Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 66)

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Introduction: Chemical weapons (chemical warfare agents), such as sarin and soman, were developed to kill or injure humans by their toxic actions. They are called “nuclear weapon of the poor”, because the weapons are relatively stable during storage, cheap for production and relatively easily synthesized with basic knowledge on organic chemistry. Main advanced countries are making efforts to reduce chemical weapons existing in the world on the basis of the Chemical Weapons Convention (CWC), after the Iran-Iraq War and the Gulf War. In 1990, an incident of human injury using mustard (yperite) took place at a usual residence at Komagome,...

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Nội dung Text: Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 66)

8.1 II.8.1 Sarin and its decomposition products by Hiroaki Ando and Yoshihiko Miyata Introduction Chemical weapons (chemical warfare agents), such as sarin and soman, were developed to kill or injure humans by their toxic actions. They are called “nuclear weapon of the poor”, because the weapons are relatively stable during storage, cheap for production and relatively easily synthesized with basic knowledge on organic chemistry. Main advanced countries are making efforts to reduce chemical weapons existing in the world on the basis of the Chemical Weapons Convention (CWC), after the Iran-Iraq War and the Gulf War. In 1990, an incident of human injury using mustard (yperite) took place at a usual residence at Komagome, Tokyo. In 1994 and 1995, unprecedented sarin poisoning terrorism took place in Matsumoto and Tokyo, Japan and surprised the whole world in the fear that similar chemical terrorism would be reproduced in other countries. Also in 1994, an attorney-at-low and his family were killed using VX in Osaka, Japan. The above sarin and VX incidents were found committed by the same cult group. These incidents show that chemical weapons can be used not only for wars, but also can be convenient means of crimes. To cope with such crimes using chemical weapons, such as yperite and sarin, various pre- ventive measures should be taken on the basis of the Revised Poisonous and Deleterious Sub- stances Control Law and the Chemical Weapons Banning Law of Japan; when such an incident happens, proper and rapid actions should be taken to minimize the damages. In the list of scheduled chemicals being defined by CWC, there are toxic chemicals and precursors for each of Schedules 1–3. In this chapter the word “chemicals” is used for such scheduled chemicals for simplicity. Before analysis of the chemicals, it is essential to get to know their histories, methods of synthesis, properties, directions for use, toxicities, therapeutic methods, stabilities and analytical methods. The chemicals directly act on organisms (animals and plants) and exert their toxicities; they are classified into the following 3 groups [1, 2]: • Poisonous chemicalsa: they directly exert toxic effects and kill or injure humans and animals. • Incapacitating chemicals: they neither cause severe injuries nor fatalities, but incapacitate people temporarily. • Chemicals for plants: they are used as defoliants using their herbicidal action. In this chapter, the methods for qualitative analysis of sarin and its decomposition products, which the authors experienced, are presented [3–6]. The chemical name of sarin is methyl- phosphonofluoridic acid isopropyl ester or O-isopropylmethylphosphonofluoridate (US code: GB, CAS registration No.: 107-44-8). Sarin is an unstable compound and easily decomposed into nonpoisonous isopropylmethyl- phosphonic acid, followed by further decomposition into methylphosphonic acid b. The above © Springer-Verlag Berlin Heidelberg 2005
610 Sarin and its decomposition products two products stably exist in soils and water for relatively a long period around the spot, where sarin has been sprayed; if isopropyl methyl phosphonic acid is identified, it can be verified that sarin has been used. Reagents and specimens • Sarin: a plastic bag containing about 600 mL of light-brown fluid, which had been obtained at Kasumigaseki Station of the Chiyoda subway line, was carefully opened, and used as the original specimen of sarin. • VX: the compound hidden by a cult group and seized by police was used. • Other compounds: N,N-diethylaniline (DEA), trimethyl phosphate, methylphosphonic acid, dimethyl methylphosphonate, methyl phosphonic