Pesticides and their metabolites in human urine: development of multi-analyte method by LC-MS/MS and GC-MS/MS.

The objective of this study consists of being able to develop a precise, reliable, easy, cheap and quick method to identify and quantify the presence of pesticide metabolites and their parents in human urine. In order to reach our purpose we selected the pesticides and their metabolites with intended uses on permanent crops such as orchards and vineyard. The activity planning started with the identification of the target list carried out by UHPLC-MS/MS and GC-MS/MS, succeeded by several tests oriented to determine the best sample treatment having recourse to instrumental analysis in the range 5-100 ng/mL. Several purifications were also investigated combining different adsorbents (PSA, EMR-lipid and final polish pouch). The use of formic acid during the extraction step has no impact on the recoveries, whereas the PSA adsorbent in the cleanup step negatively affects the results for all investigated metabolites. Any substantial differences were not observed in urine matrix for parent compounds achieving recoveries higher than 80% and RSD less than 20%. The final polish in combination or not with Enhanced Matrix Removal EMR-lipid did not show statistically significant difference in term of trueness and precision for both metabolites and parents, as evaluated by one-way ANOVA. The 3-OH THPI was the most critical compound with not acceptable results for linearity, trueness and precision.

Modified QuEChERS extraction method followed by simultaneous quantitation of nine multi-class pesticides in human blood and urine by using GC-MS.

Organophosphate, carbamate and pyrethroid pesticides are the most common insecticides used worldwide. They may cause chronic poisoning in farmers and acute poisoning in homicidal or suicidal cases. The determination of trace levels of these pesticides in human blood and urine is very challenging. This study focuses on a simultaneous quantitation method that was developed and validated for multi-class nine pesticides belonging to organophosphate, carbamate and pyrethroid classes in human blood and urine. Target pesticides were extracted from blood and urine using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction method. Capillary column DB-35 ms (15 m × 0.25 mm, 0.25 µm) was used for chromatography with a 0.079 ml/min flow rate of carrier gas at constant pressure mode. Quantitation of sulfotep, phorate, carbofuran, chlorpyriphos, profenophos, triazophos, pyriproxyfen, lambda-cyhalothrin and permethrin was performed by mass spectrometer equipped with electron impact ionization source using selected ion monitoring (SIM) mode. The lower and upper limits of quantitation for all nine pesticides were 0.01 mg/L and 2.0 mg/dL respectively. The proposed method was proved to be simple, fast, sensitive, and robust. It has been applied to the analysis of 9 pesticides samples.

Production of a specific monoclonal antibody for 1-naphthol based on novel hapten strategy and development of an easy-to-use ELISA in urine samples.

1-naphthol (1-NAP) is the main metabolite of pesticide carbaryl and naphthalene, and is also a genotoxic and carcinogenic intermediate in the synthesis of organic compound, dyes, pigment and pharmaceutical industry. In this work, two novel haptens were designed and synthesized for developing a competitive indirect enzyme-linked immunosorbent assay (ciELISA) method for 1-NAP in urine samples. The assay showed a limit of detection of 2.21 ng/mL and working range from 4.02 ng/mL to 31.25 ng/mL for 1-NAP in optimized working buffer. The matrix effect of samples was eliminated via 15-fold dilution of optimized working buffer. Good average recoveries (102.4%-123.4%) with a coefficient of variation from 11.7% to 14.7% was obtained for spiked urine samples. Subsequent instrument verification test showed good correlation between the results of ciELISA and high-performance liquid chromatography. The developed ciELISA is a high-throughput tool to monitor 1-NAP in urine, which can provide technical support for the establishment of biological exposure level for the exposure to carbaryl, naphthalene and other related pollutants.

Urine glyphosate level as a quantitative biomarker of oral exposure.

