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.

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.

Dietary administration of diquat for 13 weeks does not result in a loss of dopaminergic neurons in the substantia nigra of C57BL/6J mice.

Male and female C57BL/6J mice were administered diquat dibromide (DQ∙Br2) in their diets at concentrations of 0 (control), 12.5 and 62.5 ppm for 13 weeks to assess the potential effects of DQ on the nigrostriatal dopaminergic system. Achieved dose levels at 62.5 ppm were 6.4 and 7.6 mg DQ (ion)/kg bw/day for males and females, respectively. A separate group of mice was administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) ip as a positive control. The comparative effects of DQ and MPTP on the substantia nigra pars compacta (SNpc) and/or striatum were assessed using neurochemical, neuropathological and stereological endpoints. Morphological and stereological assessments were performed by investigators who were "blinded" to dose group. DQ had no effect on striatal dopamine concentration or dopamine turnover. There was no evidence of neuronal degeneration, astrocytic or microglial activation, or a reduction in the number of tyrosine hydroxylase positive (TH(+)) neurons in the SNpc or neuronal processes in the striatum of DQ-treated mice. These results are consistent with the rapid clearance of DQ from the brain following a single dose of radiolabeled DQ. In contrast, MPTP-treated mice exhibited decreased striatal dopamine concentration, reduced numbers of TH(+) neurons in the SNpc, and neuropathological changes, including neuronal necrosis, as well as astrocytic and microglial activation in the striatum and SNpc.

Prognostic value of plasma diquat concentration in patients with acute oral diquat poisoning: a retrospective study.

Objectives: Diquat poisoning is an important public health and social security agency. This study aimed to develop a prognostic model and evaluate the prognostic value of plasma diquat concentration in patients with acute oral diquat poisoning, focusing on how its impact changes over time after poisoning. Methods: This was a retrospective cohort study using electronic healthcare reports from the Second Hospital of Hebei Medical University. The study sample included 80 patients with acute oral Diquat poisoning who were admitted to the hospital between January 2019 and May 2022. Time-to-event analyses were performed to assess the risk of all-cause mortality (30 days and 90 days), controlling for demographics, comorbidities, vital signs, and other laboratory measurements. The prognostic value of plasma DQ concentration on admission was assessed by computing the area under a time-dependent receiver operating characteristic curve (ROC). Results: Among the 80 patients, 29 (36.25%) patients died, and 51 (63.75%) patients survived in the hospital. Non-survivors had a median survival time (IQR) of 1.3(1.0) days and the longest survival time of 4.5 days after DQ poisoning. Compared with non-survivors, survivors had significantly lower amounts of ingestion, plasma DQ concentration on admission, lungs injury within 24 h after admission, liver injury within 24 h after admission, kidney injury within 24 h after admission, and CNS injury within 36 h after admission, higher APACHE II score and PSS within 24 h after admission (all p < 0.05). Plasma Diquat concentration at admission (HR = Exp (0.032-0.059 × ln (t))) and PSS within 24 h after admission (HR: 4.470, 95%CI: 1.604 ~ 12.452, p = 0.004) were independent prognostic factors in the time-dependent Cox regression model. Conclusion: Plasma DQ concentration at admission and PSS within 24 h after admission are independent prognostic factors for the in-hospital case fatality rate in patients with acute oral DQ poisoning. The prognostic value of plasma DQ concentration decreased with time.

Monolithic spin column extraction and GC-MS for the simultaneous assay of diquat, paraquat, and fenitrothion in human serum and urine.

We present a method based on monolitic spin column extraction and gas chromatography-mass spectrometry as an analytical method for screening diquat (DQ), paraquat (PQ), and fenitrothion in serum and urine. This method is useful for clinical and forensic toxicological analyses. Recovery of DQ, PQ, and fenitrothion from serum and urine, spiked at concentrations between 0.1, 2.5, 20, and 45 µg/ml, ranged from 51.3% to 106.1%. Relative standard deviation percentages were between 3.3% and 14.8%. Detection and quantitation limits for serum and urine were 0.025 and 0.05 µg/ml, respectively, for DQ, 0.1 and 0.1 µg/ml, respectively, for PQ, and 0.025 and 0.05 µg/ml, respectively, for fenitrothion. Therefore, these compounds can be detected and quantified in the case of acute poisoning.

