An immunoassay for a urinary metabolite as a biomarker of human exposure to the pyrethroid insecticide permethrin

Permethrin is the most popular synthetic pyrethroid insecticide used in agriculture and public health. For the assessment of human exposure to permethrin, a competitive indirect enzyme-linked immunosorbent assay (ELISA) for the detection of the glycine conjugate of a major metabolite, cis-/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (DCCA), of permethrin was developed based on a polyclonal antibody. An assay based on an antibody with a high sensitivity was optimized and characterized. The IC₅₀ value and the detection range for trans-DCCA–glycine, in the assay buffer were 1.2 and 0.2−7.0 μg/L, respectively. The antibody recognized trans-DCCA–glycine and the mixture of cis-/trans-DCCA–glycine with an isomer range from 30:70 to 50:50 nearly equally. Little or no cross-reactivity to permethrin and its other free metabolites or glycine conjugates was measured. The integration of the ELISA and solid-phase extraction which was used to reduce the matrix effect from human urine samples provided for analysis of total cis-/trans-DCCA–glycine at low parts per billion levels in the samples. The limit of quantitation of the target analyte was 1.0 μg/L in urine with a limit of detection of 0.1 μg/L in buffer. This assay might be a useful tool for monitoring human exposure to permethrin.     

Ibuprofen interference in the determination of 3-phenoxybenzoic acid in urine

Pyrethroid insecticides are widely used in agriculture and private households. Analysis of urine for pyrethroid metabolites is one way to detect human exposure to these insecticides and is carried out regularly as part of the Occupational and Environmental Medicine Monitoring Program recommended by the Deutsche Forschungsgemeinschaft (DFG). Samples are analyzed using GC-MS (selected ion monitoring) following acid hydrolysis, solid phase extraction, esterification with methanol/sulfuric acid, and liquid-liquid extraction. The metabolite, 3-phenoxybenzoic acid (3-PBA), can be derived from several pyrethroids and is, therefore, a useful diagnostic analyte; however, the presence of the over-the-counter drug, ibuprofen ((R,S)-2-(4-isobutylphenyl)propionic acid), interferes with this determination, even after the ingestion of only one 200-mg tablet. The interfering analyte is carboxy-ibuprofen which is not removed by the cleanup step. Experimental work shows that it takes two days for most of the ibuprofen to clear the body before 3-PBA can reliably be determined in urine.     

Thin-layer chromatographic detection of pyrethroid insecticides containing a nitrile group.

A spray reagent for the detection of pyrethroid insecticides containing a nitrile group by thin-layer chromatography is described. These insecticides on alkaline hydrolysis yield cyanide ion, which in turn reacts with copper(II) acetate and o-tolidine in an acetic acid medium to give a blue colour. Organochlorine, organophosphorus and carbamate insecticides do not interfere. The limit of detection is about 1 microgram.     

Pyrethroids as household insecticides: analysis, indoor exposure and persistence

Summary Natural pyrethrins from insecticidal pyrethrum extract and pyrethroids (e.g. allethrin, tetramethrin, permethrin, cyphenothrin, cypermethrin, cyfluthrin) are active ingredients in insecticidal formulations such as powder, sprays, impregnated paper for electro-evaporators, mosquito coils, and solutions for wood treatment, all mainly intended for indoor use. Some commercial preparations contain also non-pyrethroid insecticides, such as dichlorovos, propoxur, or phoxim, and piperonyl butoxid as synergist. High-resolution gas-chromatography with oncolumn injection and FID and ECD detection is employed for the analysis of these insecticides in commercial formulations, in air during and after indoor application, and as residues on surfaces. The total input of pyrethroids into a large room amounts to 1 to 30 mg. The concentrations of the pyrethrins and pyrethroids in air (2 to 300 g/m³) and their deposition on surfaces (up to 1000 g/m²) reveal possible exposure of humans by inhalation (e.g. 30 g allethrin or 60 g tetramethrin) or by skin resorption (e.g. 200 g allethrin and up to 1000 g tetramethrin). The insecticides deposited on surfaces and some readily formed transformation products persist for 60 h or longer.Partly presented at the 7th International Congress of Pesticide Chemistry in Hamburg, August 5–10, 1990     

Development of a class selective immunoassay for the type II pyrethroid insecticides