BACKGROUND: Since the classification of glyphosate as a Group 2A substance "probably carcinogenic to humans" by the IARC in 2015, human health concerns have been raised regarding the exposure of operators, bystanders, and consumers. Urine measurement studies have been conducted, but since toxicokinetic data on glyphosate in humans is lacking, a meaningful interpretation of this data regarding exposure is not possible. OBJECTIVE: This study aims to determine the fraction of glyphosate and AMPA excretion in urine after consuming ordinary food with glyphosate residue, to estimate dietary glyphosate intake. METHODS: Twelve participants consumed a test meal with a known amount of glyphosate residue and a small amount of AMPA. Urinary excretion was examined for the next 48 h. RESULTS: Only 1% of the glyphosate dose was excreted in urine. The urinary data indicated the elimination half-life was 9 h. For AMPA, 23% of the dose was excreted in urine, assuming that no metabolism of glyphosate to AMPA occurred. If all of the excreted AMPA was a glyphosate metabolite, this corresponds to 0.3% of the glyphosate dose on a molar basis. CONCLUSION: This study provides a basis for estimating oral glyphosate intake using urinary biomonitoring data.

A sensitive and accurate vortex-assisted liquid-liquid microextraction-gas chromatography-mass spectrometry method for urinary triazoles.

The development and validation of an innovative, simple, rapid, selective and sensitive vortex-assisted liquid-liquid microextraction-gas chromatography- mass spectrometry (VALLME-GC/MS) method to seven triazole fungicides in urine is reported. The confidence parameters were exhaustive evaluated and linearity (r > 0.99), precision (below 15%, to quality controls, and 20%, to lower limit of quantification), accuracy and robustness were adequate for the analysis of cyproconazole, epoxiconazole, metconazole, tebuconazole, triadimenol, propiconazole and triadimefon. To assess the applicability of the method, analyses were performed in volunteers exposed to triazoles in the last 30 days. The method demonstrated satisfactory analytical performance, with sufficient detectability to be applied as a potential tool to biomonitoring individuals exposed to the seven evaluated fungicides.

Development of a Highly Sensitive Direct Competitive Fluorescence Enzyme Immunoassay Based on a Nanobody-Alkaline Phosphatase Fusion Protein for Detection of 3-Phenoxybenzoic Acid in Urine.

3-Phenoxybenzoic acid (3-PBA) is a human urinary metabolite of many pyrethroid insecticides and can be used as a biomarker to monitor human exposure to these pesticides. A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for detecting 3-PBA on the basis of a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The anti-3-PBA Nb-AP fusion protein was expressed and purified. The 50% inhibitory concentration (IC50) and linear range of dc-FEIA were 0.082 and 0.015-0.447 ng/mL, respectively, with a detection limit of 0.011 ng/mL. The IC50 of dc-FEIA was improved by nearly ten times compared with those of one-step and three-step direct competitive enzyme-linked immunosorbent assay (dc-ELISA). Spiked urine samples were detected by both dc-FEIA and liquid chromatography-mass spectrometry (LC-MS), and the results showed good consistency between the two analysis methods, indicating the reliability of dc-FEIA based on the Nb-AP fusion protein for detecting 3-PBA in urine.

An effective and high-throughput analytical methodology for pesticide screening in human urine by disposable pipette extraction and gas chromatography - mass spectrometry.

In this paper, the use of disposable pipette extraction (DPX) for the determination of pesticides in human urine in possible cases of poisoning is proposed for the first time. The pesticides studied were oxamyl, propoxur, carbofuran, 3­hydroxycarbofuran, carbaryl, methiocarb, terbufos, parathion methyl, malathion, chlorpyrifos and endosulfan. The pipette tip used for the extraction of these compounds was commercially acquired. It has a capacity of 5 mL and contains 20 mg of sorbent material (styrene-divinylbenzene). The optimization of the main parameters that can influence the extraction efficiency of this sample preparation technique was performed with univariate and multivariate approaches. The analytes were separated and identified by gas chromatography coupled to mass spectrometry (GC-MS). The optimal extraction conditions were 5 extraction cycles of 30 s and 5 desorption cycles of 15 s with 250 µL of ethyl acetate. Elution of the extract was performed in a vial containing 100 mg of anhydrous sodium sulfate. The method developed was validated, providing correlation coefficients higher than 0.9955 for all analytes, limits of detection (LOD) of 0.76 to 1.52 µg L-1, limits of quantification (LOQ) of 2.5 to 5.0 µg L-1, relative recoveries of 63 to 118%, intra-day precision of 0.7 to 15.3% and inter-day precision of 4.9 to 13.1%. An effective and rapid method for the analysis of human urine for the identification of possible cases of poisoning by pesticides was successful developed.

