Rapid determination of glyphosate and glufosinate in human blood by probe electrospray ionization tandem mass spectrometry.

In forensic science, glyphosate (GLYP) and glufosinate (GLUF), a class of non-selective broad-spectrum herbicides, have been frequently encountered in many fatal poisoning and suicide cases due to their widespread availability. Therefore, it is essential to develop an effective method for detecting these compounds. Some conventional methods, such as gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), have been reported to detect these compounds. However, these methods are not ideal for their time-consuming and non-sensitive feature. Herein, probe electrospray ionization (PESI) tandem mass spectrometry (MS/MS), a fast and sensitive technique, was applied for the determination of GLYP and GLUF in human blood, which can obtain analytical results within 0.5 min without derivatization and chromatographic separation. After protein precipitation of blood samples, the supernatant was mixed with isopropanol and ultra-pure water (1:1 v/v). Then, 8 µL of the mixture was introduced into the plastic sample plate for PESI-MS/MS analysis. The limits of detection (LODs) of the method were 0.50 µg/mL and 0.25 µg/mL for two analytes, and the limits of quantitation (LOQs) were both 1.00 µg/mL, which are higher than the concentration of reported poisoning and fatal cases. In the linear range of 1-500 µg/mL, the regression coefficients (r2) for GLYP and GLUF were over 0.99. The matrix effects ranged from 94.8 % to 119.5 %, and the biases were below 4.3 %. The recoveries ranged between 84.8 % and 107.4 %, and the biases were below 7.6 %. Meanwhile, the method was effectively utilized to detect and quantify the blood, urine, and other samples. Consequently, the results suggest that PESI-MS/MS is a straightforward, fast, and sensitive method for detecting GLUF and GLYP in forensics. In the future, PESI-MS/MS will become an indispensable technique for polar substances in grassroots units of public security where rapid detection is essential.

A highly efficient and selective rapid detection method applied to the detection of amide herbicides in fish serum.

Misuse of amide herbicides in the fisheries environment can pose unpredictable harm to aquatic products and ultimately human health. Thus, the development of a real-time, rapid on-site detection method is crucial. This study proposes for the first time, a paper-based visual detection method for amide herbicides in fish serum, by coating the molecularly imprinted polymer layer onto quantum dots, prepared fluorescent sensing materials (QDs@MIPs) for the detection of amide herbicides in aquatic products. These materials specifically cause fluorescence quenching in the presence of amide herbicides resulting in a color change. For practical application, this research designed a rapid test strip based on QDs@MIPs, meanwhile, incorporate a smartphone or a fluorescence spectrophotometer for qualitative and quantitative measurements, the limit of detection ranges of 0.061-0.500 µM. The method can be used for on-site evaluation of aquatic products, providing new technology for monitoring the safety of aquatic products.

Simultaneous determination of diquat, paraquat, glufosinate, and glyphosate in plasma by liquid chromatography/tandem mass spectrometry: from method development to clinical application.

Diquat (DQ), paraquat (PQ), glufosinate (GLU), and glyphosate (GLYP) are commonly used herbicides that have been confirmed to be toxic to humans. Rapid and accurate measurements of these toxicants in clinical practice are beneficial for the correct diagnosis and timely treatment of herbicide-poisoned patients. The present study aimed to establish an efficient, convenient, and reliable method to achieve the simultaneous quantification of DQ, PQ, GLU, and GLYP in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS) without using derivatization or ion-pairing reagents. DQ, PQ, GLU, and GLYP were extracted by the rapid protein precipitation and liquid-liquid extraction method and then separated and detected by LC-MS/MS. Subsequently, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, extraction recovery, matrix effect, dilution integrity, and stability were evaluated to validate the method based on the FDA criteria. Finally, the validated method was applied to real plasma samples collected from 166 Chinese patients with herbicide poisoning. The results showed satisfactory linearity with low LOD (1 ng/mL for DQ and PQ, 5 ng/mL for GLU, and 10 ng/mL for GLYP, respectively) and low LOQ (5 ng/mL for DQ and PQ, 25 ng/mL for GLU and GLYP, respectively). In addition, the precision, accuracy, extraction recovery, and stability of the method were acceptable. The matrix effect was not observed in the analyzed samples. Moreover, the developed method was successfully applied to determine the target compounds in real plasma samples. These data provided reliable evidence for the application of this LC-MS/MS method for clinical poisoning detection.