dichloride, acetonitrile-d3, deuterated chlo- roform (CDCl3), diisopropyl phosphorofluoridate (DFP) and N-methyl-N-(tert-butyl-dimeth- ylsilyl)trifluoroacetamide (MTBSTFA) can be all purchased from Aldrich (Milwaukee, WI, USA); triisopropyl phosphate, isopropyl hydrogenmethylphosphonate and diisopropyl methyl- phosphonate were synthesized in our laboratories according to the literature [7]. GC/MS analysis GC/MS conditions GC column: an HP-5MS fused silica capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm, Agilent Technologies, Palo Alto, CA, USA). GC/MS conditions; injection temperature: 250 °C; injection pressure: 1.05 kg/cm2; column (oven) temperature: 50 °C (2 min) → 20 °C/min → 250 °C (10 min); carrier gas: He (13 psi); split ratio, 50; ion source temperature: 250 °C; EI electron energy: 70 eV; CI mode reagent gas: isobutanec; CI electron energy: 230 eV; ionization current: 300 µA. Procedure i. Direct analysis A part of the original sarin specimen solution is diluted 10–50 fold with hexane (or acetone) and injected into GC/MS. ii. Analysis of decomposition products i. About 1 g of the above original sarin specimen solution is mixed with 12 mL of 5 % KOH solution, and left for about 24 h at room temperature. Using the headspace vapor of the mixture, the absence of undecomposed sarin is confirmed by GC/MS. ii. The above aqueous solution is extracted with chloroform (30 mL × 3 times). iii. After each centrifugation, the chloroform layers are combined, and dehydrated with anhy- drous Na2SO4; the clear supernatant chloroform extract is condensed under reduced pres- sure (sample A).
GC/MS analysis 611 iv. The aqueous layer is also condensed under reduced pressure (sample B). v. Parts of the samples A and B are placed in screw-cap glass vials respectively, and equally evaporated to dryness under streams of nitrogen. Each residue is mixed with 30 µL aceto- nitrile and 30 µL MTBSTFA, heated at 60 °C for 1 h for tert-butyldimethylsilyl (TBDMS) derivatization and injected into GC/MS. Assessment of the method > Figure 1.1 shows a TIC obtained by GC/MS for the diluted original sarin specimen ob- tained from the Tokyo Subway Sarin Incident. By measuring mass spectra and retention times, the peaks except for sarin were identified as DFP, diisopropyl methylphosphonate, triisopropyl phosphate and DEA. The big peak appearing at the retention time of 4 min in > Figure 1.1 is due to sarin. The EI and CI mass spectra of sarin are shown in > Figures 1.2 and 1.3, respectively. In the EI mass spectrum, no molecular peak (m/z 140) appeared; but a peak of the desmethylated form appeared at m/z 125. > Figure 1.4 shows a TIC and EI mass spectra for two peaks appearing in the TIC. Peaks 1 and 2 correspond to TBDMS derivatives of isopropyl methyl phosphonic acid and methylphos- phonic acid, respectively. For both compounds, neither molecular nor quasi-molecular peak appears. In the CI mode, both compounds showed the base peaks of their protonated molecular ions at m/z 252 and 325, respectively. In this connection, > Figure 1.5 shows an EI mass spectrum of underivatized VX. No molecular peak (m/z 267) appeared; a fragment ion at m/z 114 was the base peak. > Figure 1.6 ⊡ Figure 1.1 TIC by GC/MS for the original sarin specimen obtained at the Tokyo Subway Sarin Incident.
612 Sarin and its decomposition products ⊡ Figure 1.2 EI mass spectrum of sarin. ⊡ Figure 1.3 CI mass spectrum of sarin.
GC/MS analysis 613 ⊡ Figure 1.4 TIC and mass spectra for Peaks 1 and 2 obtained by GC/MS for TBDMS derivatives of hydrolyzed products of sarin. ⊡ Figure 1.5 EI mass spectrum of VX.
614 Sarin and its decomposition products ⊡ Figure 1.6 CI mass spectrum of VX. shows a CI mass spectrum of VX; an intence protonated peak appeared at m/z 268 together with fragment peaks at m/z 252, 128 and 114. NMR analysis NMR conditions i. NMR instruments JNM-EX270 and JNM-EX90A (with the tunable module) FT-NMR spectrometers (JEOL, Tokyo, Japan) were used. ii. Analytical conditions A sample tube with 5 mm i. d. was used. For 13C, the 1H decoupling mode was employed; for 19F, the 1H non-decoupling mode; and for 31P, both 1H decoupling and non-decoupling modes. The conditions for the JNM-EX270 instrument were: measurement frequency, 109 MHz; mode, 1H decoupling; data points, 32 K; pulse width, 6.9 µs; pulse delay time, 5 s; integration times, 4; measurement temperature, 25 °C; and spectral width for chemical shifts, 40,000 Hz. The parameters for NMR measurements using the JNM-EX90A instrument are summa- rized in > Table 1.1.