Analysis of metabolites of organophosphate and pyrethroid pesticides in human urine from urban and agricultural populations (Catalonia and Galicia).

Isotope dilution solid phase extraction UPLC-MS/MS has been used to develop a robust and rapid methodology for the determination of eight specific metabolites of organophosphate and pyrethroid pesticides in human urine. The use of methanol:acetone (25:75v/v) affords an improvement in extraction efficiency in comparison to these individual solvents. The use of synthetic urine improves selectivity and limits of detection for the calibration straight lines. The method provides detection limits of 14-69pg/ml and 18-19pg/ml for the organophosphate and pyrethroid metabolites, respectively. Urine analyses of these metabolites in urban non-occupationally exposed individuals and farm workers shows that ingestion of these pesticides occurred in both populations. The concentrations of organophosphate pesticide metabolites in the latter were twofold than those from non-exposed populations.

Multi-residue analysis of organic pollutants in hair and urine for matrices comparison.

Urine being currently the most classically used matrix for the assessment of human exposure to pesticides, a growing interest is yet observed in hair analysis for the detection of organic pollutants. The aim of the present work was to develop and to validate multi-residue analytical methods, as similar as possible, in order to determine pesticides and their metabolites in these two biological matrices despite their different nature. The list of parent compounds and their metabolites investigated here consisted of 56 compounds, including organochlorines, organophosphates, pyrethroids, carbamates, other pesticides and polychlorinated biphenyls (PCBs). Two different approaches were necessary for the analysis of non-polar compounds (mainly parents) on one hand and polar analytes (mainly metabolites) on the other hand. In the final procedure, extraction from hair was carried out with acetonitrile/water after sample decontamination and pulverization. Extract was split into two fractions, which were analyzed directly with solid phase microextraction (SPME) injection for non-polar compounds and after derivatization with liquid injection for polar compounds. In urine, non-polar compounds were analyzed directly using SPME. Polar compounds were analyzed after acidic hydrolysis, liquid-liquid extraction with acetonitrile-cyclohexane-ethyl acetate, derivatization and liquid injection. Analysis was performed with gas chromatography tandem mass spectrometry operating in negative chemical ionization (GC-MS/MS-NCI) for all the compounds (non-polar and polar) in the two matrices. In hair, limits of quantification (LOQ) ranged from 0.02 pg/mg for trifluralin to 5.5 pg/mg for diethylphosphate. In urine, LOQ ranged from 0.4 pg/mL for α-endosulfan to 4 ng/mL for dimethyldithiophosphate. The analysis of samples supplemented with standards and samples collected from an animal previously submitted to chronic exposure to pesticides confirmed that all the compounds were analyzable in both hair and urine. In addition, the levels of sensitivity reached with these methods were quite satisfactory with regard to previously published studies, and also considering the number of compounds investigated.

A review of methods for the analysis of orphan and difficult pesticides: glyphosate, glufosinate, quaternary ammonium and phenoxy acid herbicides, and dithiocarbamate and phthalimide fungicides.

This article reviews the chromatography/MS methodologies for analysis of pesticide residues of orphan and difficult chemical classes in a variety of sample matrixes including water, urine, blood, and food. The review focuses on pesticide classes that are not commonly included in multiresidue analysis methods such as highly polar or ionic herbicides including glyphosate, glufosinate, quaternary ammonium, and phenoxy acid herbicides, and some of their major degradation or metabolite products. In addition, dithiocarbamate and phthalimide fungicides, which are thermally unstable and have stability issues in some solvents or sample matrixes, are also examined due to their special needs in residue analysis.

Comparisons of metabolic and physiological changes in rats following short term oral dosing with pesticides commonly found in food.