Determination of twenty herbicides in blood by ultrapressure liquid chromatography-tandem mass spectrometry.

A sensitive and rapid method for the simultaneous determination of twenty herbicides (aclonifen, lactofen, terbutryn, butylate, carbetamide, fluazifop-P-butyl, propanil, prometryn, isoproturon, terbumeton, pretilachlor, pendimethalin, cycloxydim, tri-allate, metolachlor, diuron, alloxydim, prosulfuron, triflusulfuron-methyl, and acetochlor) in human blood is reported herein. Liquid-liquid extraction coupled with ultra-pressure liquid chromatography-tandem mass spectrometry was employed for the simultaneous analysis of all compounds in 15 min. Validation parameters were studied through the estimation of the limits of detection and quantification, calibration curves, sensitivity, spiked recovery and precision. The limits of detection ranged from 0.1 to 1.0 ng/mL. The limits of quantification ranged from 0.5 to 2.0 ng/mL. Good linearity was obtained for all compounds with R2> 0.99 in all cases. Furthermore, interday precision (< 15%) and intraday precision (< 15%) were shown to be satisfactory. Recoveries in spiked blood samples were evaluated, and acceptable values (88.0%~108.8%) were found. Finally, this method was successfully applied to the determination of fluazifop-P-butyl, isoproturon and acetochlor in blood samples from real forensic cases. These results suggest that this method is reliable for rapid forensic and clinical diagnosis.

Paraquat pharmacokinetics in primates and extrapolation to humans.

By extending our Paraquat (PQ) work to include primates we have implemented a modelling and simulation strategy that has enabled PQ pharmacokinetic data to be integrated into a single physiologically based pharmacokinetic (PBPK) model that enables more confident extrapolation to humans. Because available data suggested there might be differences in PQ kinetics between primates and non-primates, a radiolabelled study was conducted to characterize pharmacokinetics and excretion in Cynomolgus monkeys. Following single intravenous doses of 0.01 or 0.1 mg paraquat dichloride/kg bw, plasma PQ concentration-time profiles were dose-proportional. Excretion up to 48 h (predominantly urinary) was 82.9%, with ca. 10% remaining unexcreted. In vitro blood binding was similar across Cynomolgus monkeys, humans and rat. Our PBPK model for the rat, mouse and dog, employing a single set of PQ-specific parameters, was scaled to Cynomolgus monkeys and well represented the measured plasma concentration-time profiles over 14 days. Addition of a cartilage compartment to the model better captured the percent remaining in the monkeys at 48 h, whilst having negligible effect on model predictions for the other species. The PBPK model performed well for all four species, demonstrating there is little difference in PQ kinetics between non-primates and primates enabling a more confident extrapolation to humans. Scaling of the PBPK model to humans, with addition of a human-specific dermal submodel based on in vitro human dermal absorption data, provides a valuable tool that could be employed in defining internal dosimetry to complement human health risk assessments.

Integration of paraquat pharmacokinetic data across species using PBPK modelling.

