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VX, a well-known organophosphorus nerve agent (OPNA), poses a significant threat to public safety if employed by terrorists. Obtaining complete metabolites is critical to unequivocally confirm its alleged use/exposure and elucidate its whole-molecular metabolism. However, the nitrogenous VX metabolites containing 2-diisopropylaminoethyl moiety from urinary excretion remain unknown. Therefore, this study applied a newly developed untargeted workflow platform to discover and identify them using VX-exposed guinea pigs as animal models. 2-(N,N-diisopropylamino)ethanesulfonic acid (DiPSA) was revealed as a novel nitrogenous VX metabolite in urine, and 2-(Diisopropylaminoethyl) methyl sulfide (DAEMS) was confirmed as another in plasma, indicating that VX metabolism differed between urine and plasma. It is the first report of a nitrogenous VX metabolite in urine and a complete elucidation of the VX metabolic pathway. DiPSA was evaluated as an excellent VX exposure biomarker. The whole-molecule VX metabolism in urine was characterized entirely for the first time via the simultaneous quantification of DiPSA and two known P-based biomarkers. About 52.1% and 32.4% of VX were excreted in urine as P-based and nitrogenous biomarkers within 24 h. These findings provide valuable insights into the unambiguous detection of OPNA exposure/intoxication and human and environmental exposure risk assessment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: The comprehensive detection of environmental chemicals in biospecimens, an indispensable task in exposome research, is advancing. This study aimed to develop an exposomic approach to identify urinary metabolites of organophosphate (OP) pesticides, specifically cadusafos and prothiofos metabolites, as an example chemical group, using an original metabolome dataset generated from animal experiments., Methods: Urine samples from 73 university students were analyzed using liquid chromatography-high-resolution mass spectrometry. The metabolome data, including the exact masses, retention time (t R ), and tandem mass spectra obtained from the human samples, were compared with the existing reference databases and with our original metabolome dataset for cadusafos and prothiofos, which was produced from mice to whom two doses of these OPs were orally administered., Results: Using the existing databases, one chromatographic peak was annotated as 2,4-dichlorophenol, which could be a prothiofos metabolite. Using our original dataset, one peak was annotated as a putative cadusafos metabolite and three peaks as putative prothiofos metabolites. Of these, all three peaks suggestive of prothiofos metabolites, 2,4-dichlorophenol, 3,4,5-trihydroxy-6-(2,4-dichlorophenoxy) oxane-2-carboxylic acid, and (2,4-dichlorophenyl) hydrogen sulfate were confirmed as authentic compounds by comparing their peak data with both the original dataset and peak data of the standard reagents. The putative cadusafos metabolite was identified as a level C compound (metabolite candidate with limited plausibility)., Conclusions: Our developed method successfully identified prothiofos metabolites that are usually not a target of biomonitoring studies. Our approach is extensively applicable to various environmental contaminants beyond OP pesticides., (© 2021 The Authors. Journal of Occupational Health published by John Wiley & Sons Australia, Ltd on behalf of The Japan Society for Occupational Health.)

A protocol created for acephate detection on particulates and vapors surrounding farmworkers as well as in urine samples is reported. Acephate is detected to the low parts-per-billion (ppb) range using surface-enhanced Raman spectroscopy (SERS). Optimal SERS sensor metal choice and post-production treatments to improve sensor stability in aqueous solutions containing acephate are presented. Acephate is detected in the vapor phase and can be differentiated from urine components and structurally similar pesticides, including the acephate metabolite-degradation product methamidophos. Protocol evaluation and preliminary field tests from North Carolina farms are discussed.

A sensitive screening method based on fast liquid chromatography tandem mass-spectrometry (RSLC-MS-MS) has shown the feasibility of separation and detection of low concentration β-lyase metabolites of sulfur mustard and of nerve agent phosphonic acids in urine. The analysis of these compounds is of interest because they are specific metabolites of the chemical warfare agents (CWAs), sulfur mustard (HD), sarin (GB), soman (GD), VX and Russian VX (RVX). The 'dilute-and-shoot' RSLC-MS-MS method provides a sensitive and direct approach for determining CWA exposure in non-extracted non-derivatized samples from urine. Chromatographic separation of the metabolites was achieved using a reverse phase column with gradient mobile phases consisting of 0.5% formic acid in water and acetonitrile. Identification and quantification of species were achieved using electrospray ionization-tandem mass-spectrometry monitoring two precursor-to-product ion transitions for each compound. The method demonstrates linearity over at least two orders of magnitude and had detection limits of 0.5 ng/mL in urine., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A novel method for rapid and sensitive quantification of the nerve agent metabolites ethyl, isopropyl, isobutyl, cyclohexyl, and pinacolyl methylphosphonic acid has been established by combining salting-out assisted liquid-liquid extraction (SALLE) and online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS). The procedure allows confirmation of nerve agent exposure within 30 min from receiving a sample, with very low detection limits for the biomarkers of 0.04-0.12 ng/mL. Sample preparation by SALLE was performed in less than 10 min, with a common procedure for both serum and urine. Analyte recoveries of 70-100% were obtained using tetrahydrofuran as extraction solvent and Na2SO4 to achieve phase separation. After SALLE, selective analyte retention was obtained on a ZrO2 column by Lewis acid-base and hydrophilic interactions with acetonitrile/1% CH3COOH (82/18) as the loading mobile phase. The phosphonic acids were backflush-desorbed onto a polymeric zwitterionic column at pH 9.8 and separated by hydrophilic interaction liquid chromatography. The method was linear (R(2) ≥ 0.995) from the limits of quantification to 50 ng/mL, and the within- and between-assay repeatability at 20 ng/mL were below 5% and 10% relative standard deviation, respectively.