A general and broad class selective enzyme-linked immunosorbent assay was developed for the type II pyrethroid insecticides, such as cypermethrin, deltamethrin, cyhalothrin, cyfluthrin, fenvalerate, esfenvalerate and fluvalinate. Polyclonal antibodies were generated by immunizing with a type II pyrethroid immunogen ((RS)-α-cyano-3-phenoxybenzyl (RS)-cis,trans-2,2-dimethyl-3-carboxyl-cyclopropanecarboxylate) conjugated with thyroglobulin. Antisera were screened against nine different coating antigens. The antibody-antigen combination with the most selectivity for type II pyrethroids such as cypermethrin was further optimized and tested for tolerance to co-solvent, pH and ionic strength changes. The IC₅₀s of the optimized immunoassay were 78 μg l⁻¹ for cypermethrin, 205 μg l⁻¹ for cyfluthrin, 120 μg l⁻¹ for cyhalothrin, 13 μg l⁻¹ for deltamethrin, 6 μg l⁻¹ for esfenvalerate, 8 μg l⁻¹ for fenvalerate and 123 μg l⁻¹ for fluvalinate. No cross-reactivity was measured for the type I pyrethroids such as permethrin, bifenthrin, phenothrin, resmethrin and bioresmethrin. This assay can be used in monitoring studies to distinguish between type I and II pyrethroids.     

Biological monitoring of 3-phenoxybenzoic acid in urine by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) method was employed for determination of the pyrethroid biomarker, 3-phenoxybenzoic acid (3-PBA) in human urine samples. The optimized coating antigen concentration was 0.5 ng/mL with a dilution of 1:4000 for the 3-PBA antibody and 1:6000 for the enzyme conjugate. Urine samples were hydrolyzed with concentrated hydrochloric acid; extracted with dichloromethane and solvent-exchanged into a methanol/buffer solution, prior to analysis in a 96-microwell plate immunoassay. Quantitative recoveries of 3-PBA were obtained for fortified urine samples by ELISA (92 ± 18%) as well as by gas chromatography/mass spectrometry (GC/MS) (90 ± 13%). The overall method precision of these samples was within ±20% for both the ELISA and GC/MS methods. Analytical results from over one hundred urine samples showed that the ELISA and GC/MS data were highly correlated, with a correlation coefficient of 0.95. At the 10 ng/mL comparative concentration level, the false positive rate was 0% and the false negative rate was 0.8% for ELISA when using GC/MS as the reference method. The ELISA method has a suitable low detection limit for 3-PBA to assess pyrethroid exposures in non-occupational settings.     

Validated TLC-densitometry method for the simultaneous analysis of pyrethroid insecticides in agricultural and domestic products

Background

For therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND), the potent drug for treatment of multiple myeloma (MM), a specific antibody was required for the development of a sensitive immunoassay system for the accurate determination of LND in plasma.

Results

In this study, a hapten of LND (N-glutaryl-LND) was synthesized by introducing the glutaryl moiety, as a spacer, into the primary aromatic amine site of the LND molecular structure. The structure of the hapten (G-LND) was confirmed by mass, ¹H-NMR, and ¹³C spectrometric techniques. G-LND was coupled to each of bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) proteins by ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling reagent. LND-KLH conjugate was used as an immunogen. Four female 2-3 months old New Zealand white rabbits were immunized with an emulsion of LND-KLH with Freund`s adjuvant. The immune response of the rabbits was monitored by direct enzyme-linked immunosorbent assay (ELISA) using LND-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to LND was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on protein A column. The specificity of the purified antibody for LND was evaluated by indirect competitive ELISA using dexamethasone as a competitor as it is used with LND in a combination therapy.

Conclusions

The high affinity of the antibody (IC₅₀ = 10 ng/mL) will be useful in the development of an immunoassay system for the determination of plasma LND concentrations. Current research is going to optimize the assay conditions and validate the procedures for the routine application in clinical laboratories.     

Validated TLC-densitometry method for the simultaneous analysis of pyrethroid insecticides in agricultural and domestic products