Paraquat dichloride (PQ) is a non-selective herbicide which has been the subject of numerous toxicology studies over more than 50 years. This paper describes the development of a physiologically-based pharmacokinetic (PBPK) model of PQ kinetics for the rat, mouse and dog, firstly to aid the interpretation of studies in which no kinetic measurements were made, and secondly to enable the future extension of the model to humans. Existing pharmacokinetic data were used to develop a model for the rat and mouse. Simulations with this preliminary model were then used to identify key data gaps and to design a new blood binding study to reduce uncertainty in critical aspects of the model. The new data provided evidence to support the model structure, and its predictive performance was then assessed against dog and rat datasets not used in model development. The PQ-specific model parameters are the same for all three species, with only the physiological parameters varying between species. This consistency across species provides a strong basis for extrapolation to other species, as demonstrated here for the dog. The model enables a wide range of PQ data to be linked together to provide a broad understanding of PQ pharmacokinetics in rodents and the dog, showing that the key aspects of PQ kinetics in these species are understood and adequately encapsulated within the model.

Simultaneous determination of paraquat and diquat in human plasma by HPLC-DAD: Its application in acute poisoning patients induced by these two herbicides.

BACKGROUND: Paraquat and diquat are widely used in agricultural production in many countries, which are very toxic to human beings. Paraquat can be detected in some diquat solution sold in the market. The blood concentration of paraquat or diquat is an important indicator for clinical diagnosis of paraquat or diquat poisoning. So, it is very meaningful to develop a method for simultaneous determination of paraquat and diquat in human plasma. OBJECTIVE: To develop and validate a HPLC-DAD method for simultaneous determination of paraquat and diquat in human plasma and to apply it in the acute poisoning patients by these two herbicides. METHODS: Paraquat and diquat were simultaneously determined by HPLC-DAD. The plasma was treated using Waters OASIS® Column and then separated on a Thermo Hypersil GOLD (250 × 4.6 mm, 5 µm) Column with the mobile phase consisted of 75 mmol/L sodium heptane sulfonate (containing 0.1 mol/L phosphoric acid, pH 3.0) and acetonitrile (87:13, v:v) at a flow rate of 1.0 mL/min. The full-wavelength scanning was 200-400 nm, and the detection wavelength of paraquat and diquat was 257nm and 310nm, respectively. 120 and 30 plasma samples from patients with paraquat and diquat poisoning were collected and analyzed by the established method. RESULTS: The standard curve for paraquat and diquat ranged from 0.05 to 20 µg/mL, and the precision of LLOQ for paraquat was 16.49%, which was required to be less than 20%. The precision of other concentrations was less than 14.14%. The recovery of paraquat and diquat was 95.38%-103.97% and 94.79%-98.40%, respectively. The results showed that paraquat and diquat were stable under various storage conditions. 120 plasma samples of paraquat poisoning patients and 30 plasma samples of diquat poisoning patients were determined by the established method. The blood concentration of paraquat ranged from 0.10 to 20.62 µg/mL, with an average of 3.61 µg/mL, while for diquat, the concentration ranged from 0 to 26.59 µg/mL, with an average of 2.00 µg/mL. Among the diquat suspected poisoning samples, 5 samples were detected not only diquat but also paraquat, and 2 samples were detected only paraquat, no diquat. CONCLUSION: The HPLC-DAD method established in this study was high throughput, high sensitivity, simple operation, and wide linear ranges. It can be used for the screening analysis and quantitative detection of paraquat and diquat in acute poisoning patients, which can provide basis for the treatment and prognosis of these two herbicides poisoning patients.

[Simultaneous determination of paraquat and diquat in plasma and urine by ion chromatography-triple quadrupole mass spectrometry].