[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.

Micelle-dominated distribution strategy for non-matrix matched calibration without an internal standard: "Extract-and-shoot" approach for analyzing hydrophilic targets in blood and cell samples.

The analysis of trace hydrophilic targets in complex aqueous-rich matrices is considerably challenging, generally requiring matrix-matched calibration, internal standard, or time-and-labor-intensive sample preparation. To address this analytical bottleneck, a non-matrix-matched calibration strategy without using internal standard was reported for the first time to analyze complicated biosamples such as whole blood, plasma, serum, and cell samples. This strategy, termed micelle-dominated distribution, also aimed at realizing the simple "extract-and-shoot" analytical process for such complex matrices. The micelle-matrix interaction was found to efficiently eliminate the matrix effect by dominating phase separation and analyte distribution between the extraction and matrix phases. Thus, calibration linear curves prepared in water were applicable to the analysis of all the above-mentioned sample types. Rapid distribution equilibrium within 4 min was achieved. This strategy could tolerate direct large volume injection, thereby providing two-order-of-magnitude enhancement in the sensitivity of ion-pair chromatography. The analytical method integrated cell rupture, matrix cleanup, analyte extraction, and on-column preconcentration into a fast and high-throughput operation. The successful application to the determination of exogenous pesticides and endogenous glutathione exhibited low limits of detection (0.0085-0.015 µg mL-1 for pesticides; 0.52 µg mL-1 for glutathione), wide linear ranges (0.028-50 µg mL-1 and 0.049-50 µg mL-1 for pesticides; 1.7-1000 µg mL-1 for glutathione), good linearies (R2 = 0.9994-0.9999), excellent accuracy (recoveries of 91.3-105.2%), and good precision (0.7-6.2% at the levels of 0.028 (or 0.049), 0.1, 0.5, and 50 µg mL-1 for pesticides; 0.5-8.7% at 1.7, 500, and 1000 µg mL-1 for glutathione).

Gas chromatographic-mass spectrometric method for the determination of the herbicides paraquat and diquat in plasma and urine samples.

In the present work, a method was developed and optimized aiming to determinate the herbicides paraquat (PQ) and diquat (DQ) in human plasma and urine samples. An initial procedure of chemical reduction of the analytes by adding NaBH4 directly in the buffered samples (pH 8.0) was performed. This procedure was necessary to convert the quaternary ammonium substances into more volatile compounds for gas chromatographic analysis. The reduction compounds were extracted with C18 cartridges (solid-phase extraction). Ethyl paraquat (EPQ) was used as internal standard (IS). Gas chromatography-mass spectrometry (GC-MS) was used to identify and quantify the analytes in selected ion monitoring (SIM) mode. The limits of detection were 0.05 mg/l for both PQ and DQ. By using the weighted least squares linear regression (1/x1/2 for plasma and 1/y for urine), the accuracy of the analytical method was improved at the lower end of the calibration curve (from 0.1 to 50 mg/l; r>0.98). This method can be readily utilized as an important tool to confirm the suspicion of PQ and/or DQ poisoning and evaluate the extent of the intoxication.

Rapid and sensitive HPLC method for the simultaneous determination of paraquat and diquat in human serum.

A rapid and sensitive HPLC method for the simultaneous determination of paraquat and diquat in human serum has been developed. After deproteinization of the serum with 10% trichloroacetic acid, the samples were separated on a reversed-phase column, and subsequently reduced to their radicals with alkaline sodium hydrosulfite solution. These radicals were monitored with a UV detector at 391 nm. This method permitted the reliable quantification of paraquat over linear ranges of 50 ng - 10 microg/ml and 100 ng - 10 microg/ml for diquat in human serum. The within- and between-day variations are lower than 2.3 and 2.2%, respectively. This technique was also utilized to determine the paraquat and diquat serum levels in a patient who had ingested herbicide (Prigrox L) containing paraquat and diquat.

