Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 62)

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Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 62)

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Introduction: Since 1965, when the paraquat herbicide had started to be sold, its poisoning cases increased year by year. However, in 1986, mixture products of paraquat plus diquat with lower toxicity appeared; just after this year, the numer of cases of poisoning by paraquat (plus diquat) decreased suddenly, followed by the gradual decrease until now, but the paraquat (plus diquat) poisoning cases still count as much as about 40 % of the total number of pesticide poisoning [1]. It is necessary to separetely detect paraquat and diquat, when specimens obtained from a victim of poisoning by a paraquat-containing product are analyzed....

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  1. 7.5 II.7.5 Paraquat and diquat by Chiaki Fuke Introduction Since 1965, when the paraquat herbicide had started to be sold, its poisoning cases increased year by year. However, in 1986, mixture products of paraquat plus diquat with lower toxicity appeared; just after this year, the numer of cases of poisoning by paraquat (plus diquat) decreased suddenly, followed by the gradual decrease until now, but the paraquat (plus diquat) poisoning cases still count as much as about 40 % of the total number of pesticide poisoning [1]. It is necessary to separetely detect paraquat and diquat, when specimens obtained from a victim of poisoning by a paraquat-containing product are analyzed. For simultaneous analysis of paraquat and diquat, the methods by HPLC [2–5], GC [6], double wavelength spectropho- tometry [7] and second-derivative spectrophotometry [8] were reported. In this chapter, the methods for analysis of paraquat and diquat by simple and rapid second-derivative spectro- photometry and by highly sensitive HPLC are described. Second-derivative spectrophotometry Regents and their preparation • A 13.8-mg amount of paraquat dichloridea (Sigma, St. Louis, MO, USA and other manu- facturers) is dissolved in 10 mL distilled water (1 mg/mL as paraquat ion). • A 18.7-mg amount of diquat dibromidea (Wako Pure Chemical Industries, Ltd., Osaka, Japan) is also dissolved in 10 mL distilled water (1 mg/mL as diquat ion). • Deproteinization reagent: 10 g sulfosalicylic acid is dissolved in 100 mL distilled water. • Chromogenic reagentsb: for plasma use, 87 mg of sodium hydrosulfite (Na2S2O4) is dis- solved in 10 mL of 5 M NaOH solution; for urine use, 435 mg sodium hydrosulfite dis- solved in 10 mL of 0.5 M NaOH solution. Analytica conditions Instrumentc: a UV-260 spectrophotometer with a differential analyzing system (Shimadzu Corp., Kyoto, Japan); cell: quartz-made semimicro-cell; Measurements: a zero-order spectrum (360–500 nm; wavelength space ∆λ=0.5 nm) is first measured and then it is second-differentiated (derivative wavelength space ∆λ=4 nm). © Springer-Verlag Berlin Heidelberg 2005
  2. 572 Paraquat and diquat ⊡ Figure 5.1 Pretreatment procedures for paraquat and diquat in urine and plasma before the second- derivative spectrophotometric analysis. Procedures The procedures for urine and blood plasma specimens are shown in > Figure 5.1. i. Procedure for plasma i. A 1.0-mL volume of blood plasma is mixed well with 1.0 mL of the deproteinization rea- gent solution. ii. The mixture is centrifuged at 2,000 g for 5 min. iii. A 1.0-mL volume of the supernatant solution is mixed with 0.25 mL of the chromogenic reagent for plasma, and analyzed immediatelyd. ii. Procedure for urine A 1.0-mL volume of urine is mixed with 0.25 mL of the chromogenic reagent solution for urine and centrifuged at 2,000 g for 1 min; the supernatant solution is subjected to ana- lysisd.