Paraquat (PQ) and diquat (DQ) are widely used as non-selective contact herbicides. Several cases involving accidents, suicide, and homicide by PQ or DQ poisoning have been reported. Poising by PQ, which is mainly concentrated in the lungs, causes acute respiratory distress syndrome and leads to multiple organ toxicity. The toxic effects of DQ are similar to those of PQ but relatively less intense. The mortality rates in PQ and DQ poisoning are high. Simultaneous monitoring of the PQ and DQ concentrations in plasma and urine can provide valuable information for early clinical diagnosis and prognosis. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is the main analytical method used to detect PQ and DQ in plasma and urine. As both these compounds are highly polar and water soluble, they cannot be retained effectively on a reversed-phase column with conventional mobile phases. The separation of PQ and DQ by ion-pair chromatography or hydrophilic chromatography has been reported. The use of an ion-pairing reagent helps in improving the retention capabilities of PQ and DQ. However, the sensitivity of MS detection is noticeably decreased because of ion suppression caused by the ion-pairing reagent in the mobile phase; furthermore, ion-pairing reagents may contaminate the MS system. The separation of PQ and DQ by hydrophilic chromatography is easily affected by matrix components in the sample, and their retention times are not stable. Considering PQ and DQ are bicharged cation species in solution, they are more suitable for separation by cation-exchange chromatography. A method based on ion chromatography-triple quadrupole mass spectrometry was established for the determination of PQ and DQ in plasma and urine. The plasma and urine samples were diluted with water, and then purified on a solid-phase extraction column containing a polymer-reversed phase and weak ion-exchange mixed-mode adsorbent (Oasis WCX). PQ and DQ were separated on an IonPac CS 18 analytical column (250 mm×2.0 mm, 6.0 µm) with gradient elution using a methylsulfonic acid solution electrolytically generated from an on-line eluent generation cartridge. An in-line suppressor was used to remove methylsulfonate and other anions from the eluent before the eluent entered the mass spectrometer. Between the suppressor and the ion source in MS, the addition of 3% (v/v) formic acid in acetonitrile as an organic modifier (using an auxiliary pump and a T-piece) aided desolvation in the ion source, resulted in a one-or two-fold improvement of the response, and eliminated the residual effects of the adsorption of PQ and DQ caused by ion source. The analytes were detected by triple quadrupole tandem mass spectrometry using positive electrospray ionization in the multiple reaction monitoring (MRM) mode. PQ-d8 and DQ-d4 were used as internal standards. The calibration curves for PQ and DQ showed good linear relationships in the ranges of 1.0-150 µg/L and 0.5-75 µg/L, respectively, and the correlation coefficients were > 0.999. The average matrix effects of PQ and DQ in plasma were 84.2%-89.3% and 84.7%-91.1%, while the average matrix effects of PQ and DQ in urine were 50.3%-58.4% and 51.9%-59.4%. The average recoveries of PQ and DQ in plasma were 93.5%-117% and 91.7%-112%, respectively, with relative standard deviations (RSDs) of 3.4-16.7% and 2.8%-13.2%, and that in urine were 90.0%-118% and 99.2%-116%, with relative standard deviations of 5.6%-14.9% and 2.4%-17.3% (n=6). The limits of detection of PQ and DQ in plasma and urine were 0.3 µg/L and 0.2 µg/L, respectively, with the corresponding limits of quantification being 1.0 µg/L and 0.5 µg/L. This method is sensitive and accurate, and it can be used to determine PQ and DQ for clinical diagnosis and prognosis in patients.

A fast paraquat quantitation method in human serum using probe electrospray ionization-tandem mass spectrometry for emergency settings.

INTRODUCTION: Paraquat (PQ) is one of the most toxic herbicides to humans. However, it is still in use in many countries, including Japan, and many incidents, such as homicides, intentional ingestions, and occupational accidents, have been reported thus far. In PQ poisoning cases, it is possible to predict severity and prognosis using nomograms. Therefore, if the serum PQ level is determined immediately, a treatment plan can be rapidly established. However, most known analytical methods are time-consuming and therefore hardly ever contribute to patient treatment. METHODS: We developed a new method for PQ quantitation in serum by combining a probe electrospray ionization technique with mass spectrometry. This method requires virtually no serum pretreatment and can yield quantitation values in 18 s. RESULTS: We applied the proposed method to samples from real poisoning cases and compared the results with those obtained via liquid-chromatography-tandem mass spectrometry, revealing the absence of any significant differences at the 5% significance level (t(8) = 1.000, p > .05). The limits of detection and quantitation were 0.004 and 0.015 µg/L, respectively, and the calibration curve exhibited good linearity over the concentration range of 0.015-4.0 µg/mL (r2 = 0.998). DISCUSSION: As the proposed method is fast and easy to perform, it should be useful in emergency medical settings.