An oral dose of the organophosphate insecticide methamidophos was administered to six volunteers at the acceptable daily intake (ADI, 0.004 mg/kg). Urine was collected from the volunteers at timed intervals for 24 h post-exposure. Methamidophos itself was quantified in urine using liquid/liquid extraction and LC-MS-MS analysis (detection limit 7 nmol/L/1 μg/L). Methamidophos exhibited a rapid elimination half-life of 1.1h, (range 0.4-1.5 h). Mean metabolite levels found in 24h total urine collections (normalised for a 70 kg volunteer) were 9.2 nmol/L (range 1.0-19.1). One volunteer was anomalous; excluding this result the range was 6.7-19.1 nmol/L, with a mean of 10.9 nmol/L. Individual urine samples collected during the first 24 h ranged from below the detection limit (ND) to 237 nmol/L. The mean dose recovery excreted as methamidophos in urine was 1.1% (range 0.04-1.71%). Three environmental studies have been reported in the literature with levels ranging from ND to 66 nmol/L. The number of positive results in all three studies was low (<1.5% of total samples analyzed). When compared with our results (ND - 237 nmol/L), the studies suggest general population exposures are within the ADI. However, the very short half-life makes determining intermittent environmental exposures difficult., (Crown Copyright © 2014. Published by Elsevier Ireland Ltd. All rights reserved.)

A simple and sensitive method has been developed and validated for determining ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA), isobutyl methylphosphonic acid (iBuMPA), and pinacolyl methylphosphonic acid (PMPA) in human urine using gas chromatography-tandem mass spectrometry (GC-MS/MS) coupled with solid phase derivatization (SPD). These four alkyl methylphosphonic acids (AMPAs) are specific hydrolysis products and biomarkers of exposure to classic organophosphorus (OP) nerve agents VX, sarin, RVX, and soman. The AMPAs in urine samples were directly derivatized with pentafluorobenzyl bromide on a solid support and then extracted by liquid-liquid extraction. The analytes were quantified with isotope-dilution by negative chemical ionization (NCI) GC-MS/MS in a selected reaction monitoring (SRM) mode. This method is highly sensitive, with the limits of detection of 0.02 ng/mL for each compound in a 0.2 mL sample of human urine, and an excellent linearity from 0.1 to 50 ng/mL. It is proven to be very suitable for the qualitative and quantitative analyses of degradation markers of OP nerve agents in biomedical samples.

Rationale: Although use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. High-resolution mass spectrometry (HRMS) was compared to tandem mass spectrometry (MS/MS) analysis for the quantitation of five urinary metabolites specific to VX, Russian VX, soman, sarin and cyclosarin nerve agents. The HRMS method was further evaluated for qualitative screening of metabolites not included in the test panel., Methods: Nerve agent metabolites were extracted from urine using solid-phase extraction, separated using hydrophilic interaction chromatography and analyzed using both tandem and high-resolution mass spectrometry. MS/MS results were obtained using selected reaction monitoring with unit resolution; HRMS results were obtained using a mass extraction window of 10 ppm at a mass resolution of 50 000. The benchtop Orbitrap HRMS instrument was operated in full scan mode, to measure the presence of unexpected nerve agent metabolites., Results: The assessment of two quality control samples demonstrated high accuracy (99.5-104%) and high precision (2-9%) for both HRMS and MS/MS. Sensitivity, as described by the limit of detection, was overlapping for both detectors (0.2-0.7 ng/mL). Additionally, the HRMS method positively confirmed the presence of a nerve agent metabolite, not included in the test panel, using the accurate mass and relative retention time., Conclusions: The precision, accuracy, and sensitivity were comparable between the current MS/MS method and this newly developed HRMS analysis for five nerve agent metabolites. HRMS showed additional capabilities beyond the current method by confirming the presence of a metabolite not included in the test panel., (Published in 2013. This article is a U.S. Government work and is in the public domain in the USA.)

Objective: This study assessed pesticide exposure of children in rural Nicaragua in relation to parental pesticide use, from around conception to current school age, as part of an epidemiological evaluation of neurodevelopment effects., Methods: We included 132 children whose parents were subsistence farmers or plantation workers, or had an agricultural history. As proxies for children's long-term exposures, we constructed cumulative parental pesticide-specific use indices for periods before and after the child's birth from data obtained using an icon-calendar-based questionnaire, of application hours (h) for plantation workers and subsistence farmers, and of kilograms of active ingredients (ai) only for subsistence farmers. Pesticide residues of TCPY, 3-PBA and 2,4-D were analysed in children's urine as indicators for current exposures., Results: Life-time indices were highest for the organophosphates chlorpyrifos (median 114 h (min 2; max 1584), 19.2 kg ai (min 0.37; max 548)) and methamidophos (84 h (6; 1964), 12.2 kg ai (0.30; 780)). The P50 values of children's urinary residues were 3.7 μg/g creatinine for TCPY, 2.8 for 3-PBA and 0.9 for 2,4-D; TCPY values are comparable with those in other countries, but 3-PBA and 2,4-D are considerably higher. The maximum levels for all three pesticides are the highest reported for children. Residues increased on days after application, but most high residue levels were unrelated to parental pesticide applications., Conclusion: Urinary pesticide residues reveal high environmental exposure among children in rural Nicaragua. The quantitative parental pesticide use indices as proxies for children's exposures during different periods may be useful for the evaluation of developmental health effects.