  3. Second-derivative spectrophotometry 573 ⊡ Figure 5.2 Zero-order and second-derivative spectra after deproteinization and reduction. 1: blank plasma; 2: diquat-spiked plasma; 3: paraquat-spiked plasma; 4: paraquat plus diquat-spiked plasma. The concentration of the compounds was 10 µg/mL each. Assesemet of the method By this method, the analytical results can be obtained in a relatively short time; it is actually useful for analysis in emergency rooms. However, when high concentrations of hemoglobin and/or bilirubin are present, the measurements become difficult due to their interference. > Figure 5.2 shows zero-order and second-derivative absorption spectra for blood plas- ma, into which paraquat and/or diquat (10 µg/mL each) had been spiked. The qualitative analysis is made by observing the presence of inflection points at about 396 and 403 nm for paraquat and at 437, 445, 454 and 464 nm for diquat. The quantitation is made with ampli- tudes measurable between 396 and 403 nm for paraquat and between 454 and 464 nm for diquat ( > Figure 5.2). The calibration curves are constructed by spiking various concentrations of paraquat or diquat into blank specimens, and processing in the same way as above. The quantitative ranges for paraquat and diquat in blood plasma obtainable by this method are 0.5–10.0 and 1.0–10.0 µg/mL, respectively; those in urine are 0.25–5.0 and 0.5–5.0 µg/mL, respectively.
  4. 574 Paraquat and diquat HPLC analysis In this section, the analysis for diquat-monopyridone and diquat-dipyridone, the metabolites of diquat, are described together with that for paraquat and diquat. Reagents and their preparation • Standard solutions of paraquat and diquat (1 mg/mL each) are prepared as described in the second-derivative spectrophotometry section. • Diquat-monopyridone was extracted from rat liver homogenate, which had been incubat- ed with diquat at 37 °C for 24 h, and crystallized in methanol [9]. • Paraquat-dipyridone and diquat-dipyridone were synthesized according to Calderbank et al. [10]. • Ethyl paraquat diiodide (ethyl viologen)e was synthesized by the method of Philips et al. [11]; 10 mg of the compound is dissolved in 10 mL distilled water (1 mg/mL) as a stock solution. It is diluted 10-fold with distilled water to prepare 100 µg/mL solution (internal standard solution-1, IS-1). • A 10-mg aliquot of 2-acetamidophenol (Aldrich, Milwaukee, WI, USA and other manufac- turers) is dissolved in 1 mL methanol and diluted 10-fold with distilled water (internal standard solution-2, IS-2). HPLC conditions i. Conditions for paraquat, diquat and diquat-monopyridone Instruments; pump: LC-10 AS; detectorsf: SPD-10A and RF-10AXL (all from Shimadzu Corp.); column: Puresil C18 (150 × 4.6 mm i.d., particle size 5 µm, Waters, Milford, MA, USA); guard column: Guard-Pak Puresil C18 (Waters); column temperature: room temperature; mobile phaseg: 10 mM sodium octanesulfonate, 10 mM triethylamine and 500 mM potassium bromide aqueous solution is adjusted to pH 3.0 with phosphoric acid; its flow rate: 1 mL/min; detection wavelength: 290 nm for the UV detector; fluorescence detector: Ex = 350 nm, Em = 460 nm; injection volume: 20 µL. ii. Conditions for diquat-dipyridone The instruments and columns used are the same as described above; column temperature: room temperature; mobile phase: acetonitrile/distilled water (6:94, v/v); its flow rate: 1 mL/ min; detection wavelength: 250 nm for the UV detector; fluorescence detector: Ex = 350 nm, Em = 430 nm; injection volume: 20 µL.