Application of probe electrospray ionization-tandem mass spectrometry to ultra-rapid determination of glufosinate and glyphosate in human serum.

Glufosinate and glyphosate, which are non-selective herbicides that include an amino acid moiety in their structures, are frequently used worldwide to control unwanted vegetation. Unfortunately, these readily available herbicides are also used by people to commit suicide, and thus represent important chemicals of interest in the fields of clinical medicine and forensics. Because of the high water solubility of these herbicides, most analytical methods for their detection require a derivatization step, which results in longer analysis times. Therefore, derivatization-based methods do not currently contribute to judgements on treatment decisions in emergency medicine. In this study, we addressed this limiting factor by developing an ultra-rapid and simple analytical technique using a combination of probe electrospray ionization (PESI) and tandem mass spectrometry (MS/MS), which gives quantitative results within 0.3 min. Herbicide standards were added to human serum that was then subjected to analysis (N = 5 per concentration). The analysis was repeated daily over eight consecutive days. The limit of detection (LOD) was 0.59 µg/mL for glufosinate and 0.20 µg/mL for glyphosate. The limit of quantitation (LOQ), i.e., the lowest point on the calibration curves, was 1.56 µg/mL for both the herbicides. The matrix effects were observed at three different concentrations (between 95.7%-104% for glufosinate, and between 90.7%-95.7% for glyphosate). When applied to samples taken from actual poisoning cases (six samples for each herbicide), the present method gave almost the same quantitative values as those obtained by conventional high-performance liquid chromatography with fluorescence detection. Thus, we believe that PESI-MS/MS could emerge as a rapid diagnosis method in the clinical emergency field.

Serial measurement of glyphosate blood concentration in a glyphosate potassium herbicide-intoxicated patient: A case report.

INTRODUCTION: This report describes changes in blood and urine concentrations of glyphosate potassium over time and their correlations with clinical symptoms in a patient with acute glyphosate potassium poisoning. CASE REPORT: A 67-year-old man visited the emergency center after ingesting 250 mL of a glyphosate potassium-based herbicide 5 h before. He was alert but presented with nausea, vomiting, and bradyarrhythmia with atrial fibrillation (tall T waves). Laboratory findings revealed a serum potassium level of 6.52 mEq/L. After treatment with an injection of calcium gluconate, insulin with glucose, bicarbonate, and an enema with polystyrene sulfonate, the patient's serum potassium level normalized and the bradyarrhythmia converted to a normal sinus rhythm. During admission, the blood and urine concentration of glyphosate and urine aminomethylphosphonic acid (AMPA, a glyphosate metabolite) was measured at regular time intervals. The patient's glyphosate blood concentration on admission was 11.48 mg/L, and it had decreased rapidly by 16 h and maintained about 1mgl/L by 70 h after admission. Urine glyphosate and AMPA levels had also decreased rapidly by 6 h after admission. DISCUSSION: Glyphosate potassium poisoning causes hyperkalemia. Blood concentrations of glyphosate were decreased rapidly by 16 h after admission, and urine concentrations were also decreased by 6 h after admission.

Biocompatible chitosan-pectin polyelectrolyte complex for simultaneous electrochemical determination of metronidazole and metribuzin.