¹H Nuclear Magnetic Resonance spectroscopy has been used to profile urinary metabolites in male Fischer F344 rats in order to assess the metabolic changes induced by oral exposure to two benzimidazole fungicides (carbendazim and thiabendazole) and two bipyridyllium herbicides (chlormequat and mepiquat). Exposure levels were selected to be lower than those expected to cause overt signs of toxicity. We then compared the sensitivity of the metabolomics approach to more traditional methods of toxicity assessment such as the measurement of growth and organ weights. Separate, acute exposure experiments were conducted for each pesticide to identify potential metabolic markers of exposure across four doses (and a control). Growth, organ weights and feeding/drinking rates were not significantly affected by any compounds at any dose levels tested. In contrast, metabolic responses were detected within 8 and 24h for chlormequat and mepiquat, and after 24h for carbendazim and thiabendazole. These results demonstrate the potential for the use of metabolomics in food toxicity testing.

[Method establishment for the determination of 6 organophosphorus pesticides metabolites in human urine using LC-MS/MS].

OBJECTIVE: To establish a method to determine the presence and concentration of DEP, DMDTP, DEDTP, PNP, CMHC and TCPY organophosphorus pesticides metabolites in human urine by LC-MS/MS. METHOD: 2.0 ml of urine was enzymatically hydrolyzed in water bath 37 degrees C for 4 hours, then the urine samples were solid-phase extracted by Oasis HLB and eluted with methanol, after centrifugation and concentration the urine samples were separated on waters symmetry C18 5 microm (2.1 mm x 150 mm) column with ammonium acetate buffer solution acetonitrile-water as mobile phase to constant gradient elution. Electrospray ionization source (ESI) was applied and operated in positive ion mode, and multiple reaction monitoring (MRM) mode was adopted. RESULTS: Within concentration ranging from 0 - 1000 ng/ml the correlation coefficient for 5 compounds of 6 were greater than 0.990, recoveries were between 79% - 130%, RSD < 8.2%. The RSD of intra-day and inter-day were less than 15% (n = 6). CONCLUSION: The procedure of samples collection is simple. This is a accurate and specific method for testing organophosphorus pesticides metabolites in human urine.

Detection of herbicides in the urine of pet dogs following home lawn chemical application.

Exposure to herbicide-treated lawns has been associated with significantly higher bladder cancer risk in dogs. This work was performed to further characterize lawn chemical exposures in dogs, and to determine environmental factors associated with chemical residence time on grass. In addition to concern for canine health, a strong justification for the work was that dogs may serve as sentinels for potentially harmful environmental exposures in humans. Experimentally, herbicides [2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxypropionic acid (MCPP), dicamba] were applied to grass plots under different conditions (e.g., green, dry brown, wet, and recently mowed grass). Chemicals in dislodgeable residues were measured by LC-MS at 0.17, 1, 24, 48, 72 h post treatment. In a separate study, 2,4-D, MCPP, and dithiopyr concentrations were measured in the urine of dogs and in dislodgeable grass residues in households that applied or did not apply chemicals in the preceding 48 h. Chemicals were measured at 0, 24, and 48 h post application in treated households and at time 0 in untreated control households. Residence times of 2,4-D, MCPP, and dicamba were significantly prolonged (P<0.05) on dry brown grass compared to green grass. Chemicals were detected in the urine of dogs in 14 of 25 households before lawn treatment, in 19 of 25 households after lawn treatment, and in 4 of 8 untreated households. Chemicals were commonly detected in grass residues from treated lawns, and from untreated lawns suggesting chemical drift from nearby treated areas. Thus dogs could be exposed to chemicals through contact with their own lawn (treated or contaminated through drift) or through contact with other grassy areas if they travel. The length of time to restrict a dog's access to treated lawns following treatment remains to be defined. Further study is indicated to assess the risks of herbicide exposure in humans and dogs.

Predictors of exposure to organophosphate pesticides in schoolchildren in the Province of Talca, Chile.