LC/MS/MS analysis of quaternary ammonium drugs and herbicides in whole blood.

Quaternary ammonium drugs (atracurium, bretylium, edrophonium, ipratropium, mivacurium, neostigmine, pancuronium and rocuronium) and herbicides (difenzoquat, diquat and paraquat) in human whole blood were analysed by LC/MS/MS with positive electrospray ionisation (ESI), following extraction with Bond Elut LRC-CBA cartridges. Internal standards were benzyldimethylphenylammonium chloride monohydrate and ethyl viologen for drug and herbicide analysis, respectively. Ion-pair chromatography used heptafluorobutyric acid (15 mM)-ammonium formate (20 mM) buffer adjusted to pH 3.30 with formic acid and a linear gradient from 5 to 90% methanol run over 18 min. Recoveries ranged from 79.7 to 105.1%, detection limits were between 3.6 and 20.4 ng/ml and the intra- and inter-day precisions were less than 18.6% at a concentration of 10 ng/ml. The method was applied to a case of accidental paraquat poisoning in which the concentration of paraquat in blood was 0.64 mg/l, which is within the range associated with fatal paraquat poisoning.

Dietary arginine and N-carbamylglutamate supplementation enhances the antioxidant statuses of the liver and plasma against oxidative stress in rats.

N-Carbamylglutamate (NCG), an effective precursor of arginine (ARG), can enhance ARG synthesis, increase intestinal growth, and improve reproductive performance. However, the antioxidant effect of NCG remains largely unknown. This study aims to survey the effects of ARG and NCG supplementation on the antioxidant statuses of the liver and plasma in rats under oxidative stress. Rats were fed for 30 days with one of the three iso-nitrogenous diets: basal diet (BD), BD plus 1% ARG, and BD plus 0.1% NCG. On day 28, half of the rats fed with BD were intraperitoneally injected with 12 mg per kg body weight of diquat (diquat group) and the other half was injected intraperitoneally with sterile 0.9% NaCl solution (control group). The other diet groups also received an intraperitoneal injection of 12 mg per kg body weight of diquat, as follows: diquat + 1% ARG (DT + ARG), and diquat + 0.1% NCG (DT + NCG). Rat liver and plasma samples obtained 48 h after diquat injection were analyzed. Results indicated that diquat significantly affected the plasma conventional biochemical components (relative to the controls), which were partially alleviated in both the DT + ARG and DT + NCG groups (P < 0.05). Diquat also significantly decreased the glutathione (GSH) content (by 30.0%), and decreased anti-superoxide anion (ASA; by 13.8%) and anti-hydroxyl radical (AHR; by 38.9%) abilities in the plasma, and also decreased catalase (CAT) activity both in the liver (by 17.5%) and plasma (by 33.4%) compared with the control group. By contrast, diquat increased the malondialdehyde (MDA) content (by 23.0%) in the plasma (P < 0.05) compared with the control group. Relative to those of the diquat group, higher CAT activity and GSH content were noted in the plasma of the DT + ARG group and in the liver of both DT + ARG and DT + NCG groups (P < 0.05). Furthermore, the DT + ARG group exhibited significantly enhanced plasma ASA activity (P < 0.05). The DT + NCG group showed significantly improved total antioxidant capacity (T-AOC) in the liver and plasma (P < 0.05). Increased GSH content and elevated ASA and AHR activities were also found, but the MDA content in the plasma was depleted (P < 0.05). Compared with the DT + ARG group, the DT + NCG group showed increased liver and plasma T-AOC, enhanced plasma AHR activity, increased liver ASA activity, and decreased plasma MDA content (P < 0.05). Overall, supplementation of 1% ARG and 0.1% NCG can partially protect the liver and plasma from oxidative stress. Furthermore, compared with 1% ARG, 0.1% NCG more effectively alleviated oxidative stress.

Simultaneous Determination and Quantitation of Paraquat, Diquat, Glufosinate and Glyphosate in Postmortem Blood and Urine by LC-MS-MS.