Development of novel biocompatible sensor material suitable for modest, cost-effective, and rapid practical application is a demanding research interest in the field of electroanalytical chemistry. In this context, for the first time, we utilized biocompatible chitosan-pectin biopolyelectrolyte (CS-PC BPE) complex for the simultaneous electroreduction of an important antibiotic drug (metronidazole-MNZ) and herbicide (metribuzin-MTZ). This sensor reveals an attractive welfares such as simplicity, biocompatibility, and low production cost. Under optimized experimental conditions, the electroanalytical investigation confirmed that CS-PC BPE modified glassy carbon electrode (CS-PC BPE/GCE) was found to sense MNZ and MTZ in the nanomolar range. Moreover, as-prepared CS-PC BPE/GCE exhibited prominent selectivity, stability, and reproducibility. Additionally, the possible MNZ and MTZ sensing mechanism of CS-PC BPE/GCE have been discussed in detail. Lastly, real sample analysis was also carried out and revealed from several investigations that the CS-PC BPE/GCE is a good electrochemical sensor system for the detection of targeted analytes.

Metabolitic profiling of amino acids in paraquat-induced acute kidney injury.

BACKGROUND: The herbicide paraquat (1, 1'-dimethyl-4, 4'-bipyridylium dichloride; PQ) is a poison well-known to cause delayed mortality due to acute kidney injuries (AKI). This study examines the changes in serum amino acids (AAs) metabolite profiles as surrogate markers of renal cell metabolism and function after paraquat poisoning. METHODS: To identify the metabolic profiling of free serum AAs and its metabolites, serum from 40 paraquat-poisoned patients with or without AKI is collected. LC-MS/GC-MS is performed to analyze AA molecules. A Cox proportional hazard model was used to assess for incidence of AKI. Receiver operating characteristic (ROC) curve is applied to evaluate AKI occurrence and prognosis. RESULTS: A total of 102 serum AAs and its metabolites were identified. Compared with non-AKI patients, 37 varied significantly in AKI patients. The univariate Cox proportional hazard model analysis revealed that the estimated PQ amount, plasma PQ concentration, urine PQ concentration, APACHE, SOFA scores and 16 amino acids correlated with the incidence of AKI. Further analyses revealed that 3-methylglutarylcarnitine, 1-methylimidazoleacetate, and urea showed higher cumulative hazard ratios for the occurrence of AKI during follow-up (P < 0.05). The area under the curve (AUC) of 3-methylglutarylcarnitine, 1-methylimidazoleacetate and urea were 0.917, 0.857, 0.872, respectively. CONCLUSION: 3-methylglutarylcarnitine, 1-methylimidazoleacetate and urea were associated with AKI in patients with paraquat intoxication.

Neonatal exposure to a glyphosate-based herbicide alters the histofunctional differentiation of the ovaries and uterus in lambs.

The aim of the present study was to compare the effect of oral and subcutaneous exposure to a glyphosate-based herbicide (GBH) on the female reproductive system, specifically in the ovaries and uterus of prepubertal lambs. To this end, ewe lambs were exposed to a s.c. (n: 5) or an oral (n: 5) environmentally relevant dose of GBH (2 mg/kg/day) or to vehicle (controls, n: 12), from postnatal day (PND) 1 to PND14. Serum glyphosate and aminomethylphosphonic acid (AMPA) concentrations were measured on PND15 and PND45. The ovaries and uterus were obtained and weighed on PND45. Ovarian follicular dynamics and uterine morphological features were determined by picrosirius-hematoxylin staining. The proliferation marker Ki67 was evaluated by immunohistochemistry in ovarian and uterine samples. Glyphosate but not AMPA was detected in serum of exposed lambs on PND15, whereas neither glyphosate nor AMPA were detected on PND45. Controls were negative for glyphosate and AMPA on PND15 and PND45. GBH exposure did not affect ovarian or uterine weight. However, on PND45, the ovary of GBH-exposed lambs showed altered follicular dynamics, increased proliferation of granulosa and theca cells, and decreased mRNA expression of FSHR and GDF9, whereas their uterus showed decreased cell proliferation but no alterations in the histomorphology or gene expression. In conclusion, GBH exposure altered the ovarian follicular dynamics and gene expression, and the proliferative activity of the ovaries and uterus of lambs. It is noteworthy that all the adverse effects found in the ovaries and uterus of both GBH-exposed groups were similar, independently of the administration route.