Acephate, methamidophos, o-methoate, and dimethoate are organophosphorus pesticides, and ethylenethiouria and propylenethiourea are two metabolites from the bisdithiocarbamate fungicide family. They are some of the most widely used pesticides and fungicides in agriculture both domestically and abroad. The existing high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) method for the measurement of these compounds in human urine was improved by using a 96-well plate format sample preparation; the use of HPLC-MS/MS was comparable with a concentration range of 0.125 to 50 ng/ml. Deuterium-labeled acephate, ethylenethiouria, and methamidophos were used as internal standards. The sample preparation procedure, in the 96-well format with a 0.8-ml urine sample size, uses lyophilization of samples, followed by extraction with dichloromethane. The analytes were chromatographed on a Zorbax SB-C3 (4.6 × 150 mm, 5.0-μm) column with gradient elution by using 0.1% formic acid in aqueous solution (solvent A) and 0.1% formic acid in methanol (solvent B) mobile phase at a flow rate of 1 ml/min. Quantitative analysis was performed by atmospheric pressure chemical ionization source in positive ion mode using multiple-reaction monitoring of the precursor-to-product ion pairs for the analytes on a TSQ Quantum Ultra HPLC-MS/MS. Repeated analyses of urine samples spiked with high (15 ng/ml), medium (5 ng/ml), and low (1 ng/ml) concentrations of the analytes gave relative SDs of <13%. The limits of detection were in the range of 0.004-0.01 ng/ml. The method also has high accuracy, high precision, and excellent extraction recovery. Furthermore, the improved sample preparation method decreased the cost and labor required while effectively doubling the analytic throughput with minimal matrix effect.

Organophosphorus (OP) pesticides are used in agriculture and several are registered for home use. As young children age they may experience different pesticide exposures due to varying diet, behavior, and other factors. We measured six OP dialkylphosphate (DAP) metabolites (three dimethyl alkylphosphates (DMAP) and three diethyl alkylphosphates (DEAP)) in urine samples collected from ~400 children living in an agricultural community when they were 6, 12, and 24 months old. We examined bivariate associations between DAP metabolite levels and determinants such as age, diet, season, and parent occupation. To evaluate independent impacts, we then used generalized linear mixed multivariable models including interaction terms with age. The final models indicated that DMAP metabolite levels increased with age. DMAP levels were also positively associated with daily servings of produce at 6- and 24-months. Among the 6-month olds, DMAP metabolite levels were higher when samples were collected during the summer/spring versus the winter/fall months. Among the 12-month olds, DMAP and DEAP metabolites were higher when children lived ≤ 60 meters from an agricultural field. Among the 24-month-olds, DEAP metabolite levels were higher during the summer/spring months. Our findings suggest that there are multiple determinants of OP pesticide exposures, notably dietary intake and temporal and spatial proximity to agricultural use. The impact of these determinants varied by age and class of DAP metabolite.

Background: Limited data document the multiple and repeated pesticide absorption experienced by farmworkers in an agricultural season or their risk factors., Methods: Data were collected from 196 farmworkers four times at monthly intervals in 2007. Urine samples were tested for 12 pesticide urinary metabolites. Questionnaire data provided measures of exposure risks., Results: Farmworkers had at least one detection for many pesticide urinary metabolites; for example, 84.2% had at least one detection for acephate, 88.8% for 3,5,6-trichloro-2-pyridinol. Most farmworkers had multiple detections for specific metabolites; for example, 64.8% had two or more detections for acephate, 64.8% for 3,5,6-trichloro-2-pyridinol, 79.1% for 3-phenoxybenzoic acid, and 86.7% for 2,4-dichlorophenoxyacetic acid. Housing type had a consistent significant association with metabolite detections., Conclusions: Farmworkers are exposed to multiple pesticides across an agricultural season, and they experience repeated exposures to the same pesticides. Reducing farmworker pesticide exposure and delineating the health outcomes of this exposure require more detailed data. Am. J. Ind. Med. 53:802-813, 2010. (c) 2010 Wiley-Liss, Inc., (Copyright 2010 Wiley-Liss, Inc.)

Introduction: Acephate (AP) is a widely available organophosphorus (OP) insecticide considered to have low mammalian toxicity. In plants and insects, AP is metabolized extensively to methamidophos (MP), a more potent OP insecticide. The limited mammalian metabolism of AP to MP has been studied in laboratory rat models and suggests that initial formation of MP from AP may inhibit further formation. No case reports of human ingestion with urine AP and MP levels have been previously published., Case Report: A 4-year-old male being evaluated for altered mental status and head trauma was noted to have muscarinic and nicotinic cholinergic signs. Further history suggested possible ingestion of a commercial AP product at an unknown time. Ingestion of AP was confirmed by the presence of urinary AP and MP and severely depressed red blood cell (RBC) cholinesterase and pseudocholinesterase activity levels. The patient initially received atropine in two 0.02 mg/kg IV boluses, then was started on 0.05 mg/kg IV per hour and titrated accordingly to clinical signs of cholinergic toxicity. Pralidoxime was also given at 20 mg/kg IV bolus, followed by an infusion of 10 mg/kg per hour. The patient required mechanical ventilation for 18 days and atropine infusion for 20 days. After a complicated intensive care unit course, he recovered and was discharged after a total of 32 days of hospitalization., Methods: Four urine samples collected at different times were analyzed for AP and MP by using high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry. Kinetic calculations were performed by using standard equations., Results: Suspected ingestion was confirmed by the presence of AP and MP in urine. The amount of MP found in urine suggests some limited human metabolism to this more toxic compound., Conclusions: Urinary elimination kinetics of AP demonstrates low metabolic conversion of AP to MP in humans.

