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8.1
8.1
© Springer-Verlag Berlin Heidelberg 2005
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. ey 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
e orts to reduce chemical weapons existing in the world on the basis of the Chemical Weapons
Convention ( CWC), a er 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. e above sarin and VX incidents were found committed by the same cult group.
ese 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 de ned 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.
e chemicals directly act on organisms (animals and plants) and exert their toxicities;
they are classi ed into the following 3 groups [1, 2]:
• Poisonous chemicals
a
: they directly exert toxic e ects 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]. e chemical name of sarin is methyl-
phosphono uoridic acid isopropyl ester or O-isopropylmethylphosphono uoridate (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
. e above
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 identi ed, it can be veri ed that
sarin has been used.
Reagents and specimens
• Sarin: a plastic bag containing about 600 mL of light-brown uid, 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-d
3
, deuterated chlo-
roform (CDCl
3
), diisopropyl phosphoro uoridate ( DFP) and N-methyl-N-(tert-butyl-dimeth-
ylsilyl)tri uoroacetamide (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., lm thickness
0.25 µm, Agilent Technologies, Palo Alto, CA, USA).
GC/MS conditions; injection temperature: 250 °C; injection pressure: 1.05 kg/cm
2
; 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:
isobutane
c
; 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 le for about 24 h at room temperature. Using the headspace vapor of the
mixture, the absence of undecomposed sarin is con rmed by GC/MS.
ii. e above aqueous solution is extracted with chloroform (30 mL × 3 times).
iii. A er each centrifugation, the chloroform layers are combined, and dehydrated with anhy-
drous Na
2
SO
4
; the clear supernatant chloroform extract is condensed under reduced pres-
sure (sample A).
611
iv. e 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 identi ed as DFP, diisopropyl methylphosphonate, triiso propyl
phosphate and DEA.
e big peak appearing at the retention time of 4 min in
> Figure 1.1 is due to sarin. e
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
TIC by GC/MS for the original sarin specimen obtained at the Tokyo Subway Sarin Incident.
⊡ Figure 1.1
GC/MS analysis
612 Sarin and its decomposition products
EI mass spectrum of sarin.
⊡ Figure 1.2
CI mass spectrum of sarin.
⊡ Figure 1.3
613
TIC and mass spectra for Peaks 1 and 2 obtained by GC/MS for TBDMS derivatives of hydrolyzed
products of sarin.
⊡ Figure 1.4
⊡ Figure 1.5
EI mass spectrum of VX.
GC/MS analysis
614 Sarin and its decomposition products
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
13
C, the
1
H decoupling mode was employed;
for
19
F, t he
1
H non-decoupling mode; and for
31
P, b o t h
1
H decoupling and non-decoupling
modes.
e conditions for the JNM-EX270 instrument were: measurement frequency, 109 MHz;
mode,
1
H 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 shi s, 40,000 Hz.
e parameters for NMR measurements using the JNM-EX90A instrument are summa-
rized in
> Table 1.1.
CI mass spectrum of VX.
⊡ Figure 1.6
615
Procedures
• For direct NMR analysis, the original sarin specimen was diluted with acetonitrile-d
3
,
placed and sealed in the sample tube for NMR measurements using the JNM-EX270
instrument.
• e original sarin specimen was puri ed by vacuum distillation. A major fraction distilled
at 60–61 °C/25 mm Hg was collected and diluted with deuterated chloroform (CDCl
3
) to
make solution at 95 mg/g. e NMR measurements were carried out on the JNM-EX 90A
instrument.