ABSTRACT: BACKGROUND: Pyrethroids are widely used for the control of pests and insects, as pyrethroids are believed to pose very little risk to human health and environment. However, exposure to the pyrethroids exceeding the label directions might adversely affect human health and environment. Hence a careful selection of environment friendly household product is required that must contain exactly the label claimed pyrethroids amount. RESULTS: A sensitive and robust TLC-densitometric method for simultaneous quantification of commonly used synthetic pyrethroids including esbiothrin, alpha-cypermethrin and cis/trans permethrin in agricultural and domestic products has been developed and validated. TLC aluminum plates, precoated with 0.2 mm thick layer of silica gel 60 F-254, were used for chromatographic process. Densitometric analysis of chromatoplates was carried out in absorbance mode at corresponding lamdamax of each pyrethroid. Equally valid common mobile phase for all pyrethroids consisted of hexane-dichloromethane-ethylacetate-formic acid (8:1.5:0.4:0.1 v/v/v/v) which provided sharp and symmetrical peaks of esbiothrin, alpha-cypermethrin, trans-permethrin and cis-permethrin, at Rf 0.31, 0.53, 0.6 and 0.65, respectively. Linear regression data for respective calibration curves showed a good linearity for all pyrethroids with r = 0.991-0.996. Limits of detection (LOD) and limits of quantification (LOQ) for all pyrethroids were found in the range of 1.6-2.8 and 4.9-8.5 ng/spot, respectively. CONCLUSIONS: The developed method is applicable for separating the mixture of pyrethroids and at the same time, it is also valid for separating their isomers. The method is reproducible, precise and accurate for the quantitative determination of pyrethroids in agricultural and domestic products.     

Gas-chromatography mass-spectrometry determination of phthalic acid in human urine as a biomarker of folpet exposure

Agricultural workers are exposed to folpet, but biomonitoring data are limited. Phthalimide (PI), phthalamic acid (PAA), and phthalic acid (PA) are the ring metabolites of this fungicide according to animal studies, but they have not yet been measured in human urine as metabolites of folpet, only PA as a metabolite of phthalates. The objective of this study was thus to develop a reliable gas chromatography–tandem mass spectrometry (GC–MS) method to quantify the sum of PI, PAA, and PA ring-metabolites of folpet in human urine. Briefly, the method consisted of adding p-methylhippuric acid as an internal standard, performing an acid hydrolysis at 100 °C to convert ring-metabolites into PA, purifying samples by ethyl acetate extraction, and derivatizing with N,O-bis(trimethylsilyl)trifluoro acetamide prior to GC–MS analysis. The method had a detection limit of 60.2 nmol/L (10 ng/mL); it was found to be accurate (mean recovery, 97%), precise (inter- and intra-day percentage relative standard deviations <13%), and with a good linearity (R ² > 0.98). Validation was conducted using unexposed peoples urine spiked at concentrations ranging from 4.0 to 16.1 μmol/L, along with urine samples of volunteers dosed with folpet, and of exposed workers. The method proved to be (1) suitable and accurate to determine the kinetic profile of PA equivalents in the urine of volunteers orally and dermally administered folpet and (2) relevant for the biomonitoring of exposure in workers.     

Development of a high-throughput method for the determination of organochlorinated compounds, nitromusks and pyrethroid insecticides in indoor dust

Investigation of chemical exposure inside the homes and offices where people spend the majority of their lives has only recently begun. These chemicals are degraded much more slowly than outdoor because they are more protected from sunlight, severe environmental conditions and microbial activity. Hence, indoor dust has been recognized as an important exposure pathway for organic contaminants. Pyrethroids are synthetic insecticides widely used in domestic environment for numerous applications and also in agriculture. Chlorobenzenes are a family of compounds used as intermediates in the production of a wide range of household consumer products. Nitromusks are a kind of synthetic musks used in the production of cleaning agents, detergents, and personal care products. A high-throughput method for the determination of these compounds in indoor dust samples has been developed. Microwave-assisted solvent extraction was used as the extraction technique whereas quantification of compounds was carried out by gas chromatography with micro-electron-capture detection. Several cleanup procedures were tested and finally a non-classical "on batch" procedure was selected, which allows increasing the throughput of the analysis while decreasing sample manipulation. Extraction conditions were optimized using a multifactorial experimental design approach. Quantitative recovery (84-103%) was achieved for all compounds and method precision was satisfactory. Limits of detection ranged from 0.22 ng g(-1) for lindane to 40 ng g(-1) for 1,4-dichlorobenzene. Standard reference material SRM 2585 was analyzed and the obtained values were in good agreement with the reported reference values for organochlorinated compounds and nitromusks. Pyrethroids and polychlorobenzenes have been analyzed for the first time in this reference material and some of them have been found. In addition, real samples collected in houses of north-western Spain have been analyzed by the proposed method and 17 of the 22 target compounds have been detected in the samples.     

Clean-up of some organochlorine and pyrethroid insecticides by automated solid-phase extraction cartridges coupled to capillary GC-ECD

The ASPEC (Automatic Sample Preparation with Extraction Columns) system has been coupled on-line to capillary GC-ECD by means of a loop-type interface equipped with a solvent vapour exit. Both ASPEC and GC conditions have been optimized leading to an effective clean-up of the extracts analyzed. By means of solid-phase extraction cartridges filled with silica, it has been possible to analyze concentrated surface water extracts for a group of 18 electron-capturing compounds present in the water at ppt levels. ASPEC-GC has also been applied to the determination of synthetic pyrethroids at ppt levels in surface water. The complete analytical procedure is greatly facilitated by automation and miniaturization. Miniaturization results in a considerable decrease in the sample volume required. The potential of the method for the analysis of other pesticides is estimated.     

