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

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

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

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-

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)