Because of increasing concern about widespread use of insecticides and fungicides, we have developed a highly sensitive analytical method to quantify urine-specific urinary biomarkers of the organophosphorus pesticides acephate, methamidophos, omethoate, dimethoate, and two metabolites from the fungicides alkylenebis-(dithiocarbamate) family: ethylenethiourea and propylenethiourea. The general sample preparation included lyophilization of the urine samples followed by extraction with dichloromethane. The analytical separation was performed by high-performance liquid chromatography (HPLC), and detection by a triple quadrupole mass spectrometer with and atmospheric pressure chemical ionization source in positive ion mode using multiple reaction monitoring and tandem mass spectrometry (MS/MS) analysis. Two different Thermo-Finnigan (San Jose, CA, USA) triple quadrupole mass spectrometers, a TSQ 7,000 and a TSQ Quantum Ultra, were used in these analyses; results are presented comparing the method specifications of these two instruments. Isotopically labeled internal standards were used for three of the analytes. The use of labeled internal standards in combination with HPLC-MS/MS provided a high degree of selectivity and precision. Repeated analysis of urine samples spiked with high, medium and low concentration of the analytes gave relative standard deviations of less than 18%. For all compounds the extraction efficiency ranged between 52% and 63%, relative recoveries were about 100%, and the limits of detection were in the range of 0.001-0.282 ng/ml.

Determination of dialkylphosphates (DAPs) in urine is useful for assessing human exposure to organophosphates (OPs). An improved method for the determination of four DAPs based on a strong anion exchange (SAX) disk extraction and in-vial derivatization was presented in this study. The matrix effect of urine components such as chloride ion and phosphate ion by using a SAX disk to extract DAPs in urine analysis was carefully evaluated. It was observed that the chloride ion mainly affected the extraction of diethylphosphate (DEP), dimethylthiophosphate (DMTP), and diethylthiophosphate (DETP) in urine. The addition of silver hydroxide could significantly improve the extraction efficiencies of these three DAPs, but it decreases the extraction efficiencies of dimethyldithiophosphate (DMDTP) and diethyldithiophosphate (DEDTP). The LOD of this method for DMTP, DETP, DMDTP, and DEDTP are 5, 5, 11, and 5 microg/L, respectively. A pretreatment strategy for the determination of DMTP, DMDTP, DETP, and DEDTP in urine was proposed which can provide reliable and prompt determination of routine urine analysis.

Background: Organophosphorus (OP) pesticide urinary metabolite levels in a sample of farmworker children in North Carolina are documented and compared to national reference data. The relative importance of para-occupational, residential, and environment risk factors are delineated., Methods: Urine samples were collected from 60 farmworker children 1-6 years of age, and interviews were completed by their mothers. Urine samples were analyzed for the dialkylphosphate (DAP) metabolites of OP pesticides. Summed molar concentrations of the diethyl and dimethyl DAP metabolites provided summary measures., Results: The farmworker children had relatively high levels of OP pesticide urinary metabolites compared to national reference data; for example, participating children had higher geometric means for diethylphosphate (DEP), diethylthiophosphate (DETP), and the summed diethyl metabolites. However, analyses found no pattern of significant associations between predictors and metabolite levels., Conclusions: Future research requires greater precision in sampling and measurement to determine the risk factors for pesticide exposure among farmworker children.

A new method based on direct solid-phase microextraction (DI-SPME) followed by gas chromatography-mass spectrometry was developed for the purpose of determining quinalphos in blood and urine. Two types of coated fibre have been assayed and compared: carbowax/divinylbenzene (CW/DVB 65 microm) and polydimethylsiloxane (PDMS 100 microm). The main parameters affecting the SPME process such as temperature, salt addition, pH, stirring and adsorption/desorption time profiles were optimized to enhance the sensitivity of the procedure. The method was developed using only 100 microL of blood and urine. Limits of detection of the method for blood and urine matrices were, respectively, 10 and 2 ng/mL. Linearity was established over concentration ranges from 0.05 to 50 microg/mL for blood, and 0.01 to 50 microg/mL for urine, with regression coefficients ranging between 0.9991 and 0.9999. Intra- and interday precision values were less than 13%, and accuracy was within +/-15% of the nominal concentration for all studied levels in both matrices. Absolute recoveries were 14 and 26% for blood and urine, respectively.