The photochemical behaviour of five household pyrethroid insecticides and a synergist as studied by photo-solid-phase microextraction

In the present study, solid-phase microextraction in photochemical studies was used to investigate UV light induced photodegradation of five pyrethroids (empenthrin, transfluthrin, allethrin, phenothrin and cyphenothrin) and a synergist (piperonyl butoxide), which are common ingredients of household insecticides. Gas chromatography coupled with mass spectrometry was used to separate and tentatively identify the parent compounds and their corresponding photoproducts generated in the same polydimethylsiloxane fibre. Kinetics curves were obtained and apparent first-order rate constants and half-lives were estimated. Twenty-six photoproducts were tentatively identified and photodegradation pathways for the compounds investigated were proposed. It is a matter of some concern that three of the photoproducts identified [(3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-phenoxybenzaldehyde and (3-phenoxyphenyl)methanol] have been reported to be endocrine disruptors. There is no record of previous studies of cyphenothrin and empenthrin photodegradation, and therefore the present study represents the first attempt to elucidate the photochemical behaviour of these compounds. Figure Photo-SPME for Pyrethroids     

Immunochemical analysis of a photoreceptor protein using anti-IP3 receptor antibody in the unicellular organism, Blepharisma

The blepharismin-200 kD protein complex of the ciliated protozoan Blepharisma is a novel type of photosensor responsible for the step-up photophobic response of the cell. In immunoblotting assays, the 200 kD protein is weakly cross-reacted with anti-inositol triphosphate receptor antibody (anti-IP3 R antibody). Indirect immunofluorescence assays show that the pigment granules in which the blepharismin-200 kD protein complex is localized are labelled by anti-IP3 R antibody. When the anti-IP3 R antibody or antisense oligonucleotide for IP3 receptor is introduced into the living cells of Blepharisma, both the photosensitivity of the cells and content of blepharismin-200 kD protein are reduced. The results suggest that the photoreceptor 200 kD protein is possibly an IP3 receptor-like protein.     

Performance of gas chromatography/tandem mass spectrometry in the analysis of pyrethroid insecticides in environmental and food samples

The performance of gas chromatography coupled with tandem mass spectrometry (GC/MS/MS) was tested for the simultaneous determination of twelve pyrethroid insecticides. First, a comparison of two different ionization modes, electron ionization (EI) and negative chemical ionization (NCI), was carried out using MS and MS/MS. NCI-MS/MS provided the best results in terms of selectivity and sensitivity giving very low detection limits of 0.11 to 450 fg injected. The reliability of the method was confirmed through the evaluation of quality parameters such as accuracy (70-100%), and repeatability and reproducibility, with coefficients of variation below 15% and 10%, respectively. The applicability of the GC/MS/MS method to real samples and influence of matrix effects were evaluated through the analysis of spiked water, sediment and milk at 0.25 ng L(-1) , 5 ng g(-1) dry weight (dw) and 25 ng g(-1) (dw), respectively, of each pyrethroid insecticide considered. Using GC/NCI-MS/MS, matrix spectral interferences were minimized providing method limits of detection (MLODs) of 0.05-2.59 ng L(-1) , 0.10-87.7 pg g(-1) dw, 2.29-1071 pg g(-1) lipid weight (lw) for water, sediment and milk, respectively. To the best of our knowledge, the MLOD values found in our study were better than those reported in previous studies; in particular for sediment and food samples, they were one order of magnitude lower.     

An optimized sampling and GC-MS analysis method for benzene in exhaled breath, as a biomarker for occupational exposure

Benzene is known to be toxic and carcinogenic: therefore, in case of exposure to benzene vapours, a reliable biological monitoring procedure is needed, particularly in the field of occupational hygiene. The determination of the concentration of benzene in the exhaled air 8 h after the exposure has been demonstrated to be a significant biomarker, even for low concentrations of airborne benzene vapours. This work presents a sampling and analysis method that optimizes previously described procedures: in the sampling phase, a double-step sample collection in Tedlar bags is used, in order to remove the breath moisture and to standardise the sample volumes. The analytical phase uses a cryogenic trap for the concentration of the air samples to be injected in the GC-MS, without the need for trapping materials, significantly reducing time and costs of the analysis and improving sensitivity. The presented method has been successfully applied to the biological monitoring of a mixed population (occupationally exposed and not exposed subjects, smokers and non-smokers), with a lower detection limit of 1.5 ng of benzene per litre of exhaled air, that is 1/200 of the biological exposure index recommended by the American Conference of Governmental Hygienists.     

