II. Chapters on specific toxins
1.1
1.1
© Springer-Verlag Berlin Heidelberg 2005
II.1.1 Carbon monoxide
By Keizo Sato
Introduction
 e incidence of carbon monoxide ( CO) poisoning is highest among those of various poison-
ings in forensic science practice; about 2/3 of the total accidental poisoning deaths is due to CO
poisoning in Japan [1]. Previously, suicides, homicides and accidental deaths frequently took
place using city gas containing about 9% CO. However, during recent years, city gas is being
replaced by natural gas containing no CO, resulting in drastic decrease of the number of CO
poisoning cases. Nevertheless, many incidents of CO poisoning are occurring due to imperfect
combustion,  re, exhaust gas of automobiles and other causes. For a victim found at the scene
of a  re, the saturation ratio of carboxyhemoglobin ( COHb) can be an indicator
a
for judging
whether the victim has died in a  re or had been already killed before the  re.
For measurements of COHb saturation ratios in blood, spectrophotometric and GC meth-
ods are available. Since methemoglobin ( Met-Hb) is contained in many of blood specimens in
forensic science practice for measurements of COHb saturation [2], it is important to use a
method
b
, which is not in uenced by the presence of Met-Hb. In this chapter, simple and reli-
able spectrophotometric [3] and GC [4] methods for measurements of COHb saturation not to
be in uenced by Met-Hb are presented.
Spectrophotometric method
See [3].
Reagents and their preparation
• 0.1% Na
2

) are read against
distilled water in another cuvette as a blank.
vi.  e percentage of HbCO can be calculated by the following equation:
COHb %=(2.44–A
558
/A
532
) × 67
Assessment and some comments on the method
 e above spectrophotometric assay for HbCO saturation ratio well meets the needs in forensic
science practice. To perform accurate measurements of low ranges of COHb by this method,
the following modi cation of the method is recommended.
About 20–30 specimens of fresh blood obtained from healthy subjects is analyzed accord-
ing to the above procedure. A specimen with the lowest COHb value is taken as 0%, which can
be used as the blank test. When the blood specimen with the lowest COHb value is processed
through 1–4 described in the above procedure, the absorbance spectrum 1 due to reduced Hb
can be obtained as shown in
> Figure 1.1; when CO gas is then bubbled in the same cuvette,
the absorbance spectrum 2 shown in the same  gure can be obtained. In the spectrum 1, the
isobestic point around 532 nm and the absorbance maximum around 558 nm appear; the exact
wavelengths are re-examined for both points and small shi s of their wavelengths according to
instrumental conditions can be corrected. Even when the absorptions at corrected wavelengths
⊡ Figure 1.1
Absorption spectra of reduced Hb (1) and COHb (2) in the presence of NaOH.
93
are used, it is not necessary to change the coe cient values in the above equation.  e method
using a blank test of a healthy and fresh blood and using corrected wavelengths enables the
accurate measurements of COHb contents less than 10% [3, 5].
For measurements of COHb in bloody  uids in the thoracic and abdominal cavities, a
special care should be taken. Kojima et al. [6] reported that 2.3–44.1% of COHb could be de-

Reagents and their preparation
• Solution of saponin and potassium ferricyanide: 500 mg of saponin (obtainable from many
manufacturers) and 2 g of potassium ferricyanide are dissolved in distilled water to make
10 mL solution.
GC analysis
94 Carbon monoxide
• Plastic disposable syringes (5 mL, Terumo, Tokyo, Japan or any other manufacturer)
• CO standard gas: 50 L (GL Sciences, Tokyo, Japan).

• Cyanmethemoglobobin reagent: Hemoglobin Test Wako (Wako Pure Chemical Industries,
Ltd., Osaka, Japan).
• Cyanmethemoglobin reagent (by Sato et al. [11]): 20 g of potassium ferricyanide is dis-
solved in 500 mL of 1/15 M phosphate bu er solution (pH 7.1), followed by the addition of
50 mg potassium cyanide and 100 mL of 1% Triton X-100 (obtainable from every manu-
facturer) with gentle mixing.  e mixture is made to 1 L by adding distilled water.  e  nal
pH of this reagent is about 7.2.
GC conditions
Column: Molecular Sieve 5A (60–80 mesh, 2 m × 3 mm i.d., Shimadzu Corp., Kyoto, Japan)
GC condition: a common GC instrument for packed columns with an FID is used. Carrier
gas is hydrogen at a  ow-rate of 40 mL/min; the column temperature is 80° C.  e above sepa-
ration column (Molecular sieve 5A) is connected with a stainless steel column (40 cm × 3 mm
i.d.) packed with a nickel catalyst ( Shimalite-Ni, Shimadzu).  e stainless column is heated
at 650° C in a reaction furnace (RAF-1A, Shimadzu Corp.), in which CO is converted into
methane to be detected with an FID.  e temperature of the injection port and detector is
150° C.
Procedure
i.  e plunger of a plastic disposable syringe (5 mL, Terumo) is drawn to make a 3-mL
space as shown in
> Figure 1.2.
ii.  e tip of the syringe is capped with a silicone rubber plug (the detailed structure also