Acephate is a commonly used organophosphate insecticide applied on agricultural crops and in residential communities. Because very little acephate is metabolized prior to excretion, the parent pesticide compound can be measured in human urine. The residue method must be sensitive enough to determine human exposure and potential health risk for both agricultural workers and their families who may be exposed by pesticide drift or by inadvertent carry-home residues. A reliable and sensitive method was developed to measure acephate concentrations in human urine. Urine was diluted with water and acetone, adjusted to a neutral pH, and partitioned twice in acetone-methylene chloride (1 + 1, v/v), with NaCl added to aid separation. The solvent-reduced organic phase extracts were clarified by activated charcoal solid-phase extraction and then adjusted to a final volume with the addition of a D-xylose analyte protectant solution to reduce matrix enhancement effects. Acephate concentrations in urine were determined by gas chromatography using pulsed flame photometric detection. The method limit of detection was established at 2 microg/L, with a method limit of quantitation of 10 microg/L. The average recovery from urine fortified with 10-500 microg/L was 102 +/- 12% (n = 32).

Organophosphorous pesticides (OPs) are suspected of altering reproductive function by reducing brain acetylcholinesterase activity and monoamine levels, thus impairing hypothalamic and/or pituitary endocrine functions and gonadal processes. Our objective was to evaluate in a longitudinal study the association between OP exposure and serum levels of pituitary and sex hormones. Urinary OP metabolite levels were measured by gas-liquid chromatography, and serum pituitary and sex hormone levels by enzymatic immunoassay and radioimmunoassay in 64 men. A total of 147 urine and blood samples were analyzed for each parameter. More than 80% of the participants had at least one OP metabolite in their urine samples. The most frequent metabolite found was diethylthiophosphate (DETP; 55%), followed by diethylphosphate (DEP; 46%), dimethylthiophosphate (DMTP; 32%), and dimethyldithiophosphate (DMDTP; 31%). However, the metabolites detected at higher concentrations were DMTP, DEP, DMDTP, and dimethylphosphate. There was a high proportion of individuals with follicle-stimulating hormone (FSH) concentrations outside the range of normality (48%). The average FSH serum levels were higher during the heavy pesticide spraying season. However, a multivariate analysis of data collected in all periods showed that serum FSH levels were negatively associated with urinary concentrations of both DMTP and DMDTP, whereas luteinizing hormone (LH) was negatively associated with DMTP. We observed no significant associations between estradiol or testosterone serum levels with OP metabolites. The hormonal disruption in agricultural workers presented here, together with results from experimental animal studies, suggests that OP exposure disrupts the hypothalamic-pituitary endocrine function and also indicates that FSH and LH are the hormones most affected.

A rugged and sensitive method was developed to monitor urinary concentrations of O,S-dimethyl hydrogen phosphorothioate (O,S-DMPT), a specific biomarker of exposure to the organophosphate insecticide methamidophos. After pH adjustment and C18 solid phase extraction column cleanup, the urine was lyophilized at a low temperature to prevent loss of possibly highly volatile and unstable O,S-DMPT metabolite. The dried residue was derivatized using N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide and 1% tert-butyldimethylchlorosilane (MTBSTFA + 1% TBDMCS) in acetonitrile. After it was filtered, the derivatized product was analyzed and quantified by gas chromatography using a pulse flame photometric detector specific for phosphorus compounds. The limit of detection for this method was 0.004 ppm with a limit of quantitation of 0.02 ppm of urine. The mean recovery value for O,S-DMPT from 17 urine samples fortified at varying concentrations was 108% with a standard deviation of 12%.

This research evaluated exposure pathways across work tasks for three organophosphate pesticides in a group of greenhouse workers. During reentry in ornamental plant greenhouses, five male workers were monitored for five consecutive days. Skin contamination (excluding hands) was evaluated with nine pads of filter paper placed on the skin. Hand contamination was assessed by washing with 95% ethanol. Respiratory exposure was evaluated by personal air sampling. The respiratory dose was based on a lung ventilation of 20 L/min. The doses absorbed were estimated assuming 10% skin penetration and 100% lung retention. Urinary alkylphosphates were assayed in the 24-hour urine samples of the days on which exposure was evaluated. Respiratory exposure was usually less than skin contamination, being 4.5 +/- 8.4%, 9.9 +/- 10.0%, and 49.5 +/- 26.6% (mean +/- standard deviation) of total exposure for omethoate, tolclofos-methyl, and fenitrothion, respectively. Multiple regression analysis showed that urinary alkylphosphate (nmol/24 hours) (y) was significantly correlated (r = 0.716, p < 0.001) with the respiratory doses of the three active ingredients absorbed the same day (x1) and with the cutaneous dose absorbed the previous day (x2). The relationship was expressed by the equation y = 0.592x2 + 0.117x, + 156.364. The doses of omethoate absorbed by one worker were more than 45 times the acceptable daily intake (ADI) of 1.41 nmol/kg body weight (b.w.) The ADI for fenitrothion and tolclofos-methyl (10.8 and 212.6 nmol/kg body weight, respectively) were never exceeded. High absorption by one worker underlines the importance of correct use of protective clothing. In this study the hands were always a source of contact with the pesticides. Greater precautions should be taken to reduce contamination (clean gloves, constant use of gloves).

A simple gas chromatography-mass spectrometry (GC-MS) procedure has been developed for the main metabolites of organophosphorus nerve agents, alkylmethylphosphonic acids (AMPAs; alkyl = Et, i-Pr, and pinacolyl) in biofluids via extractive pentafluorobenzylation. The derivatization was carried out under liquid-liquid-solid-phase-transfer conditions using a polymer-bound tri-n-butylmethylphosphonium bromide as a catalyst. AMPAs in aqueous samples were semiquantitatively extracted into a small-volume organic layer as their pentafluorobenzyl derivatives at pH 4.5 (85 degrees C). Sample pretreatments for urine, serum, and saliva were each examined to minimize matrix interference. The detection limits of APMAs by electron-impact ionization GC-MS were around 50 ng/mL and 2.5-10 ng/mL in the full-scan and selected-ion monitoring modes, respectively. In order to detect trace-level AMPAs, negative-ion chemical ionization (NICI) was also employed to enhance sensitivity. The detection limits of AMPAs in biofluids were typically 60 pg/mL by GC-NICI-MS.

