Revealing nitrogenous VX metabolites and the whole-molecule VX metabolism in the urine of guinea pigs.

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.

Development of a strategic approach for comprehensive detection of organophosphate pesticide metabolites in urine: Extrapolation of cadusafos and prothiofos metabolomics data of mice to humans.

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 (tR ), 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.

Detection of Pesticides and Metabolites Using Surface-Enhanced Raman Spectroscopy (SERS): Acephate.

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.

'Dilute-and-shoot' RSLC-MS-MS method for fast detection of nerve and vesicant chemical warfare agent metabolites in urine.

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 ß-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.

Quantification of nerve agent biomarkers in human serum and urine.

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.

Biological monitoring for exposure to methamidophos: a human oral dosing study.

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.

Determination of nerve agent metabolites in human urine by isotope-dilution gas chromatography-tandem mass spectrometry after solid phase supported derivatization.

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.

Comparison of high-resolution and tandem mass spectrometry for the analysis of nerve agent metabolites in urine.

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.

Assessment of long-term and recent pesticide exposure among rural school children in Nicaragua.

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.

Determinants of organophosphorus pesticide urinary metabolite levels in young children living in an agricultural community.

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.

High-throughput sample preparation for the quantitation of acephate, methamidophos, omethoate, dimethoate, ethylenethiourea, and propylenethiourea in human urine using 96-well-plate automated extraction and high-performance liquid chromatography-tandem mass spectrometry.

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.

Repeated pesticide exposure among North Carolina migrant and seasonal farmworkers.

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.

Urinary elimination kinetics of acephate and its metabolite, methamidophos, in urine after acute ingestion.

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.

Improved analysis of dialkylphosphates in urine using strong anion exchange disk extraction and in-vial derivatization.

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.

Method for determination of acephate, methamidophos, omethoate, dimethoate, ethylenethiourea and propylenethiourea in human urine using high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry.

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.

Organophosphorus pesticide urinary metabolite levels of children in farmworker households in eastern North Carolina.

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.

Determination of quinalphos in blood and urine by direct solid-phase microextraction combined with gas chromatography-mass spectrometry.

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.

Pesticide exposure alters follicle-stimulating hormone levels in Mexican agricultural workers.

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.

Determination of acephate in human urine.

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).