Assessment of the method
> Figure 1.7 shows a
31
P-NMR spectrum obtained from the original sarin specimen. e
main doublet signals were judged due to sarin, because they (δ:29.62 ppm, J
PF
= 1037 Hz)
were
almost identical with those of sarin (δ: 28.44 ppm, J
PF
= 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 methylphosphono uoridate appears in ad-
dition (
> Figure 1.7).
e
1
H-,
13
C-,
31
P- and
19
F-NMR data for the puri ed sample a er distillation are shown
in
> Table 1.2; the
31
P-NMR data for decomposition products and by-products of sarin shown
in
> Table 1.3.
e composition of sarin and contaminants in the original specimen solution of the Tokyo
Subway Sarin Incident was carefully examined by
31
P-NMR, using trimethyl phosphate as a
standard, because it did not overlap any sarin-related compound. e results were (w/w): sarin,
35 %; hydrogen methylphosphono uoridate
d
, 10 %; diisopropyl methylphosphonate
e
, 1 %; and
⊡ Table 1.1
Parameters for NMR measurements of sarin
Nuclear species
1
H
13
C
19
F
31
P
Measurement frequency
(MHz)
89.56 22.52 84.26 36.25
standard
internal
external
tetramethyl-
silane (TMS)
TMS
trifluoroacetic acid
(δ
F
= –76.5 ppm)
85 % phos-
phoric acid
measurement temperature 26 °C
data point 16 K 16 K 32 K 32 K
NMR lock deuterated chloroform (CDCl
3
)
spectral width (Hz) 1,800.5 7,507.5 26,041.7 8,000.0
pulse width 6.5 µs
(45° pulse)
3.5 µs
(45° pulse)
14.5 µs
(90° pulse)
12.6 µs
(90° pulse)
integration time
(repetition time)
32
(7 µs)
2,400
(3 µs)
64
(3 µs)
256
(5 µs)
NMR analysis
616 Sarin and its decomposition products
DFP, trace (0.1 %). e content (w/w) of organic solvents (DEA
f
plus hexane) measured by GC
a er 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 clari ed
[8]. In its mild poisoning, rhinorrhea, darkness of eyeshot and di culty 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/m
3
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/m
3
[8].
31
P- NMR spectrum in the
1
H decoupling mode for the original sarin specimen solution obtained
at the Tokyo Subway Sarin Incident.
⊡ Figure 1.7
617
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 e ect 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 ve senses of humans
⊡ Table 1.2
1
H-,
13
C-,
31
P- and
19
F-NMR data obtained from sarin [5]
Nucleus Chemical shift (ppm) Coupling constants (Hz)
1
H
1-H 1.62 (dd, 3H)
2
J
HP
= 18.5
3
J
HF
= 5.7
2-H 4.90 (m, 1H)
3
J
HH
= 6.3
3
J
HP
= 7.3
3-H 1.38 (d, 6H)
3
J
HH
= 6.3
13
C
1-C 10.42
1
J
CH
= 129.2
1
J
CP
= 150.3
2
J
CF
= 27.5
2-C 72.70
1
J
CH
= 151.2
2
J
CP
= 6.4
3-C 23.74 23.90
1
J
CH
= 126.4
31
P 29.62
2
J
HP
= 18.5
3
J
HP
= 7.3
1
J
PF
= 1045.4
19
F –58.07
3
J
HF
= 5.7
1
J
PF
= 1045.4
⊡ Table 1.3
Chemical shift values for sarin, its related compounds and trimethyl phosphate [6].
Compound Chemical shift
(ppm)*
Coupling constant
(Hz)
trimethyl phosphate 2.39
DFP –10.42 J
PF
= 967
diisopropylmethylphosphonate 29.32
sarin J
PF
= 1037
methylphosphonic acid 31.51
isopropyl hydrogenmethylphosphonate 32.80
* 85% phosphoric acid external standard
Poisoning symptoms, and toxic and fatal concentrations
618 Sarin and its decomposition products
and can be treated for protection. e 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 e ects and
own away.
• ere are types of poisons with early (within several hours) and delayed onset of poi-
soning symptoms.
• ere 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) is compound is an impurity produced by decomposition of methylphosphonic di uo-
ride, the precursor of sarin, or of methylphosphonic chloro uoride, 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
31
P-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)
. 18:261–267
6) Miyata Y, Ando H (2001) Examination of an internal standard substance for the quantitative analysis of sarin
using
31
P-NMR. J. Health Sci 47:75–77
. 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
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