7. 5 © Springer-Verlag Berlin Heidelberg 2005 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 a er 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 dichloride a (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 dibromide a (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 reagents b : for plasma use, 87 mg of sodium hydrosul te (Na 2 S 2 O 4 ) is dis- solved in 10 mL of 5 M NaOH solution; for urine use, 435 mg sodium hydrosul te dis- solved in 10 mL of 0.5 M NaOH solution. Analytica conditions Instrument c : a UV-260 spectrophotometer with a di erential 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  rst measured and then it is second-di erentiated (derivative wavelength space ∆λ=4 nm). 572 Paraquat and diquat Procedures  e 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.  e 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 immediately d . 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- lysis d . Pretreatment procedures for paraquat and diquat in urine and plasma before the second- derivative spectrophotometric analysis. ⊡ Figure 5.1 573 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 di cult 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.  e qualitative analysis is made by observing the presence of in ection points at about 396 and 403 nm for paraquat and at 437, 445, 454 and 464 nm for diquat.  e 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).  e calibration curves are constructed by spiking various concentrations of paraquat or diquat into blank specimens, and processing in the same way as above.  e 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. 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. ⊡ Figure 5.2 Second-derivative spectrophotometry 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; detectors f : SPD-10A and RF-10A XL (all from Shimadzu Corp.); column: Puresil C 18 (150 × 4.6 mm i.d., particle size 5 µm, Waters, Milford, MA, USA); guard column: Guard-Pak Puresil C 18 (Waters); column temperature: room temperature; mobile phase g : 10 mM sodium octanesulfonate, 10 mM triethylamine and 500 mM potassium bromide aqueous solution is adjusted to pH 3.0 with phosphoric acid; its  ow rate: 1 mL/min; detection wavelength: 290 nm for the UV detector;  uorescence detector: Ex = 350 nm, Em = 460 nm; injection volume: 20 µL. ii. Conditions for diquat-dipyridone  e instruments and columns used are the same as described above; column temperature: room temperature; mobile phase: acetonitrile/distilled water (6:94, v/v); its  ow rate: 1 mL/ min; detection wavelength: 250 nm for the UV detector;  uorescence detector: Ex = 350 nm, Em = 430 nm; injection volume: 20 µL. 575 Procedures i. Paraquat, diquat and diquat-monopyridone i. A 1-mL or 1-g amount of a specimen h is mixed with 10 µL of the IS-1 solution. ii. For a body  uid specimen, the above solution is mixed with 1 mL of 10 % trichloroacetic acid solution with stirring i . 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 stirring i . iii. Each protein-denatured solution is centrifuged at 2,000 g for 10 min to obtain clear su- pernatant solution. iv.  e sediment is again extracted twice with 1 mL each of 10 % trichloroacetic acid solu- tion (with stirring and centrifugation). v.  e supernatant solutions are combined and adjusted to about pH 11 j with 2 M NaOH solution. vi. It is poured into a Sep-Pak C 18 cartridge k (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.  e 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.  e eluate is evaporated to dryness l under a stream of nitrigen using a boiling water bath. ix.  e 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 specimen h 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.  e sediment is again extracted with 1 mL methanol (with stirring and centrifugation). v.  e supernatant solutions are combined. vi.  e combined methanolic extract is washed with 2 mL hexane twice m (with vortex-mix- ing and centrifugation). vii.  e methanolic layer is evaporated to dryness under a stream of nitrogen with heating at 50 °C in a water bath. viii.  e 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.  e quantitation is made by the internal calibration method as described in the last part of the above (1) section using IS-2. HPLC analysis 576 Paraquat and diquat 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  uorescence having its maximum at 462 nm. Diquat-dipyridone shows the absorption maximum at 365 nm and emits intense  uorescence having its maximum at 429 nm.  ese compounds are detectable with high sensitivity using a  uorescence 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.  ere 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.  e detection limit for both paraquat and diquat is 0.5 ng on-column; that for diquat-monopyridone 0.02 ng on-column.  e recovery rates were not lower than 80 % for paraquat, diquat and ethyl paraquat and not lower than 60 % for diquat-monopyridone using Sep-Pak C 18 cartridges. Diquat-dipyri- done is not recovered by the solid-phase extraction. Structures of diquat-monopyridone and diquat-dipyridone. ⊡ Figure 5.3 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. ⊡ Figure 5.4 577 > 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.  ere was excellent linearity for diquat-dipyridone in the range of 0.01–1 µg/mL.  e 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 hydrosul te reaction, his specimens were subjected to HPLC analysis.  e results obtained are summarized in > Table 5.1. A er paraquat is absorbed into a human body, its majority is excreted into urine in 48 h; blood paraquat concentrations rapidly decrease according to times a er ingestion.  ese phe- nomena are also true for diquat.  e blood concentration in this case was equally 0.6 µg/mL for both paraquat and diquat at the femoral vein.  e concentration is relatively lower than those reported in fatal cases, in which the mixtures of paraquat and diquat had been ingested.  erefore, the antemortem time a er 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- 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 Poisoning case 578 Paraquat and diquat quat are almost equal to those of diquat within 24 h a er ingestion of a mixture herbicide product of paraquat and diquat. A er 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.  e 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 a er ingestion using  ve 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). ⊡ 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- monopyridone Diquat- dipyridone blood left heart right heart femoral vein 0.8 1.0 0.6 0.6 0.5 0.6 0.07 0.10 0.05 0.13 0.12 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 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. ⊡ Figure 5.6 579 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. A er cooling to room tempera- ture, the weighing of the compounds should be made under dry conditions very rapidly. b)  e chromogenic reagent should be prepared just before use and be consumed within 2 h. c) Any spectrophotometric instrument equipped with the second-di erential 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.  is compound is useful for correcting the errors produced during the extrac- tion procedure. Ethyl paraquat is being sold as ethyl viologen (Aldrich). f)  e detectors should be connected in series (a UV detector  rst followed by a  uorescence detector). g) Since the pro le for separation of paraquat from diquat is di erent in each column, the composition of the mobile phase should be optimized for each column. h)  e organ tissue is crushed with a homogenizer into a paste state at the  rst step. i) Without stirring, the surface layer may be clotted, resulting in insu cient mixing. j) A er alkalization, the test solution should be immediately poured into the Sep-Pak C 18 cartridge, followed by washing with distilled water, because paraquat and diquat are easily decomposed under alkaline conditions; this is more marked for diquat. k)  e  ow rate through the Sep-Pak C 18 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 a er use. m)  e 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 Poisoning case 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 . specimens obtained from a victim of poisoning by a paraquat-containing product are analyzed. For simultaneous analysis of paraquat and diquat, the methods. 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