globlin completely using the reagent solution of the commerciable kit [11]. To shorten the
analysis time, a hand-made reagent of Sato et al. [11] containing a larger amount (20 g/L)
of potassium ferricyanide is recommendable to be used. To 5 ml of the Sato’s solution
(without dilution), 20 µL of the test whole blood is added, mixed well and le only for
5 min; the following procedure is the same as described above.
x. On the basis of the fact that 1.36 mL of CO can be bound with 1 g of hemoglobin, the
COHb saturation percentage is calculated by the following equation:
⊡ Figure 1.2
Handling procedure for liberating CO from a blood specimen.
1: microsyringe; 2: silicone rubber plug; 3: silicone rubber tube; 4: plastic disposable syringe.
GC analysis
where A is the CO concentration (ppm) measured by the GC method; B the total Hb concentration (g/dL).
96 Carbon monoxide
Assessment and some comments on the method
> Figure 1.4 shows a typical gas chromatogram for the authentic CO gas. Usually a single peak
due to CO appears, but when CO
2
or methane coexists, multiple peaks are detected. Both CO and
CO
2
are converted into methane by nickel catalysis to be detected by an FID, but CO is neither
contaminated by CO
2
nor methane, because they are well separated by the Molecular Sieve 5A
column before their conversion into methane. Since hydrogen at a constant  ow rate of 40 mL/
min is used in this method, care should be taken for su cient ventilation of the laboratory.
⊡ Figure 1.3
Calibration curve for CO measurements by GC using the authentic standard gas.
⊡ Figure 1.4
Gas chromatogram for CO. A 200-µL volume of 500 ppm CO was injected into GC.

by Hb in human body. CO does not only cause hypoxia in tissues, but
also causes inhibition of enzymes, such as cytochrome oxidase [1].  e poisoning symptoms as
a function of blood COHb percentage are shown in
> Table 1.1. However, the toxicity of CO
depends upon both CO concentration in the air and duration of CO inhalation.  e table
shows only an outline of its toxicity; 50% or more of COHb in blood is an indicator of fatality.
⊡ Table 1.1
COHb saturation percentages in blood and symptoms in CO poisoning [1]
COHb in blood (%) Poisoning symptom
0–10 No symptoms
10–20 Tense feeling of the forehead, slight headache
20–30 Headache, pulse feeling in the temporal region
30–40 Severe headache, general fatigue, dizziness, impairment of visual acuity,
vomiting
40–50 Hyperventilation, coma with convulsion, Cheyne-Stokes breathing
60–70 Coma with convulsion, cardiac disfunction
70–80 Death
Notes
a) When the percentage of COHb in heart blood is more than 10%, it is probable that the
victim has died in the  re.
b) A spectrophotometric method for COHb using separate measurements of COHb and
O
2
Hb [14] and a GC method using a ratio of CO peak areas before and a er complete
saturation with CO [15] are in uenced by the presence of Met-Hb, while the spectropho-
tometric [3, 5, 16] and GC [4, 17] methods presented in this chapter are not.
c)  e amount of the homogenate should be increased to 40–50 µL.
d)  e saponin serves to hemolyze erythrocytes, while the potassium ferricyanide converts
COHb into Met-Hb to liberate CO.
e)  e accurate volume of the plastic disposable syringe (Terumo) at the mark of 3 mL was

15) Takahashi S, Kumabe Y, Ito N et al. (1980) Gas chromatographic analysis of carbon monoxide in blood with the
use of ultrasonic irradiation. Jpn J Legal Med 34:556–562
16) Fukui M, Kumaoka K, Ito H et al. (1971) Forensic Chemistry. Hirokawa Publishing Co., Tokyo, pp 244–245 (in
Japanese)
17) Kojima T, Nishiyama Y, Yashiki M et al. (1981) Determination of carboxyhemoglobin saturation in blood and
body cavity fluids by carbon monoxide and total hemoglobin concentrations. Jpn J Legal Med 35:305–311 (in
Japanese with an English abstract)
Toxic and fatal concentrations