Acephate is an important systemic organophosphorus insecticide with toxicity attributed to bioactivation on metabolic conversion to methamidophos (or an oxidized metabolite thereof) which acts as an acetylcholinesterase (AChE) inhibitor. The selective toxicity of acephate is considered to be due to facile conversion to methamidophos in insects but not mammals. We show in the present investigation that a carboxyamidase activates acephate in mice and in turn undergoes inhibition by the hydrolysis product, i.e., methamidophos; thus, the bioactivation is started but immediately turned off. These relationships are established by finding that 4 h pretreatment of mice with methamidophos i.p. at 5 mg/kg has the following effects on acephate action: reduces methamidophos and acephate levels in liver by 30-60% in the first 2 h after i.p. acephate dosage; inhibits the liver carboxyamidase cleaving [14CH3S]acephate to [14CH3S]methamidiphos with 50% block at approximately 1 mg/kg; strongly inhibits 14CO2 liberation from [CH3(14)C(O)]acephate in vivo; markedly alters the pattern of urinary metabolites of acephate by increasing O- and S-demethylation products retaining the carboxyamide moiety; greatly reduces the brain AChE inhibition following acephate treatment; doubles the LD50 of i.p.-administered acephate from 540 to 1140 mg/kg. Methamidophos pretreatment in rats also markedly alters the metabolism of dimethoate (another systemic insecticide) from principally carboxyamide hydrolysis to mainly other pathways. In contrast, methamidophos pretreatment of houseflies does not alter the acephate-induced toxicity and brain AChE inhibition. The safety of acephate in mammals therefore appears to be due to conversion in small part to methamidophos which, acting directly or as a metabolite, is a potent carboxyamidase inhibitor, thereby blocking further activation.

1. To examine the metabolic fate of Cyanox [O-4-cyanophenyl O, O-dimethyl phosphorothioate, cyanophos, CYAP], rats of both sexes were administered [phenyl-4C]Cyanox as a single oral dose at levels of 0.5 mg/kg (low-dose group) or 25 mg/kg (high-dose group), or as multiple doses at 50 mg/kg/day once daily for 7 days (repeat-dose group). 2. The radiocarbon was almost completely eliminated from rats within 7 days after administration in both low- and high-dose groups. 14C-recoveries (expressed as % relative to the dosed 14C) in faeces and urine were 2-3 and 95-96% respectively for the low-dose and 13-14 and 86% respectively for the high-dose. 3. 14C-tissue residues on the seventh day after a single administration were generally low. Peak 14C-concentrations in blood and kidney occurred at 0.5 h (high-dose) and decreased rapidly thereafter. 4. Sex-related differences in the amounts of metabolites were observed in both groups. With the low-dose, the major metabolite was 4-cyanophenylsulphate in both sexes. However, in the high-dose, the major metabolites were 4-cyanophenyl sulphate and desmethylcyanoxon in males, but 4-cyanophenyl sulphate and desmethylcyanox in females. These findings indicate that the amounts or the types of enzymes responsible for oxidative desulphuration or oxidative dearylation in males are different from those in females. In the male rat given repeat doses significant differences in the amounts of metabolites in excreta were observed between early and final dosing. 5. The greater formation of desmethylcyanoxon in the male rat in the high-dose case is consistent with the higher incidence of toxicity in this sex.

Biological monitoring was carried out by assaying urinary dimethylated alkylphosphates [dimethyldithiophosphate (DMDTP), dimethylthio-phosphate (DMTP), and dimethylphosphate (DMP)] in 11 workers exposed to chlorpyrifos-methyl and azinphosmethyl during operations in a previously sprayed peach orchard. The subjects were divided into groups on the basis of the protective clothing worn. The results were compared with those of a reference group of 99 subjects not occupationally exposed to organophosphorus insecticides. The hand-wash liquid of the workers was also analyzed to evaluate skin contamination. Significantly higher levels of urinary excretion of alkylphosphates were found in all groups than in unexposed controls (Student's t test). A good correlation was found between quantities of the active ingredients on the hands and urinary excretion of total dimethylated alkylphosphates (r = 0.788) and of DMTP (r = 0.749) and DMP (r = 0.790) alone. The correlation between azinphos-methyl on the hands and urinary excretion of DMDTP was poor (r = 0.069). Under the working conditions investigated, the main route of absorption seems to be via the skin. Respiratory absorption, however, also appears significant in view of the difference in urinary excretion of dimethylated alkylphosphates found between subjects with and without face masks.