  5. HPLC analysis 575 Procedures i. Paraquat, diquat and diquat-monopyridone i. A 1-mL or 1-g amount of a specimenh is mixed with 10 µL of the IS-1 solution. ii. For a body fluid specimen, the above solution is mixed with 1 mL of 10 % trichloroacetic acid solution with stirringi. For an organ tissue specimen h, the mixture at the step i) is mixed with 1 mL distilled water, followed by addition of 5 mL of 10 % trichloroacetic acid solution with stirringi. iii. Each protein-denatured solution is centrifuged at 2,000 g for 10 min to obtain clear su- pernatant solution. iv. The sediment is again extracted twice with 1 mL each of 10 % trichloroacetic acid solu- tion (with stirring and centrifugation). v. The supernatant solutions are combined and adjusted to about pH 11j with 2 M NaOH solution. vi. It is poured into a Sep-Pak C18 cartridgek (classic type, Waters), which had been activated by passing 5 mL methanol, 5 mL distilled water, 5 mL of 0.1 M hydrochloric acid solution and 5 mL distilled water through it. vii. The cartridge is washed with 5 mL water, 3 mL methanol and 5 mL distilled water; target compounds including IS are eluted with 4 mL of 0.1 M HCl solution. viii. The eluate is evaporated to drynessl under a stream of nitrigen using a boiling water bath. ix. The residue is dissolved in 100 µL of the mobile phase and centrifuged at 12,000 g for 5 min; 20 µL of the supernatant solution is injected into HPLC. x. For constructing a calibration curve, various concentrations of a target compound plus IS are added to blank specimens, and treated as above; a peak area ratio of a target com- pound to IS-1 for a specimen is applied to the above calibration curve to obtain its con- centration. ii. Diquat-dipyridone i. A 0.1-mL or 0.1-g amount of a specimenh is mixed with 5 µL of the IS-2 solution. ii. A 0.1-mL volume of distilled water and 1 mL methanol are added to the above mixture with stirring. iii. It is centrifuged at 2,000 g for 5 min to obtain supernatant solution. iv. The sediment is again extracted with 1 mL methanol (with stirring and centrifugation). v. The supernatant solutions are combined. vi. The combined methanolic extract is washed with 2 mL hexane twicem (with vortex-mix- ing and centrifugation). vii. The methanolic layer is evaporated to dryness under a stream of nitrogen with heating at 50 °C in a water bath. viii. The residue is dissolved in 100 µL mobile phase and centrifuged at 12,000 g for 5 min; 20 µL of the supernatant solution is injected into HPLC. ix. The quantitation is made by the internal calibration method as described in the last part of the above (1) section using IS-2.
  6. 576 Paraquat and diquat ⊡ Figure 5.3 Structures of diquat-monopyridone and diquat-dipyridone. Assessment of the method > Figure 5.3 shows structures of diquat-monopyridone and diquat-dipyridone. Diquat-mono- pyridone shows the absorption maximum at 363 nm and emits intense fluorescence having its maximum at 462 nm. Diquat-dipyridone shows the absorption maximum at 365 nm and emits intense fluorescence having its maximum at 429 nm. These compounds are detectable with high sensitivity using a fluorescence detector [12]. > Figure 5.4 shows HPLC chromatograms for blank blood specimens and for those spiked with 1 µg/mL each of paraquat and diquat, and spiked with 0.1 µg/mL diquat-monopyridone. In the blank blood chromatograms, there were no interfering impurity peaks. There was exellent linearity in the range of 0.1–10 µg/mL for paraquat and diquat, and in the range of 0.01–1 µg/mL for diquat-monopyridone. The detection limit for both paraquat and diquat is 0.5 ng on-column; that for diquat-monopyridone 0.02 ng on-column. The recovery rates were not lower than 80 % for paraquat, diquat and ethyl paraquat and not lower than 60 % for diquat-monopyridone using Sep-Pak C18 cartridges. Diquat-dipyri- done is not recovered by the solid-phase extraction. ⊡ Figure 5.4 HPLC chromatograms for extracts of blood in the presence and absence of paraquat, diquat and diquat-monopyridone. The concentrations were: 1 µg/mL for paraquat and diquat; 0.1 µg/mL for diquat-monopyridone.
