1.5 © Springer-Verlag Berlin Heidelberg 2005 II.1.5 Ethanol by Kanako Watanabe Introduction Alcohol usually means ethanol/ ethyl alcohol. It has a long history for the human being, and gives euphoric e ects and sometimes improves the human relationship. In contrast, there are many cases of violence, injuries, homicides and tra c accidents with drinking; close relation can be observed between ethanol and crimes/accidents. When forensic autopsies are per- formed, ethanol concentrations in blood and urine are routinely measured. GC analysis of ethanol using the conventional packed columns is described in many of literature [1, 2]. In this chapter, a quantitative method is presented for GC analysis of ethanol in blood and urine using headspace extraction and wide-bore capillary columns; an ultra-sensi- tive analysis of breath ethanol is also given. Determination of ethanol in blood and urine Reagents and their preparation • 10 µL of ethanol is dissolved in 10 mL distilled water (0.1 %, v/v). • 10 µL of n-propanol a is dissolved in 10 mL of distilled water (0.1 %, v/v). • 10 µL of tert-butanol (internal standard, IS) b is dissolved in 10 mL of distilled water (0.1 %, v/v). All above chemicals can be of reagent grade. GC conditions GC columns: a DB-1 fused silica wide-bore capillary column (30 m × 0.53 mm i. d.,  lm thick- ness 5 µm, J&W Scienti c, Folsom, CA, USA), and a Rtx-BAC2 fused silica wide-bore capillary column (30 m × 0.53 mm i. d.,  lm thickness 2.0 µm, Restek: Bellefonte, PA, USA). GC conditions [3]: an HP6890 Series gas chromatograph c (Agilent Technologies: Palo Alto, CA, USA) equipped with FID. Conditions for the DB-1 column are: Column (oven) temperature: 40 °C (isothermal); injection port and detector temperature: 170 °C; carrier gas: helium; its  ow rate: 20 mL/min. Conditions for the Rtx-BAC2 column are: Column (oven) temperature: 40 °C→ 5 °C/min→ 70 °C (5 min)→ 20 °C/min→ 280 °C; injec- tion port and detector temperature: 170 °C; carrier gas: helium; its  ow rate: 5 mL/min. 136 Ethanol Procedure i. 0.5 mL of a specimen (whole blood or urine) is placed in a 4 mL volume test tube d with a rubber cap, followed by the addition of 0.2 mL IS solution and 0.2 mL distilled water, and sealed with the rubber cap. ii. In another test tube (standard solution), 0.5 ml of 0.1 % ethanol, 0.2 mL of IS solution and 0.2 mL of 0.1 % n-propanol are placed, and sealed with the rubber cap. iii. Both test tubes are heated at 55 °C for 15 min on an aluminum block heater or in a water bath. During the above heating, a glass syringe to be used is also put on the heater to heat it simultaneously; the syringe is a 1 mL volume gas-tight grass syringe with a 23 G needle e . A er heating, 0.1 mL of the headspace vapor is withdrawn into the syringe and injected to GC swi ly. Calculation: P(ethanol)s : peak area of ethanol in the specimen P(IS)s : peak area of IS in the specimen P(ethanol)ss : peak area of ethanol in the standard solution P(IS)ss : peak area of IS in the standard solution. Assessment and some comments on the method > Figure 5.1 shows gas chromatogram for a whole blood specimen containing 0.1 % ethanol. With the DB-1 column [3], peaks of ethanol and IS appeared at short retention times at 40 °C; the Rtx-BAC2 column [4] had been developed for analysis of ethanol, and gives good shapes of the peak and high sensitivity.  e detection limits of the method are 20–50 μg/mL whole blood with the DB-1 wide-bore capillary column, and about 10 μg/mL whole blood with the Rtx-BAC2 wide-bore capillary column. For actual measurements of blood ethanol a er drinking, the GC method is su ciently sensitive with either of the capillary columns, because blood ethanol concentrations in blood a er drinking is usually 0.3–0.4 mg/mL.  e headspace method for GC analysis of ethanol gives clean background and almost no interfering peaks.  