Five metabolites were detected in the plasma and urine of a patient following ingestion of the organophosphate insecticide, phenthoate. Intact phenthoate was detected only in gastric lavage fluid. After methylation of acidic extracts of plasma and urine, phenthoate acid, demethyl phenthoate, demethyl phenthoate oxon acid, demethyl phenthoate S-isomer, and demethyl phenthoate acid S-isomer were identified with synthesized phenthoate analogues by gas chromatography and gas chromatograph-mass spectrometry. The main metabolites were phenthoate acid and demethyl phenthoate oxon acid. Although demethyl phenthoate oxon acid was a significant metabolite, no phenthoate oxon, phenthoate oxon acid or demethyl phenthoate oxon were detected. If the oxon was formed in the patient, it may have been rapidly degraded by carboxylesterase or glutathione transferase to demethyl phenthoate oxon acid.

Exposure in vinyl hothouses to DMTP (0,0-dimethyl-s-(2-methoxy-1,3,4-thiadiazol-5(4H)-onyl-(4)-m ethyl)- dithiophosphate) was studied in 5 spray-operators and one assistant worker. To determine the level of exposure to DMTP, concentrations in the atmosphere, concentration in atmosphere near the operators breathing zone, skin levels from penetration through the operator's clothing, and urinary excretion of metabolites of DMTP (2,3-dihydro-5-methoxy-3-methyl-sulphonyl methyl-1,3,4-thiadiazol-2-one and 2,3-dihydro-5-methoxy-3-methyl-sulphinyl methyl-1,3,4-thiadiazol-2-one) were measured. The results were as follows; 1. DMTP concentrations in the atmosphere were 10.0-18.0 micrograms/m3, and concentrations in the atmosphere near the operators breathing zone were 5.4-33.6 micrograms/m3 during spraying. 2. Metabolites of DMTP were detected in all sprayers. Levels were 1.5 to 31.6 micrograms/day for the sulphonyl-metabolite. But it was not detected in the assistant sprayer. 3. Urinary concentrations of the DMTP metabolite were maximum 15-20 h after spraying. 4. Amount of DMTP adherent on the skin was estimated to be 0.4 to 71.3 micrograms for spray-operators. Variations resulted from differences in condition of protective clothing. 5. DMTP appeared to be absorbed not only through the respiratory system, but also through the skin.

Tissue concentration and elimination and urinary excretion were followed after intravenous injection of 14C-ethion in 12 goats. Tissue levels were determined in two goats after 8 hr and on each of days 1, 3, 7, 14 and 28 after exposure. During the four-week period plasma and all tissues examined (liver, kidney, fat, muscle, lung, heart and brain) had detectable 14C-residues, the highest values being found in liver, kidney and fat. Elimination of 14C-residues was faster in the first 3 days than in the later part of the experiment, where the elimination half-life for most tissues was approximately 2 weeks. During the first two weeks after exposure, 77% of the administered dose was eliminated in urine (55%) and faeces (22%). TLC of urine collected over the first 15 hr after exposure showed at least 8 bands of metabolites, five of which accounted for about two thirds of the dose excreted in urine.

Phosphorothioate oligodeoxynucleotides (S-ODNs) have potential as anti-viral agents and are being investigated for the chemotherapy of AIDS. A high-performance liquid chromatographic method is described for the analysis, in urine and plasma, of a 28-unit deoxycytidine homopolymer (S-dC28) and a 28-unit S-ODN "antisense" to the rev gene of the human immunodeficiency virus. This method employs ion-pairing HPLC with a polymeric column. Tetrabutylammonium is used as the ion-pairing agent in a mobile phase of acetonitrile in pH 7.0 phosphate buffer. Analysis of the S-ODNs is relatively rapid (20 min) and sensitive (20 nm) and is accomplished by a gradient elution (22.5-30.0% acetonitrile) followed by ultraviolet (266 or 272 nm) absorption detection. This method is likely applicable, with appropriate modifications, to all S-ODNs of similar molecular weight regardless of sequence. The S-ODNs bind very strongly to plasma proteins but are readily prepared for analysis by a phenol extraction procedure. In a preliminary pharmacokinetic study in mice with S-dC28, very rapid elimination of the oligomer from plasma was observed (half-time, 11.6 min). Estimates for the apparent volume of distribution and total body clearance were 3 ml and 0.2 ml/min, respectively. It appears that the majority of the oligomer is eliminated by renal clearance (glomerular filtration), a property likely shared by all S-ODNs of similar molecular mass.

An acute poisoning in a 50-year-old man who ingested approximately 6.2 g of the phosphorus ester methidathion is described. The patient was treated with three haemoperfusions 23, 44 and 115 h after ingestion, with continuous gastric lavage, atropine and pralidoxime administration and with prolonged mechanical ventilation. Haemoperfusion was an ineffective epuration technique since it removed only 0.22% of the ingested methidathion. The clinical course wavered because of a probable redistribution of phosphorus ester from fat to blood. A plasma level higher than 100 micrograms l-1 was associated with the most serious phases. Methidathion was present in the plasma until the sixth day, in the urine until the seventh and in the gastric juice until the eighth. Its absence in the fat biopsy made on the tenth day was an aid to therapy. The phosphorus ester did not inhibit lymphocyte neuropathy target esterase (NTE), neither did it induce development of delayed polyneuropathy.