  7. Poisoning case 577 ⊡ Figure 5.5 HPLC chromatograms for extracts of blood in the presence and absence of diquat-dipyridone and paraquat-dipyridone. The concentration of each compound was 0.1 µg/mL. > Figure 5.5 shows HPLC chromatograms for blank blood specimens and for those spiked with 0.1 µg/mL diquat-dipyridone and paraquat-dipyridone. In the blank chromatograms, these were no interfering impurity peaks. There was excellent linearity for diquat-dipyridone in the range of 0.01–1 µg/mL. The re- covey rates for diquat-dipyridone and 2-acetamidophenol (IS) from blood specimens were not lower than 85 %. Poisoning case A 42-year-old male was found dead in a parking automobile. Since a positive result could be obtained for the urine specimen by a screening test using hydrosulfite reaction, his specimens were subjected to HPLC analysis. The results obtained are summarized in > Table 5.1. After paraquat is absorbed into a human body, its majority is excreted into urine in 48 h; blood paraquat concentrations rapidly decrease according to times after ingestion. These phe- nomena are also true for diquat. The blood concentration in this case was equally 0.6 µg/mL for both paraquat and diquat at the femoral vein. The concentration is relatively lower than those reported in fatal cases, in which the mixtures of paraquat and diquat had been ingested. Therefore, the antemortem time after ingestion was estimated for the above case. According to the reports by Yoshioka et al. [13] and Ameno et al. [14], the plasma concentrations of para-
  8. 578 Paraquat and diquat ⊡ Table 5.1 Concentrations of paraquat, diquat and diquat metabolites in specimens obtained at autopsy from a poisoned victim (µg/mL or g) Specimen Paraquat Diquat Diquat- Diquat- monopyridone dipyridone blood left heart 0.8 0.6 0.07 0.13 right heart 1.0 0.5 0.10 0.12 femoral vein 0.6 0.6 0.05 0.11 urine 10.1 11.2 0.82 0.12 stomach contents 3.9 2.6 0.15 0.12 liver 3.9 2.0 0.51 0.25 brain 0.5 0.5 0.03 0.10 quat are almost equal to those of diquat within 24 h after ingestion of a mixture herbicide product of paraquat and diquat. After 24 h, the diquat concentration become lower than that of paraquat. Since the blood paraquat concentration in the femoral vein was almost equal to that for diquat, the antemortem time until death may be shorter than 24 h. The diquat- monopyridone and diquat-dipyridone concentrations in the femoral vein of the above case were 0.05 and 0.11 µg/mL, respectively ( > Table 5.1). Since the blood concentration ratios of diquat to diquat-monopyridone or diquat-dipyridone were found to decrease according to times after ingestion using five poisoning cases as shown in > Figure 5.6 [15], the ratio values of the above case for the femoral vein (12 and 5.45, respectively) were applied to the de- creasing curve; it was estimated that more than 12 h had passed from the ingestion until death ( > Figure 5.6). ⊡ Figure 5.6 Concentration ratios of diquat to diquat-monopyridone or diquat-dipyridone as a function of time after ingestion. Dq/Dq-O: diquat concentration : diquat-monopyridone; Dq/Dq-O2: diquat concentration : diquat-dipyridone concentration.
  9. Poisoning case 579 Notes a) Since paraquat dichloride and diquat dibromide include water crystal, they should be heated at 100 °C for 2 h to remove water and kept in a dessicator. After cooling to room tempera- ture, the weighing of the compounds should be made under dry conditions very rapidly. b) The chromogenic reagent should be prepared just before use and be consumed within 2 h. c) Any spectrophotometric instrument equipped with the second-differential function can be used, regardless of its manufacturer. d) Distilled water is processed in the same way and used as the reference. When some of the similar specimens are analyzed successively, the same reference solution can be used with- out change. e) Ethyl paraquat is very suitable for IS and can be added at the initial step of the extraction procedure. This compound is useful for correcting the errors produced during the extrac- tion procedure. Ethyl paraquat is being sold as ethyl viologen (Aldrich). f) The detectors should be connected in series (a UV detector first followed by a fluorescence detector). g) Since the profile for separation of paraquat from