BACKGROUND: Few data exist in Latin America concerning the association between organophosphate (OP) urinary metabolites and the consumption of fruits and vegetables and other exposure risk variables in schoolchildren. METHODS: We collected samples of urine from 190 Chilean children aged 6-12 years, fruits and vegetables, water and soil from schools and homes, and sociodemographic data through a questionnaire. We measured urinary dialkylphosphate (DAP) OP metabolites and OP pesticide residues in food consumed by these 190 children during two seasons: December 2010 (summer) and May 2011 (fall). We analyzed the relationship between urinary DAP concentrations and pesticide residues in food, home pesticide use, and residential location. RESULTS: Diethylalkylphosphates (DEAP) and dimethylalkylphosphates (DMAP) were detected in urine in 76% and 27% of the samples, respectively. Factors associated with urinary DEAP included chlorpyrifos in consumed fruits (p<0.0001), urinary creatinine (p<0.0001), rural residence (p=0.02) and age less than 9 years (p=0.004). Factors associated with urinary DMAP included the presence of phosmet residues in fruits (p<0.0001), close proximity to a farm (p=0.002), home fenitrothion use (p=0.009), and season (p<0.0001). CONCLUSIONS: Urinary DAP levels in Chilean school children were high compared to previously reported studies. The presence of chlorpyrifos and phosmet residues in fruits was the major factor predicting urinary DAP metabolite concentrations in children.

National study of exposure to pesticides among professional applicators: an investigation based on urinary biomarkers.

Epidemiologic studies of pesticides have been subject to important biases arising from exposure misclassification. Although turf applicators are exposed to a variety of pesticides, these exposures have not been well characterized. This paper describes a repeated measures study of 135 TruGreen applicators over three spraying seasons via the collection of 1028 urine samples. These applicators were employed in six cities across the United States. Twenty-four-hour estimates (µg) were calculated for the parent compounds 2,4-D, MCPA, mecoprop, dicamba, and imidacloprid and for the insecticide metabolites MPA and 6-CNA. Descriptive statistics were used to characterize the urinary levels of these pesticides, whereas mixed models were applied to describe the variance apportionment with respect to city, season, individual, and day of sampling. The contributions to the overall variance explained by each of these factors varied considerably by the type of pesticide. The implications for characterizing exposures in these workers within the context of a cohort study are discussed.

Comparison of current-use pesticide and other toxicant urinary metabolite levels among pregnant women in the CHAMACOS cohort and NHANES.

BACKGROUND: We measured 34 metabolites of current-use pesticides and other precursor compounds in urine samples collected twice during pregnancy from 538 women living in the Salinas Valley of California, a highly agricultural area (1999-2001). Precursors of these metabolites included fungicides, carbamate, organochlorine, organophosphorus (OP), and pyrethroid insecticides, and triazine and chloroacetanilide herbicides. We also measured ethylenethiourea, a metabolite of the ethylene-bisdithiocarbamate fungicides. Repeat measurements of the compounds presented here have not been reported in pregnant women previously. To understand the impact of the women's regional environment on these findings, we compared metabolite concentrations from the CHAMACOS (Center for the Health Assessment of Mothers and Children of Salinas) cohort with U.S. national reference data for 342 pregnant women sampled by the National Health and Nutrition Examination Survey (1999-2002). RESULTS: The eight metabolites detected in > 50% of samples [2,4-dichlorophenol (2,4-DCP); 2,5-dichlorophenol (2,5-DCP); 1- and 2-naphthol; ortho-phenylphenol (ORTH); para-nitrophenol (PNP); 2,4,6-trichlorophenol (2,4,6-TCP); and 3,4,6-trichloro-2-pyridinol (TCPy)] may be related to home or agricultural pesticide use in the Salinas Valley, household products, and other sources of chlorinated phenols. More than 78% of women in this study had detectable levels of at least one of the OP pesticide-specific metabolites that we measured, and > 30% had two or more. The 95th percentile values of six of the most commonly detected (> 50%) compounds were significantly higher among the CHAMACOS women after controlling for age, race, socioeconomic status, and smoking [(2,4-DCP; 2,5-DCP; ORTH; PNP; 2,4,6-TCP; and TCPy); quantile regression p < 0.05]. CONCLUSIONS: Findings suggest that the CHAMACOS cohort has an additional burden of precursor pesticide exposure compared with the national sample, possibly from living and/or working in an agricultural area.

Variation across the agricultural season in organophosphorus pesticide urinary metabolite levels for Latino farmworkers in eastern North Carolina: project design and descriptive results.

