the experiment.

Acaricide residues in honeys from Galicia (N.W. Spain).

Honey samples (101) from Galicia (N.W. Spain) were analyzed by gas chromatography (electron capture and flame ionization) for the presence of acaricides (amitraz, bromopropylate, coumaphos, and fluvalinate). Seventy-three samples were free from detectable residues. Bromopropylate residues were found in 16 samples in levels ranging from 5 to 60 microg/kg. Fluvalinate residues were found in 11 samples in levels ranging from 10 to 40 microg/kg. One sample contained 100 microg of fluvalinate per kg. Neither amitraz nor coumaphos residues were detected.