NMR analysis 615 ⊡ Table 1.1 Parameters for NMR measurements of sarin 1 13 19 31 Nuclear species H C F P Measurement frequency 89.56 22.52 84.26 36.25 (MHz) internal tetramethyl- TMS standard silane (TMS) external trifluoroacetic acid 85 % phos- (δF= –76.5 ppm) phoric acid measurement temperature 26 °C data point 16 K 16 K 32 K 32 K NMR lock deuterated chloroform (CDCl3) spectral width (Hz) 1,800.5 7,507.5 26,041.7 8,000.0 pulse width 6.5 µs 3.5 µs 14.5 µs 12.6 µs (45° pulse) (45° pulse) (90° pulse) (90° pulse) integration time 32 2,400 64 256 (repetition time) (7 µs) (3 µs) (3 µs) (5 µs) Procedures • For direct NMR analysis, the original sarin specimen was diluted with acetonitrile-d3, placed and sealed in the sample tube for NMR measurements using the JNM-EX270 instrument. • The original sarin specimen was purified by vacuum distillation. A major fraction distilled at 60–61 °C/25 mm Hg was collected and diluted with deuterated chloroform (CDCl3) to make solution at 95 mg/g. The NMR measurements were carried out on the JNM-EX 90A instrument. Assessment of the method > Figure 1.7 shows a 31P-NMR spectrum obtained from the original sarin specimen. The main doublet signals were judged due to sarin, because they (δ:29.62 ppm, JPF = 1037 Hz) were almost identical with those of sarin (δ: 28.44 ppm, JPF = 1046.3 Hz) reported in literature. DFP and diisopropyl methylphosphonate could be detected together with sarin by GC/MS ( > Figure 1.1); in this NMR spectrum, hydrogen methylphosphonofluoridate appears in ad- dition ( > Figure 1.7). The 1H-, 13C-, 31P- and 19F-NMR data for the purified sample after distillation are shown in > Table 1.2; the 31P-NMR data for decomposition products and by-products of sarin shown in > Table 1.3. The composition of sarin and contaminants in the original specimen solution of the Tokyo Subway Sarin Incident was carefully examined by 31P-NMR, using trimethyl phosphate as a standard, because it did not overlap any sarin-related compound. The results were (w/w): sarin, 35 %; hydrogen methylphosphonofluoridated, 10 %; diisopropyl methylphosphonatee, 1 %; and
616 Sarin and its decomposition products ⊡ Figure 1.7 31 P-NMR spectrum in the 1H decoupling mode for the original sarin specimen solution obtained at the Tokyo Subway Sarin Incident. DFP, trace (0.1 %). The content (w/w) of organic solvents (DEAf plus hexane) measured by GC after hydrolysis of the original specimen solution was about 53 %. Poisoning symptoms, and toxic and fatal concentrations By the Tokyo Sarin Subway Incident, the poisoning symptoms provoked by sarin were clarified [8]. In its mild poisoning, rhinorrhea, darkness of eyeshot and difficulty in breathing were most common, followed by pain of the eye, dyspnea, cough, nausea, vomiting, headache and feeling of enervation. In severe poisoning, the victims are killed by paralysis of the respiration muscles. Sarin is highly volatile and shows toxicity higher than that of tabun. In the presence of sarin at 2 mg · min/m3 in the air, the darkness of the eyeshot and thus visual disturbance appear; the fatal atmospheric concentration was reported to be about 100 mg · min/m3 [8].