BACKGROUND: Community Participatory Approach to Measuring Farmworker Pesticide Exposure, PACE3, used a longitudinal design to document pesticide biomarkers among farmworkers. This article presents an overview of PACE3 and provides a descriptive analysis of participant characteristics and one set of pesticide biomarkers, the dialkylphosphate (DAP) urinary metabolites of organophosphorus (OP) pesticides. METHODS: Two hundred eighty seven farmworkers were recruited during 2007 from 44 farmworker camps in 11 eastern North Carolina counties. Participants provided interviews, urine samples, blood samples, and saliva samples up to four times at monthly intervals beginning in May. A total of 939 data points were collected. RESULTS: Farmworkers were largely men (91.3%) from Mexico (94.8%) with a mean age of 33.7 years (SE 0.82); 23.3% spoke an indigenous language. Across all data points, frequencies of detection and median urinary concentrations were 41.3% and 0.96 microg/L for dimethylphosphate (DMP), 78.3% and 3.61 microg/L for dimethylthiophosphate (DMTP), 33.3% and 0.04 microg/L for dimethyldithiophosphate (DMDTP), 40.5% and 0.87 microg/L for diethylphosphate (DEP), 32.3% and 0.17 microg/L for diethylthiophosphate (DETP), and 8.09% and 0.00 microg/L for diethyldithiophosphate (DEDTP). The frequencies of detection and urinary concentrations of the DAP metabolites increased during the season. CONCLUSIONS: More PACE3 participants were from Mexico, male, migrant workers, and spoke an indigenous language compared to national data. PACE3 participants had comparable frequencies of detection and urinary metabolite concentrations with participants in other studies. Variability in the frequencies of detection and urinary concentrations of the DAP metabolites indicates the importance of longitudinal studies of biomarkers of currently used pesticides in farmworker populations.

Determination of no-observed effect level (NOEL)-biomarker equivalents to interpret biomonitoring data for organophosphorus pesticides in children.

BACKGROUND: Environmental exposure to organophosphorus pesticides has been characterized in various populations, but interpretation of these data from a health risk perspective remains an issue. The current paper proposes biological reference values to help interpret biomonitoring data related to an exposure to organophosphorus pesticides in children for which measurements of alkylphosphate metabolites are available. METHODS: Published models describing the kinetics of malathion and chlorpyrifos in humans were used to determine no-observed effect level - biomarker equivalents for methylphosphates and ethylphosphates, respectively. These were expressed in the form of cumulative urinary amounts of alkylphosphates over specified time periods corresponding to an absorbed no-observed effect level dose (derived from a published human exposure dose) and assuming various plausible exposure scenarios. Cumulative amounts of methylphosphate and ethylphosphate metabolites measured in the urine of a group of Quebec children were then compared to the proposed biological reference values. RESULTS: From a published no-observed effect level dose for malathion and chlorpyrifos, the model predicts corresponding oral biological reference values for methylphosphate and ethylphosphate derivatives of 106 and 52 nmol/kg of body weight, respectively, in 12-h nighttime urine collections, and dermal biological reference values of 40 and 32 nmol/kg of body weight. Out of the 442 available urine samples, only one presented a methylphosphate excretion exceeding the biological reference value established on the basis of a dermal exposure scenario and none of the methylphosphate and ethylphosphate excretion values were above the obtained oral biological reference values, which reflect the main exposure route in children. CONCLUSION: This study is a first step towards the development of biological guidelines for organophophorus pesticides using a toxicokinetic modeling approach, which can be used to provide a health-based interpretation of biomonitoring data in the general population.

Analytical method developed for measurement of dialkylphosphate metabolites in urine collected from children non-occupationally exposed to organophosphate pesticides in an agricultural community in Thailand.

There has been increasing concern in regards to organophosphate (OP) pesticide exposure among farm workers and their families in Thailand's agricultural areas. Therefore, the development of an analytical method for estimating OP pesticide exposure is necessary to allow for monitoring of OP pesticide exposures within these populations. This paper describes an analytical method developed to measure dialkylphosphate (DAP) metabolites in urine. The methods in this study are important in the biological monitoring of OP metabolites in agricultural families in Thailand and can be used as an initial guidance procedure in any environmental toxicological laboratory in Thailand.