Tài liệu Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 16) doc

1.7
© Springer-Verlag Berlin Heidelberg 2005
II.1.7 Toluene, benzene, xylene
and styrene
by Xiao-Pen Lee and Keizo Sato
Introduction
Toluene, benzene, xylene and styrene are being widely used for chemical product materials,
solvents and constituents of adhesives and paints, and thus sometimes cause poisoning inci-
dents by inhaling their gas at chemical product-manufacturing factories.  e abuse of thinner
solvents, containing toluene, benzene and xylene, is now a social problem especially for young
people.  ere are many crimes and poisoning incidents involving the thinner solvent abuse.
Recently, styrene leaking from new building materials is an object of interest, because it causes
the sick house syndrome.
 e above four solvents are rapidly absorbed into human body by inhalation, per os
and percutaneously to cause various disturbances, such as suppression of the central nervous
system. In addition, benzene and styrene are being regarded as carcinogenic compounds.
For analysis of toluene, benzene, xylene and styrene, the conventional GC methods with
packed columns are being used [1–10]. In this chapter, a method of their analysis for human
blood using headspace wide-bore capillary GC is presented.
Reagents and their preparation
• Standard solutions: toluene, benzene or each isomer
a
of xylene of special grade is dissolved
in methanol to prepare each 1 mg/mL solution.
• Internal standard (IS) solution: aniline of special grade is dissolved in methanol to give
10 mg/mL solution.
GC conditions
Column: an Rtx-BAC2 wide-bore capillary column (30 m × 0.53 mm i. d.,  lm thickness
2.0 µm, Restek, Bellefonte, PA, USA).
GC conditions; instrument: a Shimazu GC-14B gas chromatograph (Shimadzu Corp.,
Kyoto, Japan); detector: FID; column (oven) temperature: 30 °C (1 min) → 15 °C/min → 240 °C

injection and detector temperature: 240 °C; carrier gas: He; its  ow rate: 5.0 mL/min.
150 Toluene, benzene, xylene and styrene
Procedure
i. A 0.5 mL volume of whole blood, 20 µL (200 µg) of IS solution, 1.5 mL of distilled water
and 0.6 g of solid NaCl
b
are placed in a 7.5 mL volume vial, stoppered with a silicone-sep-
tum cap and mixed well.
ii.  e vial is heated at 100 °C with stirring
c
on an aluminum block heater. A er 15 min of
heating, about 0.6 mL of the headspace vapor is drawn into a gas-tight syringe
d
and the
volume of the vapor is reduced to 0.3 mL
e
by pushing the plunger. It is immediately in-
jected into GC for analysis.
iii. Quantitation: to each 0.5 mL of blank whole blood, one of the known amounts of each
solvent and 200 µg of IS are added to construct a calibration curve
f
,

consisting of concen-
tration of a solvent on the horizontal axis and peak area ratio of a solvent to IS on the verti-
cal axis for enabling calculation of a concentration of a test compound in whole blood.
Assessment of the method
> Figure 7.1 shows a headspace wide-bore capillary gas chromatogram obtained from 0.5 mL
whole blood, to which toluene, benzene, 3 kinds of xylene isomers and styrene, 3 µL (3 µg) of
each, together with 20 µL (200 µg) of IS solution, had been added. Every peak was separated

well from each other except for m- and p-xylenes.  e background was clean with few impu-
rity peaks.  e extraction e ciencies (recoveries) for each organic solvent are 3.1–5.2 %. Good
linearity of the calibration curve can be obtained in the range of 0.5–5.0 µg/0.5 mL for each
compound; the detection limit is equally about 0.2 µg/0.5 mL.
It is impossible to separate m-xylene from p-xylene with the present Rtx-BAC2 wide-bore
capillary column. To separate such isomers, the use of a packed column with Bentone-34 or a
chiral capillary column, such as an α-DEX 120 capillary column (Supelco, Bellefonte, PA,
USA), is recommendable.
Toxic and fatal concentrations and poisoning symptoms
Although the poisoning e ects of toluene and benzene vary according to di erent individuals,
the oral ingestion of 15–20 mL toluene generally causes serious toxicity.  e fatal dose of ben-
zene is estimated to be about 100 mL; but there was a case in which a subject died a er oral in-
gestion of only 15 mL benzene.  e atmospheric concentration of toluene at 2,000 ppm is dan-
gerous for humans [11].  ere is a possibility of death a er single inhalation of 10,000 ppm gas
of benzene [11].  e maximum permissible atmospheric concentrations of toluene and benzene
are 50 and 0.5 ppm, respectively, according to the Japanese Society of Industrial Hygiene. In fatal
thinner poisoning cases, the blood concentrations of toluene were reported to be 6–110 µg/mL
[3]; in fatal benzene poisoning cases, blood benzene concentrations to be 0.94–38 µg/mL [12].
As symptoms of acute poisoning by exposure to toluene or benzene, immediate headache,
vertigo and coma appear, according to severity of poisoning, followed by death  nally.
As symptoms of chronic poisoning by toluene or benzene, hematological disorders, such as
aplastic anemia and acute leukemia, together with the symptoms of the central nervous system,
can take place.
151
 e toxicity of xylene is lower than that of toluene or benzene, but it also causes acute and
chronic poisoning.  e fatal atmospheric concentration of xylene is about 19,000 ppm [13]. Its
maximum permissible atmospheric concentration is 100 ppm according to the same society.
In autopsy cases of self-ignition using gasoline, the blood concentrations of the three xylene
isomers were reported to be 46.5–250 µg/mL [14].
As symptoms by styrene poisoning, the disturbances of the central nervous system appear.

