However, in 1986, mixture products of paraquat plus diquat with lower toxicity appeared; just aft er this year, the numer of cases of poisoning by paraquat plus diquat decreased suddenly
Trang 1© Springer-Verlag Berlin Heidelberg 2005
II.7.5 Paraquat and diquat
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 aft 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 spectrophospectropho-tometry [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 hydrosulfi te (Na2S2O4) is solved in 10 mL of 5 M NaOH solution; for urine use, 435 mg sodium hydrosulfi te dis-solved in 10 mL of 0.5 M NaOH solution
Analytica conditions
Instrumentc: a UV-260 spectrophotometer with a diff 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 fi rst measured and then it is second-diff erentiated (derivative wavelength space ∆λ=4 nm)
Trang 2572 Paraquat and diquat
Procedures
Th 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 Th 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 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
Pretreatment procedures for paraquat and diquat in urine and plasma before the second-derivative spectrophotometric analysis.
⊡ Figure 5.1
Trang 3Assesemet 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 diffi 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 Th e qualitative analysis is made by observing the presence of infl ection points at about 396 and 403 nm for paraquat and at 437, 445, 454 and 464 nm for diquat Th 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).
Th e calibration curves are constructed by spiking various concentrations of paraquat or diquat into blank specimens, and processing in the same way as above Th 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
Trang 4574 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 crystallizincubat-ed 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 fl ow rate: 1 mL/min; detection wavelength: 290 nm for the UV detector; fl uorescence detector: Ex = 350 nm, Em = 460 nm; injection volume: 20 µL
ii Conditions for diquat-dipyridone
Th 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 fl ow rate: 1 mL/ min; detection wavelength: 250 nm for the UV detector; fl uorescence detector: Ex = 350 nm,
Em = 430 nm; injection volume: 20 µL
Trang 5i 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 fl uid 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 Th e sediment is again extracted twice with 1 mL each of 10 % trichloroacetic acid solu-tion (with stirring and centrifugasolu-tion)
v Th e 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 Th 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 Th e eluate is evaporated to drynessl under a stream of nitrigen using a boiling water bath
ix Th 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 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 Th e sediment is again extracted with 1 mL methanol (with stirring and centrifugation)
v Th e supernatant solutions are combined
vi Th e combined methanolic extract is washed with 2 mL hexane twicem (with vortex-mix-ing and centrifugation)
vii Th e methanolic layer is evaporated to dryness under a stream of nitrogen with heating at
50 °C in a water bath
viii Th 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 Th e quantitation is made by the internal calibration method as described in the last part
of the above (1) section using IS-2
Trang 6576 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 fl uorescence having its maximum at 462 nm Diquat-dipyridone shows the absorption maximum at 365 nm and emits intense fl uorescence having its maximum at 429 nm Th ese compounds are detectable with high sensitivity using a fl 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
Th 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 Th e detection limit for both paraquat and diquat is 0.5 ng on-column; that for diquat-monopyridone 0.02 ng on-column
Th 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 C18 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
Trang 7> 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
Th ere was excellent linearity for diquat-dipyridone in the range of 0.01–1 µg/mL Th 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 hydrosulfi te reaction, his specimens were subjected to HPLC analysis Th e results obtained are summarized in > Table 5.1.
Aft 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 aft er ingestion Th ese phe-nomena are also true for diquat Th e blood concentration in this case was equally 0.6 µg/mL for both paraquat and diquat at the femoral vein Th e concentration is relatively lower than those reported in fatal cases, in which the mixtures of paraquat and diquat had been ingested
Th erefore, the antemortem time aft 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
Trang 8578 Paraquat and diquat
quat are almost equal to those of diquat within 24 h aft er ingestion of a mixture herbicide product of paraquat and diquat Aft 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 Th 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 aft er ingestion using fi 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)
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
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
Trang 9a) 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 Aft er cooling to room tempera-ture, the weighing of the compounds should be made under dry conditions very rapidly
b) Th e chromogenic reagent should be prepared just before use and be consumed within 2 h c) Any spectrophotometric instrument equipped with the second-diff 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 Th is compound is useful for correcting the errors produced during the extrac-tion procedure Ethyl paraquat is being sold as ethyl viologen (Aldrich)
f) Th e detectors should be connected in series (a UV detector fi rst followed by a fl uorescence detector)
g) Since the profi le for separation of paraquat from diquat is diff erent in each column, the composition of the mobile phase should be optimized for each column
h) Th e organ tissue is crushed with a homogenizer into a paste state at the fi rst step
i) Without stirring, the surface layer may be clotted, resulting in insuffi cient mixing
j) Aft er 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) Th e fl ow 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 aft er use
m) Th 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-high-performance liquid chromatography/mass spectrometry Jpn J
Trang 10580 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