e postmortem production of ethanol a due to putrefaction should be kept in mind in case of non-fresh specimens; in such cases the appearance of n-propanol is an indicator of the con- comitant postmortem production of ethanol. Even without drinking, so-called “ endogenous ethanol” is present, which is probably due to food and enteric bacteria; its level was reported to be as low as 0.180 ± 0.117 μg/mL [5], and thus does not interfere with the ethanol determination a er drinking. A er fresh blood is sampled into a test tube and sealed with a cap, it can be stored at 4 °C for 1–2 weeks without any change of ethanol levels.  e author et al. set the cuto level of blood ethanol to be 0.1 mg/mL, considering the postmortem production of ethanol. 137 Toxic and lethal concentrations > Figure 5.2 shows symptoms of Japanese subjects caused by ethanol according to its blood concentrations [6].  e symptoms shown in the  gure only show typical ones; there exist ex- ceptional individuals, who show blood levels of as high as 4 mg/mL, but are not drunk heavily. About 10 % of Japanese population is of homotype of ALD-type 2 for the isozymes of aldehyde dehydrogenase, and thus very weak to ethanol showing various intoxication symptoms even at low blood ethanol levels. Determination of breath ethanol A close relationship exists in ethanol concentration between breath and blood ; the breath concentration is as low as about 2,000 times less than that in blood. Because of its extremely low concentrations, it cannot be measured by the usual capillary GC method described before. When the breath ethanol is measured by the conventional GC with FID, more than 500 mL of the vapor should be passed through a column packed with an adsorbent to collect and concen- trate breath ethanol before introducing to GC or GC/MS [7–9].  e apparatus for the above pretreatment is relatively complicated and its handling also requires some training. A microcomputer controlling cryogenic oven temperature below 0 °C has become widely available for modern types of GC instruments. It was originally designed for rapid cooling of an oven to reduce analysis time. We have used it to trap volatile organic compounds contained in Detection of blood ethanol by wide-bore capillary GC. ⊡ Figure 5.1 Determination of breath ethanol 138 Ethanol gas samples, and named it “ cryogenic oven trapping ( COT)” [10, 11]. Since the COT system can be attached easily and cheaply (about 1,000 US dollars) to any modern type of GC instruments, we present a sensitive method for determination of breath ethanol by GC using the system. Materials and their preparation • Quanti cation is made by the external standard calibration method. Each aliquot of etha- nol, viz. 3.17, 6.34, 9.51 and 12.7 µL, is added to 10 mL methanol to prepare standard solu- tions of ethanol. Using a plunger-in-needle syringe g , each 0.1 µL aliquot of the standard solutions is injected into GC.  e peak areas obtained at 4 concentrations of ethanol are plotted against the amount of ethanol to make a calibration curve; the  nal amounts of ethanol injected are 25, 50, 75 and 100 ng, respectively. • Breath ethanol polyethylene bags (about 1 L volume, Komyo-Rikagaku-Kogyo, Tokyo, Japan) h GC conditions GC column: an Rtx-BAC2 fused silica wide-bore capillary column (30 m × 0.53 mm i. d.,  lm thickness 2.0 µm, Restek · Bellefonte, PA, USA) i . COT [11]: a liquid carbon dioxide (CO 2 ) tank with a siphon steel tube which enables direct introduction of liquid CO 2 into a GC oven to cool it. An electrically operated solenoid valve Correlation between blood ethanol concentration and intoxication rate. ⊡ Figure 5.2 139 (Agilent Tchnologies · Palo Alto, CA, USA) controlled by a microcomputer introduces liquid CO 2 at a rate appropriate for cooling of the oven to a temperature desired. GC conditions: an HP 6890 Series gas chromatograph equipped with FID. Column (oven) temperature: –60 °C (1 min) →10 °C/min → 40 °C (10 min) → 20 °C/min → 240 °C; injection temperature: 200 °C; detector temperature: 240 °C; carrier gas: helium; its  ow rate: 3 mL/min.  e breath gas is injected into GC at –60 °C of column (oven) temperature in the splitless mode within 