At 200–400 ppm of atmospheric concentrations of styrene, stimulation of the eye and the re-
spiratory organs can occur; by inhalation of 1 % vapor of styrene, a victim can fall into a coma-
tose state in a few minutes [15]. A er chronic exposure to styrene, the side chain of styrene is
epoxidized and causes carcinogenicity.  e maximum permissible atmospheric concentration
of the compound de ned by the same society is 50 ppm. In the survived cases of styrene poi-
soning, the blood concentrations were reported to be 0.01–1.0 µg/mL [16, 17].
Detection of toluene, benzene, xylene isomers and styrene from whole blood by headspace
capillary GC. 1: benzene; 2: toluene; 3: p-xylene; 4: m-xylene; 5: o-xylene 6: styrene; 7: aniline (IS).
Three micrograms each of the above 6 compounds and 200 µg of aniline (IS) were added to
0.5 mL whole blood.
⊡ Figure 7.1
Toluene, benzene, xylene and styrene
152 Toluene, benzene, xylene and styrene
Notes
a)  ere are 3 isomers of xylene, viz. o-, m- and p-xylene.
b) To improve extraction e ciency (recovery), the addition of NaCl is e ective, because of its
salting-out e ect.
c) By using a stirrer, the time for extraction can be shortened; the recovery rate is also in-
creased also by heating, due to enhanced mobility of molecules.
d) When a vial septum made of silicone/Te on is used, a 90°cut needle at its tip for the gas-
tight syringe is e ective to prevent the leakage of gas upon piercing through the septum.
e)  is procedure of reducing the volume is e ective for expelling atmospheric air, which has
been aspirated into the syringe just a er taking the needle out of the vial, because of the
negative pressure.
f) For drawing a calibration curve, 5–6 concentrations of a compound should be plotted to
con rm its linearity.
References
1) Wampler TP, Bowe WA, Levy EJ (1985) Dynamic headspace analyses of residual volatiles in pharmaceuticals. J
Chromatogr Sci 23:64–67
2) Zahlsen K, Rygnestad T, Nilsen OG (1985) Oral intake of a toluene-containing thinner. Effects and headspace

gas chromatographic analytical diagnosis. Arch Toxicol Suppl 8:412–415
3) Takeichi S, Yamada T, Shikata I (1986) Acute toluene poisoning during painting. Forensic Sci Int 32:109–115
4) Morinaga M, Hara K, Kageura M et al. (1990) A simple, rapid and simultaneous analysis of complex volatile
hydrocarbon mixtures in blood using gas chromatography/mass spectrometry with a wide-bore capillary
column. Z Rechtsmed 103:567–572
5) Kato K, Nagata T, Kimura K et al. (1990) Demonstration of ingested thinner. Jpn J Legal Med 44:223–226
6) Ghittori S, Fiorentino ML, Maestri L et al. (1993) Urinary excretion of unmetabolized benzene as an indicator of
benzene exposure. J Toxicol Environ Health 38:233–243
7) Kessels H, Hoogerwrf W, Lips J (1992) The determination of volatile organic compounds from EPA method
524.2 using purge and trap capillary gas chromatography, ECD and FID. J Chromatogr Sci 30:247–255
8) Otson R, Kumarathasan P (1995) An automated head space analysis method for xylenes and ethylbenzene in
blood and water. Chemosphere 30:1109–1123
9) Pantarotto C, Fanelli R, Belletti I et al. (1980) Determination of styrene in biological specimens by gas chromato-
graphy selected ion monitoring distribution in mice. Anal Biochem 105:340–347
10) Varner SL, Breder CV, Fazio T (1983) Determination of styrene migration from food-contact polymers into
margarine, using azeotropic distillation and headspace gas chromatography. J Assoc Off Anal Chem 66:1067–
1073
11) Olson KR (ed) (1994) Poisoning & Drug Overdose, 2nd edn. Appleton & Lange, Norwalk, CT, pp 298–300
12) Moffat AC, Jackson JV, Moss MS et al. (eds) (1986) Clark’s Isolation and Identification of Drugs, 2nd edn.
Pharmaceutical Press, London, p 380
13) Ishibashi H (1967) Guide to Organic Solvents. Maki-shoten, Tokyo, p 128 (in Japanese)
14) Hattori H, Iwai M, Kurono S et al. (1998) Sensitive determination of xylenes in whole blood by capillary gas
chromatography with cryogenic trapping. J Chromatogr B 718:285–289
15) Patty FA (ed) (1967) Industrial Hygiene and Toxicology, 2nd edn. Wiley, New York, p 1222
16) Cherry N, Waldron HA, Wells GG et al. (1980) An investigation of the acute behavioral effects of styrene on
factory workers. Br J Ind Med 37:234–240
17) Klaassen CD (ed) (1996) Casarett and Doull’s Toxicology, 5th edn. McGraw-Hill, New York, p 999