Rapid analysis of pyrethroid insecticides in aquaculture seawater samples via membrane-assisted solvent extraction coupled with gas chromatography-electron capture detection

A simple, efficient, and environmentally friendly membrane-assisted solvent extraction (MASE) method for the extraction and preconcentration of six pyrethroid insecticides from aquaculture seawater samples followed by gas chromatography-electron capture detection (GC-ECD) was successfully proposed. The operating conditions for MASE, such as the extraction solvent, solvent volume, NaCl concentration, stirring rate, extraction time, and temperature, were optimized. Compared to conventional Florisil-solid phase extraction (SPE), higher extraction recoveries (85.9% to 105.9%) of three spiked levels of the six pyrethroid pesticides in aquaculture seawater were obtained using MASE, and the RSD values were lower than 7.9%. The limits of detection (LOD, signal-to-noise ratio (S/N)=3) and quantification (LOQ, S/N = 10) were in the range of 0.037-0.166 and 0.12-0.55 μg L(-1), respectively. The results demonstrate the excellent applicability of the MASE method in analyzing the six pyrethroid pesticides in aqueous samples. The proposed method exhibited a high potential for routine monitoring analysis of pyrethroid insecticides in seawater samples.     

Microwave-assisted extraction of pyrethroid insecticides from semi permeable membrane devices (SPMDs) used to indoor air monitoring

A rapid and environmentally friendly methodology was developed for the extraction of pyrethroid insecticides from semi permeable membrane devices (SPMDs), in which they were preconcentrated in gas phase. The method was based on gas chromatography mass–mass spectrometry determination after a microwave-assisted extraction, in front of the widely employed dialysis method. SPMDs were extracted twice with 30 mL hexane:acetone, irradiated with 250 W power output, until 90 °C in 10 min, this temperature being held for another 10 min. Clean-up of the extracts was performed by acetonitrile–hexane partitioning and solid-phase extraction (SPE) with a combined cartridge of 2 g basic-alumina, deactivated with 5% water, and 500 mg C₁₈.

Pyrethroids investigated were Allethrin, Prallethrin, Tetramethrin, Bifenthrin, Phenothrin, λ-Cyhalothrin, Permethrin, Cyfluthrin, Cypermethrin, Flucythrinate, Esfenvalerate, Fluvalinate and Deltamethrin. The main pyrethroid synergist compound, Pyperonyl Butoxide, was also studied. Limit of detection values ranging from 0.3 to 0.9 ng/SPMD and repeatability data, as relative standard deviation, from 2.9 to 9.4%, were achieved. Pyrethroid recoveries, for spiked SPMDs, with 100 ng of each one of the pyrethroids evaluated, were from 61 ± 8 to 103 ± 7% for microwave-assisted extraction, versus 54 ± 4 to 104 ± 3% for dialysis reference method. Substantial reduction of solvent consumed (from 400 to 60 mL) and analysis time (from 48 to 1 h) was achieved by using the developed procedure.

High concentration levels of pyrethroid compounds, from 0.14 to 7.3 μg/SPMD, were found in indoor air after 2 h of a standard application.     

Conformational properties of a phototautomerizable nucleoside biomarker for phenolic carcinogen exposure

We have characterized the conformational properties of the C8-deoxyguanosine (C8-dG) nucleoside adduct, 8-(2"-hydroxyphenyl)-2'-dG (1), which is a potential biomarker for exposure to phenolic carcinogens. Adduct 1 possesses the unique ability to phototautomerize, through an excited-state intramolecular proton transfer (ESIPT) process, to generate its keto form. This tautomerization depends on the presence of an intramolecular hydrogen (H)-bond between the phenolic OH and the imine nitrogen (N7) and has permitted insight into the equilibrium ground states of adduct 1. The results of our studies demonstrate that adduct 1 undergoes an ESIPT despite preferring a nonplanar "twisted" conformation that is imposed by the deoxyribose (dR) sugar moiety. Interestingly, a planar conformation of adduct 1 is also formed in certain aprotic solvents due to the anchoring effect of the intramolecular H-bond. Our results provide a basis for future studies aimed at determining the conformations of adduct 1 within DNA that will aid in our understanding of phenol-mediated carcinogenesis.