Poisoning symptoms, and toxic and fatal concentrations 617 ⊡ Table 1.2 1 H-, 13C-, 31P- and 19F-NMR data obtained from sarin [5] Nucleus Chemical shift (ppm) Coupling constants (Hz) 1 H 2 1-H 1.62 (dd, 3H) JHP = 18.5 3 JHF = 5.7 3 2-H 4.90 (m, 1H) JHH = 6.3 3 JHP = 7.3 3 3-H 1.38 (d, 6H) JHH = 6.3 13 C 1 1-C 10.42 JCH = 129.2 1 JCP = 150.3 2 JCF = 27.5 1 2-C 72.70 JCH = 151.2 2 JCP = 6.4 1 3-C 23.74 23.90 JCH = 126.4 31 2 P 29.62 JHP = 18.5 3 JHP = 7.3 1JPF = 1045.4 19 3 F –58.07 JHF = 5.7 1JPF = 1045.4 ⊡ Table 1.3 Chemical shift values for sarin, its related compounds and trimethyl phosphate [6]. Compound Chemical shift Coupling constant (ppm)* (Hz) trimethyl phosphate 2.39 DFP –10.42 JPF = 967 diisopropylmethylphosphonate 29.32 sarin JPF = 1037 methylphosphonic acid 31.51 isopropyl hydrogenmethylphosphonate 32.80 * 85% phosphoric acid external standard Notes a) As prerequisites of being the poisonous chemicals, the following 3 items can be men- tioned. • Very high toxicity: it should have toxicity, which can kill a number of humans or ani- mals with a small amount of a poison. • Stability under certain conditions: upon the use of a poison, the poisonous effect should last for a required period; upon its storage, it should be highly stable. • Low perceptibility of a poison by humans: a compound, which gives a characteristic smell, a color or a taste, is easily detected by one or more of the five senses of humans
618 Sarin and its decomposition products and can be treated for protection. The compound should not be easily perceived by any human sense. In addition, the following items can be also mentioned as their common properties. • A poison should invade various structures and exert its homicidal action, but does not destroy or ruin the structures themselves. • A poison can be spread widely, retained for a while to exert its poisonous effects and flown away. • There are types of poisons with early (within several hours) and delayed onset of poi- soning symptoms. • There are short (within 10 min) and long-acting poisons. • Because of the low perceptibility of a poison, people are easily exposed and injured by the poison without any consciousness. b) sarin/isopropyl hydrogenmethylphosphonate/methyl phosphonic acid c) As reagent gas, ammonia or methane can be also used. d) This compound is an impurity produced by decomposition of methylphosphonic difluo- ride, the precursor of sarin, or of methylphosphonic chlorofluoride, the disproportionation reaction product. e) Methylphosphonic dichloride side-reacts with isopropyl alcohol to produce diisopropyl methylphosphonic acid. f) DEA is considered to be added to enhance the reaction of the sarin synthesis. References 1) Wakai H (2000) Defense against chemical weapon attack. Japanese Bureau of the Organization for the Prohibi- tion of Chemical Weapons (OPCW), Tokyo, pp 142–155 (in Japanese) 2) Komoto T (1995) Basic knowledge on chemical weapons. Criminal Data File of the Metropolitan Police Depart- ment (Tokyo) 46:17–36 (in Japanese) 3) Ando H (1995) Practical study on sarin analysis at the Tokyo Subway Sarin Incident. In: Abstracts of the 1995 Annual Meeting of Kanto District Society of Forensic Technology. Tokyo, pp 49–56 (in Japanese) 4) Miyata Y, Nonaka H, Yoshida T et al. (2000) Analyses of sarin and related compounds used in the Tokyo Subway Sarin Incident. Jpn J Forensic Toxicol 18:39–48 5) Miyata Y, Nonaka H, Ando H (2000) Nuclear magnetic resonance data of sarin obtained at the Tokyo Subway Sarin Incident. Jpn J Forensic Toxicol 18:261–267 6) Miyata Y, Ando H (2001) Examination of an internal standard substance for the quantitative analysis of sarin using 31P-NMR. J. Health Sci 47:75–77 7) Japanese Association of Organic Synthetic Chemistry (ed) (1971) Organophosphorus compounds. In: Modern Organic Synthesis Series (5). Gihodo, Tokyo, pp 329–330 (in Japanese) 8) Tu AT, Inoue N (2001) Overall View of Chemical and Biological Weapons. Joho Inc., Tokyo, p 27, p 82 (in Japanese)