5 seconds, and the splitter is opened a er 1 min. Procedure i.  e mouth of a subject is washed well with tap water. ii.  e breath gas is expired into the above polyethylene bag h , and its introduction tube is rapidly sealed with Para lm. iii. A needle j of a glass syringe (5 mL volume) is stabbed to the polyethylene bag, and 5 mL gas is withdrawn into the syringe. All of the volume is injected into GC at –60 °C of column (oven) temperature in the splitless mode, and analyzed as above. iv. Using the external standard calibration curve, the concentrations expressed as per mL of breath ethanol are easily calculated dividing the value obtained by 5. Assessment of the method > Figure 5.3 shows gas chromatograms of breath gas sampled at 30 and 60 min a er drinking 200 mL of common beer [12].  e big peaks of ethanol appear with low background and a few small impurity peaks. A er 10 volunteers of both sexes drank 200 mL beer each, the concen- tration of breath ethanol ware 30.0 ± 18.1 ng/mL gas at 30 min and 15.8 ± 6.72 ng/mL gas at 60 min ( > Table 5.1).  e detection limit of this method is about 1 ng/mL gas. ⊡ Table 5.1 Concentrations of breath ethanol 30 and 60 min after drinking 200 ml beer Subject (sex, age) Breath ethanol concentration (ng/mL) 30 min 60 min No. 1 (F, 29 ) 30.8 19.6 No. 2 (M, 23 ) 9.62 6.23 No. 3 (F, 23 ) 73.5 18.0 No. 4 (F, 24 ) 24.8 13.0 No. 5 (F, 20 ) 34.0 29.0 No. 6 (M, 24 ) 18.5 8.94 No. 7 (M, 35 ) 20.2 10.4 No. 8 (F, 22 ) 45.2 21.8 No. 9 (M, 23 ) 19.9 16.3 No. 10 (M, 36 ) 23.9 14.9 Mean ± SD 30.0 ± 18.1 15.8 ± 6.72 Determination of breath ethanol 140 Ethanol Notes a) In putrefying specimens, ethanol can be easily produced by the action of bacteria [13]. Together with the postmortem production of ethanol during putrefaction, n-propanol is always produced concomitantly. Since n-propanol is usually not detectable in fresh human specimens, the presence of n-propanol becomes indicative of the postmortem production of ethanol.  erefore the standard solution contains n-propanol for reference.  e concen- tration ratio of n-propanol to ethanol in putre ed specimems was reported to be about 0.05; the postmortem ethanol concentration may be 20 times as much as that of n-propa- nol.  e following equation, therefore, can be postulated. Antemortem blood ethanol concentration = postmortem ethanol concentration – n-pro- panol concentration × 20. b) Various alkyl alcohols were used in the literature as IS. n-Propanol is produced postmor- tem; isobutanol coexists with ethanol in many alcoholic beverages, though its concentra- tions are much lower [14]. tert-Butanol seems best as IS, because it does not usually exist in biological specimens. Detection of breath ethanol by GC with cryogenic oven trapping. The volunteer drank 200 mL of common beer ⊡ Figure 5.3 141 c) Every type of gas chromatographs, to which capillary columns can be attached, can be used. d) A 5–7 mL screw vial with a Te on septum cap can be also used.  e authors are using cheap test tubes with rubber caps; they give no impurity peaks due to plasticizers. e)  e syringe needle is cut obliquely; the edges of the tip is very sharp. When this type of needles is used for the headspace extraction, it is o en experienced that the needle is ob- structed with the septum debris.  e authors are using special conical needles, which can be ordered to a manufacturer (Kurita Syringe Needle Manufacturing Co., 5-25-7 Hongo, Bunkyo-ku, Tokyo, Japan). Using this type of needles, the obstruction of the needle and contamination of the injection chamber with the septum debris became not so serious. f)  e concentrations of ethanol can be expressed as % (percent, v/v), and also as ‰ (permil); 0.1 % = 1 ‰. When it should be expressed as mg/mL, 0.1 % is equal to 0.789 mg/mL, be- cause speci c gravity of ethanol at 20 °C is 0.789. g)  e plunger-in-needle syringe is designed to be used for injecting as small as 0.1 µL of sample solution with high precision.  