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. 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McGraw-Hill, New York, p 999 . 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. 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