[Determination of paraquat in serum by ultra performance liquid chromatography tandem mass spectrometry and toxicokinetics of paraquat in rat].

OBJECTIVE: To establish a simple and rapid ultra-performance liquid chromatography tandem mass spectrometry( UPLC-MS/MS) method for the determination of paraquat in serum, and to apply to the toxicokinetics of paraquat in rats. METHODS: The samples separated on ACQUITY UPLC BEH HILIC column( 2. 1 mm × 50 mm, 1. 7 µm)with acetonitrile-50 mmol/L ammonium formate( 0. 4% formic acid) as mobile phase. The analytes were analyzed using ESI operating in the positive multiple reaction monitoring( MRM) mode. The method was used in toxicokinetic study in poisoned rat. Toxicokinetic parameters were calculated by WinNonlin 7. 0 statistical software. RESULTS: Paraquat was linear in the range of 0. 3-1000. 0 µg/L, the recovery rate was 89. 0%-107. 7%, and the relative standard deviation( RSD) 1. 9%-13. 8%( n = 6). Toxicokinetic parameterswere as follows: C_(max), T_(max)and T_(1/2) were( 46. 50 ± 5. 11) mg/L, 0. 167 h, ( 63. 2 ±16. 2) h, respectively. CONCLUSION: This method is highly sensitive, high accuracy and is suitable for the analysis of paraquat in the toxicokinetic study in rats.

Comparative Pharmacokinetics of High and Low Doses of the Herbicide Propanil in Mice.

We have documented that the herbicide propanil is immunotoxic in mice, and our in vitro tissue culture experiments largely recapitulate the in vivo studies. Laboratory studies on environmental contaminants are the most meaningful when these studies are conducted using concentrations that approximate levels in the environment. Many techniques to measure the distribution and pharmacokinetics (PK) on compounds rely on techniques, such as liquid scintillation counting (LSC) of radio-labeled starting compound, that require concentrations higher than environmental levels. The aim of this study was to compare tissue PK after exposure to propanil concentrations more relevant to levels of exposure to agricultural workers and the general population to concentrations previously reported for laboratory studies. To this end, we conducted a study to measure propanil distribution in three immune organs, using ultrasensitive accelerator mass spectrometry (AMS). We used two doses: the lower dose modeled levels expected in the environment or long-term occupational exposure to low doses, while the higher dose was to model the effects of an accidental exposure. Our results showed that the distribution and PK profiles from these two different concentrations was markedly different. The profile of the high dose (concentration) exposure was indicative of saturation of the detoxifying capability of the animal. In contrast, at the lower environmentally relevant concentration, in vivo concentrations of propanil in spleen, liver, and blood dropped to a very low level by 720 min. In conclusion, these studies highlight the differences in PK of propanil at these two doses, which suggests that the toxicity of this chemical should be re-investigated to obtain better data on toxic effects at doses relevant for humans.

Evaluation of non-invasive biomonitoring of 2,4-Dichlorophenoxyacetic acid (2,4-D) in saliva.