A simple method, incorporating protein-precipitation/organic backwashing and liquid chromatography-tandem mass spectrometry (LC-MS-MS), has been successfully developed for the simultaneous analysis of four highly water-soluble and less volatile herbicides (paraquat, diquat, glufosinate and glyphosate) in ante- and postmortem blood, urine and gastric content samples. Respective isotopically labeled analogs of these analytes were adopted as internal standards. Acetonitrile and dichloromethane were used for protein precipitation and organic solvent backwashing, respectively, followed by injecting the upper aqueous phase into the LC-MS-MS system. Chromatographic separation was achieved using an Agilent Zorbax SB-Aq analytical column, with gradient elution of 15 mM heptafluorobutyric acid and acetonitrile. Mass spectrometric analysis was performed under electrospray ionization in positive-ion multiple reaction monitoring mode. The precursor ions and the two transition ions (m/z) adopted for each of these four analytes were paraquat (185; 169 and 115), diquat (183; 157 and 78), glufosinate (182; 136 and 119) and glyphosate (170; 88 and 60), respectively. Analyte-free blood and urine samples, fortified with the analytes of interest, were used for method development/validation and yielded acceptable recoveries of the analytes; interday and intraday precision and accuracy data; calibration linearity and limits of detection and quantitation. This method was successfully incorporated into an overall analytical scheme, designed for the analysis of a broad range of compounds present in postmortem samples, helpful to medical examiners' efforts to determine victims' causes of death.

Determination of paraquat and diquat in human body fluids by high-performance liquid chromatography/tandem mass spectrometry.

Paraquat (PQ) and diquat (DQ) in human whole blood and urine were analyzed by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) with positive ion electrospray ionization (ESI). The compounds were extracted with Sep-Pak C18 cartridges from whole blood and urine samples containing ethyl paraquat as an internal standard. The separation of PQ and DQ was carried out using ion-pair chromatography with heptafluorobutyric acid in 20 mM ammonium acetate and acetonitrile gradient elution for successful coupling with MS. Both compounds formed base peaks due to [M-H]+ ions by HPLC/ESI-MS and the product ions produced from each [M-H]+ ion by HPLC/MS/MS. Selective reaction monitoring (SRM) showed much higher sensitivity for both body fluids. Therefore, a detailed procedure for the detection of compounds by SRM with HPLC/MS/MS was established and carefully validated. The recoveries of PQ and DQ were 80.8-95.4% for whole blood and 84.2-96.7% for urine. The calibration curves for PQ and DQ showed excellent linearity in the range of 25-400 ng ml(-1) of whole blood and urine. The detection limits were 10 ng ml(-1) for PQ and 5 ng ml(-1) for DQ in both body fluids. The intra- and inter-day precision for both compounds in whole blood and urine samples were not greater than 13.0%. The data obtained from the determination of PQ and DQ in rat blood after oral administration of the compounds are also presented.

A short, simple method for the determination of paraquat in plasma.

Paraquat was measured in plasma after precipitation of plasma proteins by addition of a solvent/salt mixture. The paraquat remaining in the supernatant is measured by its free radical formation after addition of alkaline sodium dithionite. The time required for a single determination is approximately 20 minutes.

Purification and characterization of diquat (1,1'-ethylene-2, 2'-dipyridylium)- metabolizing enzyme from paraquat-resistant rat liver cytosol.

To establish a paraquat-resistant Wistar rat strain, we carried out continuous sister-brother mating among rats that survived high-dose intraperitoneal administration of paraquat dichloride (360 mg/kg). The percentages of paraquat-resistant rats among wild rats and among the fifth-generations were 7.1% and 20.6%, respectively. After high-dose paraquat administration, the serum paraquat concentration in sensitive rats was much higher than that in paraquat-resistant rats. The cytosol fraction of liver from paraquat-resistant rats had higher paraquat- and diquat-metabolizing activities than that of liver from paraquat-sensitive rats. By contrast, microsomal fractions from livers of paraquat-resistant and paraquat-sensitive rats had no paraquat- or diquat-metabolizing activity. This paraquat/diquat-metabolizing enzyme was partially purified from paraquat-resistant rat liver cytosol using affinity chromatography for diquat. At the end of the purification procedure, rat liver diquat-metabolizing enzyme was purified 1154-fold to a final specific activity of 32.32 mol/h/mg protein, and an overall recovery of about 0.46% was obtained. This enzyme oxidized diquat to diquat-dipyridone during overnight incubation at 37 degrees C, but only metabolized traces of paraquat. The molecular mass of the enzyme was estimated as 190 kDa, and its isoelectric point of it was 4.6-4.7. Kinetic study revealed the values of K(m) and V(max) to be 35.0 micromol/l and 0.81 micromol/h/ml, respectively.