diquat is different in each column, the composition of the mobile phase should be optimized for each column. h) The organ tissue is crushed with a homogenizer into a paste state at the first step. i) Without stirring, the surface layer may be clotted, resulting in insufficient mixing. j) After alkalization, the test solution should be immediately poured into the Sep-Pak C18 cartridge, followed by washing with distilled water, because paraquat and diquat are easily decomposed under alkaline conditions; this is more marked for diquat. k) The flow rate through the Sep-Pak C18 cartridge is preferably about 2.5 mL/min. When the air is incorporated into the cartridge, the recovery rate may be decreased. l) To dry the eluate up, an evaporator or a freeze-drier can be also used. When a large number of specimens have to be dried up, the use of the freeze-drier is convenient. Since the eluate contains hydrochloric acid, care should be taken to clean the device used for evaporation to avoid corrosion by the acid after use. m) The washing with hexane can be omitted for specimens with small amounts of lipids, such as urine and serum. References 1) National Research Institute of Police Science (ed) (2001) Annual Case Reports of Drug and Toxic Poisoning in Japan, No.43. National Police Agency, Tokyo, p 2 (in Japanese) 2) Gill R, Qua SC, Moffat AC (1983) High-performance liquid chromatography of paraquat and diquat in urine with rapid sample preparation involving ion-pair extraction on disposable cartridges of octadecyl-silica. J Chroma- togr 255:483–490 3) Fuke C, Ameno K, Shirakawa Y et al. (1992) Simultaneous determination of paraquat and diquat in biological materials using high performance liquid chromatography and its application to poisoned patients. Jpn J Toxi- col 5:387–393 (in Japanese with an English abstract) 4) Ito S, Nagata T, Kudo K et al. (1993) Simultaneous determination of paraquat and diquat in human tissues by high-performance liquid chromatography. J Chromatogr 617:119–123 5) Kage S, Kudo K, Fukushima S et al. (1998) Selective determination of paraquat and diquat in blood by high- performance liquid chromatography and high-performance liquid chromatography/mass spectrometry. Jpn J Forensic Toxicol 16:34–41
  10. 580 Paraquat and diquat 6) Kawase S, Kanno A, Ukai S (1984) Determination of the herbicides paraquat and diquat in blood and urine by gas chromatography. J Chromatogr 283:231–240 7) Tayama J, Komatsu M, Doy M et al. (1991) Simultaneous measurement of paraquat and diquat in serum by wavelength spectrophotometry. Jpn J Toxicol 4:157–162 (in Japanese with an English abstract) 8) Fuke C, Ameno K, Ameno S et al. (1987) Simultaneous analysis of paraquat and diquat in serum and urine by second-derivative spectroscopy. Igakunoayumi 143:657–658 (in Japanese) 9) Fuke C, Ameno K, Ameno S et al. (1993) In vitro studies of the metabolism of paraquat and diquat using rat liver homogenates – isolation and identification of the metabolites of paraquat and diquat. Jpn J Legal Med 47:33– 45 (in Japanese with an English abstract) 10) Calderbank A, Charlton DF, Farrington JA et al. (1972) Bipyridylium quaternary salts and related compounds. Part IV. Pyridones derived from paraquat and diquat. J Chem Soc 10:138–142 11) Phillips AP, Mentha J (1955) Synthetic hypotensive agents. III. Some 4,4,-bipiperidenes. J Am Chem Soc 77:6393–6395 12) Tsuchihashi H, Tatsuno M, Ohtsuki K et al. (1988) Simultaneous analysis of paraquat and diquat by high-perfor- mance liquid chromatography with oxidation. Eisei Kagaku 34:31–35 (in Japanese with an English abstract) 13) Yoshioka T, Hirade A, Kishikawa M et al. (1989) Effects of dilution of paraquat concentration and of mixing of diquat on lifesaving ratios – the comparison between poisoning cases with old and new pesticide products. Jpn J Toxicol 2:31–38 (in Japanese) 14) Ameno K, Fuke C, Shirakawa Y et al. (1994) Different distribution of paraquat and diquat in human poisoning cases after ingestion of a combined herbicide. Arch Toxicol 68:134–137 15) Fuke C, Ameno K, Ameno S et al. (1996) Detection of two metabolites of diquat in urine and serum of poisoned patients after ingestion of a combined herbicide of paraquat and diquat. Arch Toxicol 70:504–507

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