Acephate is a water-soluble organophosphate insecticide whose action on insects has been related to its conversion to methamidophos, a very potent anticholinesterase agent which has caused delayed neuropathy in man. Inhalation and skin exposure to acephate was evaluated in four workers engaged in 8-day campaigns with the formulation of the 97%-pure technical product. Before, during, and after exposure, the workers were monitored for the urine content of acephate and methamidophos, and for erythrocyte (AChE) and plasma (PChE) cholinesterase levels. Median air concentrations (8-hr TWA) ranged from 0.278 to 2.170 mg/m3; median total-body skin deposition ranged from 26.1 to 41.9 mg/day. Based on these values, daily workers' absorption of acephate was estimated to be in the order of 10-20 mg. Urinary excretion of unchanged acephate followed a pattern consistent with exposure, showing peak values of excretion during the workshift or in the eight hr after the end of the workshift. The urine levels of unchanged acephate were found to vary from 1 to 10 mg/L. Methamidophos was not detected in any urine sample (detection limit: 30 micrograms/L). High correlation (r = 0.78) was found between skin exposure level and urine acephate elimination. No changes in AChE or PChE were observed for the workers whose urinary concentrations of acephate were 1 or 2 mg/L. One subject who had urinary acephate excretion between 3 and 8 mg/L, showed slightly decreased values of PChE during exposure and of AChE after exposure.

Recognition of exposure to diazinon, an organophosphate insecticide, was studied in goats. Urine and milk dialkyl phosphate concentrations (DETP; O,O-diethyl phosphorothionate) and blood cholinesterase activity (ChE) and diazinon concentrations were measured. Groups (n = 3 each) given (orally) diazinon at doses of 0.5 mg/kg for 7 days (small dose) or 5 mg/kg for 7 days (large dose) were compared with goats acutely exposed to single doses of 150 mg/kg (n = 1) or 700 mg/kg (n = 1). Clinical signs of intoxication occurred only in the goat given the 700 mg/kg dose. Urinary DETP concentrations were sensitive indicators of diazinon exposure and provided quantitative differences between small, large, and acute dosage exposures. Milk DETP concentrations were not detected. Cholinesterase measurement was useful only in the acute exposure studies. Whole blood diazinon concentrations were detected only in goats given the large dose for 7 days and acutely exposed. Measurement of urinary DETP was a sensitive aid for recognition of diazinon exposure.

The absorption of malathion and phosalone was followed in occupationally exposed workers by determination of residues excreted in the urine. Because of the high concentrations found in the morning urine samples, the rates of excretion of phosalone metabolites in the urine of a volunteer experimentally exposed to phosalone during one and then again during three subsequent working days were investigated. The urinary excretion of phosalone metabolites was most intense 4--5 hours after exposure. At the beginning of the next day, the metabolites were still well measureable in the urine. Blood and plasma cholinesterase activities were only slightly reduced during exposure. The analyses of 24-hour urine samples, or of urine samples taken 4--5 hours after exposure, are not suitable for the routine control of occupationally exposed persons because of sampling difficulties. Instead, analyses of samples taken immediately before and after work hours have to be performed. A systematic increase in the concentrations of pesticide residues in the morning urine should initiate more efficient and well-timed protection measures.

Recognition of exposure to imidan was assessed in goats by dialkyl phosphate concentrations, blood cholinesterase (ChE) determinations, and blood imidan concentrations. Groups of three goats received 5.0 mg imidan/kg/day (low dose) or 10 mg imidan/kg/day (high dose) for 7 days orally. One goat received no imidan and one goat received an acute single dose (200 mg/kg). The urine of all treated goats was examined for the excretory dialkyl phosphates, O,O-dimethyl phosphorodithioate (DMDTP) and O,O-dimethyl phosphorothionate (DMTP). The overall mean DMDTP urinary concentration was 19.1 ppm (10-mg/kg treatment group) and 7.2 ppm (5-mg/kg treatment group). These metabolites rapidly disappeared following removal of the treatment except in those goats clinically affected. Milk contained no identifiable concentrations of dialkyl phosphates. Cholinesterase depression was observed in all imidan-treated goats, and a dose effect was observed. No imidan was detected in whole blood of either the 5- or 10-mg/kg treatment groups. Low blood concentrations (ppb) of imidan were measured in the acute single-dose exposed goat. Both urinary DMDTP and blood ChE provided recognition of imidan exposure. DMDTP, however, was immediately present in urine after exposure and provided stronger support for organophosphate exposure than did blood ChE.

On-column transesterification with methanol was applied for the gas chromatographic determination of N-methylcarbamates extracted from human urine. Transesterification conversion efficiencies of N-methylcarbamates dioxacarb, carbofuran and OMS-22, calculated from the amount of the on-column produced O-methyl-N-methylcarbamate (DMC), were 96, 77 and 76% with detection limits of 8, 10 and 10 ng, respectively. In the investigated concentration range of 0.2-3 micrograms/ml of urine the extraction efficiencies with methylene chloride were independent of the initial concentration of N-methylcarbamate added to urine samples of non-exposed persons. The recoveries and rel. S.D. were 74 +/- 11, 64 +/- 8 and 79 +/- 12% for dioxacarb, carbofuran and OMS-22, respectively. The procedure was applied for the gas chromatographic determination of carbofuran and its metabolites containing the N-methylcarbamic group extracted from urine samples of occupationally exposed persons in a pesticide formulating plant. The level of extracted N-methylcarbamates and the concentration of degradation products of organophosphorus pesticides detected in the urine of the same persons were correlated with the blood and plasma cholinesterase activities. Although the determination of DMC includes only a smaller part of the excreted N-methylcarbamate, a simultaneous determination of both carbamates and organophosphorus residues made it possible to distinguish the cause of depression in cholinesterase activity, indicating early and specifically the exposure to a particular group of agents hazardous to health.