e authors are using the syringe (code No. 0.5BNR-5) obtained from SGE International (Ringwood, Victoria, Australia); the shape of the needle tip is conical. h) When the special breath ethanol bags are not available, a usual polyethylene bag can be used instead.  e breath is expired into the bag which is knotted immediately, and the following procedure is the same as described before. Since there is possibility of contamina- tion of the air inside by volatile plasticizers leaked from the usual polyethylene bag, it should be checked by GC beforehand. i) For cryogenic oven trapping ( COT), it is not necessary to use a wide-bore capillary column; medium-bore capillary columns seem more suitable for COT-GC because they give sharper peaks and thus higher sensitivity. At the moment of making the present experiments, only the Rtx-BAC2 wide-bore capillary columns are commercially available; however, very recently, the Rtx-BAC2 medium-bore capillary columns have become available from the same manufacturer.  us, now the latter column can be recommended for use because of higher sensitivity. j)  e size of the syringe needle should be 23–24 G and the tip shape of the needle is prefer- ably conical as stated in e). References 1) Nanikawa R (1977) Legal Medicine. Nippon-Iji-Shinposha, Tokyo, pp 239–260 (in Japanese) 2) Phemacentical Society of Japan (ed) (1992) Standard Methods of Chemical Analysis in Poisoning, 4th edn. Nanzando, Tokyo, pp 108–116 (in Japanese) 3) Hara K, Kageura M, Hieda Y et al. (1991) Quantitative analysis of ethanol in postmortem specimens using wide- bore capillary gas chromatography. Jpn J Forensic Toxicol 9:94–95 (in Japanese with an English abstract) 4) O’Neal CL, Wolf CE, Levine B et al. (1996) Gas chromatographic procedures for determination of ethanol in postmortem blood using t-butanol and methyl ethyl ketone as internal standards. Forensic Sci Int 83:31–38 5) Watanabe-Suzuki K, Seno H, Isii A et al. (1999) Ultra-sensitive method for determination of ethanol in whole blood by headspace capillary gas chromatography with cryogenic oven trapping. J Chromatogr B 727:89–94 6) Kashimura S (2001) Drink joyfully. Farewell to tossing off. To prevent death from acute alcoholism. Jpn J Legal Med 55:42–45 (in Japanese) 7) Jones AW (1985) Excretion of low-molecular weight volatile substances in human breath, focus on endoge- nous ethanol. J Anal Toxicol 9:246–250 Determination of breath ethanol 142 Ethanol 8) Phillips M, Greenberg J (1987) Detection of endogenous ethanol and other compounds in the breath by gas chromatography with on-column concentration of sample. Anal Biochem 163:165–169 9) Ghoos Y, Hiele M, Rutgeerts P et al. (1989) Porous-layer open-tubular gas chromatography in combination with an ion trap detector to assess volatile metabolites in human breath. Biomed Environ Mass Spectrom 18: 613–616 10) Watanabe K, Seno H, Ishii A et al. (1997) Capillary gas chromatography with cryogenic oven temperature for headspace samples. Analysis of chloroform or methylene chloride in whole blood. Anal Chem 69:5178–5181 11) Watanabe-Suzuki K, Ishii A, Lee X-P et al. (2000) Analysis of volatile organic compounds by cryogenic oven trapping gas chromatography. Jpn J Forensic Toxicol 18:201–209 (in Japanese with an English abstract) 12) Watanabe-Suzuki K, Seno H, Ishii A et al. (1999) Sensitive detection of ethanol in human breath by headspace capillary gas chromatography with cryogenic oven trapping. Jpn J Forensic Toxicol 17:61–65 13) Watanabe-Suzuki K, Ishii A, Seno H et al. (2000) Stability of ethyl acetate in whole blood during storage under various conditions. Jpn J Forensic Toxicol 18:237–243 14) Hosogai Y, Nakazawa H, Nishijima M (eds) (1998) Chemical Compound Date Book for Food Hygiene. Chuohouki, Tokyo, p 580 (in Japanese) . columns; an ultra-sensi- tive analysis of breath ethanol is also given. Determination of ethanol in blood and urine Reagents and their preparation • 10 µL of. 2]. In this chapter, a quantitative method is presented for GC analysis of ethanol in blood and urine using headspace extraction and wide-bore capillary