The objective of this study was to evaluate the potential for non-invasive biomonitoring of 2,4-Dichlorophenoxyacetic acid (2,4-D) in saliva. Using an in vitro rat salivary gland epithelial cell (SGEC) system, a collection of experiments investigating chemical protein binding, temporal and directional transport, as well as competitive transport with para-aminohippuric acid (PAH), a substrate for renal organic anion transporters, was conducted to identify cellular transport parameters required to computationally model salivary transport of 2,4-D. Additionally, a physiological protein gradient was implemented to mimic physiologically relevant concentrations of protein in rat plasma and saliva, and under these conditions the transfer of 2,4-D was markedly slower, driven by increased protein binding (i.e. reduced free 2,4-D species available to cross salivary barrier). The rate of transfer was directly proportional to the amount of unbound 2,4-D and demonstrated no indication of active transport. An in vivo assessment of 2,4-D exposure in rats revealed non-linear protein binding in plasma, indicating saturated protein binding and increased levels of unbound 2,4-D species at higher doses. A strong correlation between 2,4-D concentrations in saliva and unbound 2,4-D in plasma was observed (Pearson correlation coefficient = 0.95). Saliva:plasma 2,4-D ratios measured in vivo (0.0079) were consistent within the linear protein binding range and expected 2,4-D levels from occupational exposures but were significantly different than ratios measured in vitro (physiological conditions) (0.034), possibly due to 2,4-D concentrations in saliva not being at equilibrium with 2,4-D concentrations in blood, as well as physiological features absent in in vitro settings (e.g. blood flow). We demonstrated that 2,4-D is consistently transported into saliva using both in vitro and in vivo models, making 2,4-D a potential candidate for human non-invasive salivary biomonitoring. Further work is needed to understand whether current sensor limits of detection are sufficient to measure occupationally relevant exposures.

Transplacental transfer and metabolism of diuron in human placenta.

Diuron is a broad-spectrum phenylurea derived herbicide which is commonly used across the globe. Diuron is toxic to the reproductive system of animals and carcinogenic to rat urothelium, and recently found to be genotoxic in human cells. In in vivo, it is metabolized predominately into 3-(3,4-dichlorophenyl)-1-methyl urea (DCPMU) in humans and 3-(3, 4-dichlorophenyl)urea (DCPU) in animals. Information on diuron toxicokinetics and related toxicity in human placenta is absent. We have investigated the toxicokinetics of diuron in ex vivo human placental perfusion and in in vitro human placental microsomes and human trophoblastic cancer cells (BeWo). Diuron crossed human placenta readily in placental perfusion. Furthermore, diuron was metabolized into DCPMU in perfused placenta and in in vitro incubations using microsomes from placentas of smokers. In incubations with placental microsomes from non-smokers, and in BeWo cells, metabolism to DCPMU was detected but only with the highest used diuron concentration (100 µM). Diuron metabolism was inhibited upon addition of α-naphthoflavone, a CYP1A1 inhibitor, underscoring the role of CYP1A1 in the metabolism. In conclusion, it is evident that diuron crosses human placenta and diuron can be metabolized in the placenta to a toxic metabolite via CYP1A1. This implicates in vivo fetal exposure to diuron if pregnant women are exposed to diuron, which may result in fetotoxicity.

A novel approach for determination of paraquat based on dried blood spot (DBS) extraction and UHPLC-HRMS analysis.

Paraquat is an effective herbicide chemical but a highly toxic compound for humans and animals. The measurement of paraquat concentration in blood is important to clinic or forensic practice. Herein, a method has been developed for the analysis of paraquat in human blood using dried blood spot (DBS) extraction and subsequent UHPLC-HRMS analysis. Three droplets (100 µL each) of blood were spotted on the Whatman® FTA classic card and then let dry by microwave irradiation (1200 W) for 5 min to prepare DBS. An 8 mm diameter punch was removed from the center of DBS and extracted with 190 µL of mobile phase (20 mM ammonium acetate with 0.1% formic acid and 5% acetonitrile in ultra-pure water) and 10 µL of internal standard (paraquat-d8, 100 ng/mL). After ultrasonic treatment for 10 min, the tube was centrifuged, and the supernatant was then filtered by 0.2 µm membrane and injected into the UHPLC-HRMS system. The method was validated considering the following parameters: selectivity, LOD and LLOQ, linearity, precision, accuracy. The method showed satisfactory linearity in the range of 1-1000 ng/mL, with high determination coefficient (0.9986). LOD was 0.5 ng/mL, and LLOQ was 1 ng/mL. Selectivity, intra and inter day precision and accuracy were acceptable. The validated method was then applied to authentic blood samples and has proved to be a simple, fast and reliable procedure for the determination of paraquat in blood.