Paraquat and diquat interference in the analysis of creatinine by the Jaffé reaction.

Paraquat and diquat were shown to interfere significantly in the measurement of plasma creatinine by the alkaline picrate (Jaffé) reaction in a young man who ingested a massive dose of a mixture of the 2 herbicides. It is likely that these bipyridylium compounds react in a manner similar but at different rates compared with creatinine in the Jaffé reaction.

Extraction of diquat with 1-butanol from biological materials.

Diquat can be extracted with 1-butanol from high pH solution in the presence of several moderate reductants. The red colored reduced compound of diquat in water turns to a purple compound in 1-butanol. The absorption of the purple compound is 0.105 at 383 nm and 0.119 at 520 nm in 1 microgram diquat/ml 1-butanol. The latter value is a little higher than that of the red compound at 495 nm in water. The purple compound is much more stable than the red compound in water. More than 80% of 10 ppm diquat added can be extracted from serum, blood, tissues, urine and some drinks. The extraction with 1-butanol is useful for concentration of diquat contained in large volume. The lower limit of detection is 0.1 microgram/ml 1-butanol. Paraquat is insoluble in 1-butanol under the same condition. Therefore, this method is applicable for the determination of diquat when paraquat is also contained in the solution.

Spectra interference between diquat and paraquat by second derivative spectrophotometry.

A rapid and accurate method, combining solid-phase extraction and second-order derivative spectrophotomety approaches, is developed for the simultaneous determination of diquat (DQ) and paraquat (PQ) in blood, tissue and urine samples. Supernatant resulting from the precipitation of protein (with trichloroacetic acid) in plasma and tissue or Amberlite IRA-401 resin treated urine are passed through a mini-column packed with Wakogel gel (Silica gel). Analytes are then eluted with a non-organic solvent, 0.2mol/l HCl solution containing 2mol/l NH(4)Cl. UV spectrum of the eluent in 220-350nm range provides effective screen to detect the presence of DQ and/or PQ. In the presence of DQ or PQ alone, the analyte present is quantitated by conventional zero- or second-order derivative spectrophotometry. The calibration curve in the 0.1-5.0mg/l range for either analyte obeys Beer's law. When both DQ and PQ are present, their concentrations are determined by the peak amplitudes of their respective second-derivative spectra after the addition of alkaline dithionite reagent. Interference is negligible when the DQ/PQ concentration ratio is within the 5.0-0.2 range. Using a 2-ml of sample size, the detection limits for DQ and PQ in plasma are 0.02 and 0.005mg/l. The corresponding detection limits for urine samples (10ml sample size) are 0.004 and 0.001mg/l. Recoveries of DQ and PQ in triplicate plasma and urine samples spiked with 0.5mg/l of analytes are 93 and 85%. The precision of the proposed method resulting from triplicate study of spiked urine samples varies from 3.2 to 4.6% at 0.5mg/l of DQ and PQ, respectively.

Extraction and quantitation of paraquat and diquat from blood.

Trace amounts of paraquat and diquat in blood were extracted with phenol after deproteinization of blood protein with a chloroform:ethanol mixture and ammonium sulfate. The color reaction of paraquat was achieved by addition of alkaline sodium dithionite to the phenolic extract. The blue paraquat radical produced was determined directly by measurement of the absorption of the phenol layer. The assay of paraquat (> or = 0.5 micrograms) in 1.0 ml of blood (recovery, 93.4%) could be performed within 30 min. Furthermore, simultaneous analysis of paraquat and diquat in the phenolic extract of the sample could be achieved by use of second-derivative spectroscopy within 30 min.