DOT/FAA/AM-02/15

Russell J. Lewis

Robert D. Johnson

Dennis V. Canfield

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, OK 73125

August 2002

Final Report

This document is available to the public

through the National Technical Information

Service, Springfield, VA 22161.

An Accurate Method for the

Determination of Carbon Monoxide in

Postmortem Blood Using GC/TCD Office of Aerospace Medicine

Washington, DC 20591
NOTICE

This document is disseminated under the sponsorship of

the U.S. Department of Transportation in the interest of

information exchange. The United States Government

assumes no liability for the contents thereof.
i

Technical Report Documentation Page

1. Report No. 2. Government Accession No. 3. Recipient's Catalog No.

DOT/FAA/AM-02/15

4. Title and Subtitle 5. Report Date

An Accurate Method for the Determination of Carbon Monoxide in

Postmortem Blood Using GC/TCD

August 2002

7. Author(s) 6. Performing Organization Code

Lewis, R.L., Johnson, R.D. and Canfield, D.V.

9. Performing Organization Name and Address 8. Performing Organization Report No.

FAA Civil Aerospace Medical Institute

P.O. Box 25082 10. Work Unit No. (TRAIS)

Oklahoma City, Oklahoma 73125

12. Sponsoring Agency name and Address 11. Contract or Grant No.

Office of Aerospace Medicine

Federal Aviation Administration 13. Type of Report and Period Covered

800 Independence Ave., S.W.

Washington, DC 20591

15. Supplemental Notes

This work was accomplished under the approved task AM-B-02-TOX-204. 14. Sponsoring Agency Code

16. Abstract

During the investigation of aviation accidents, postmortem samples from accident victims are submitted to the

FAA’s Civil Aerospace Medical Institute for toxicological analysis. To determine if the accident victim was exposed

to an in-flight/post crash fire or faulty heating/exhaust system, the analysis of carbon monoxide (CO) is

conducted. While our laboratory predominantly uses a spectrophotometric method for the determination of

carboxyhemoglobin (COHb), we consider it essential to confirm with a second technique based on a different

analytical principle. Our laboratory encountered difficulties with many of our postmortem samples while

employing a commonly used GC method. We believed these problems were due to elevated methemoglobin

(MetHb) concentration in our specimens. MetHb does not bind CO, thus elevated MetHb levels will result in a

loss of CO binding capacity. Since most commonly employed GC methods determine %COHb from a ratio of

unsaturated blood to CO-saturated blood, a loss of CO binding capacity will result in an erroneously high

%COHb value. Our laboratory has developed a new GC method for the determination of %COHb that

incorporates sodium dithionite, which will reduce any MetHb present to Hb. Using numerous fresh human blood

controls ranging from 1% to 67% COHb, we found no statistically significant differences between %COHb

results from our new GC method and our spectrophotometric method. We then applied our new GC method to

putrefied and non-putrefied postmortem samples. To validate the new GC method, postmortem samples were

analyzed with our existing spectrophotometric method, a GC method commonly used without reducing agent,

and our new GC method with the addition of sodium dithionite. As expected, we saw errors up to and exceeding

50% when comparing the unreduced GC results with our spectrophotometric method. With our new GC

procedure, which incorporates a reducing agent, the error was virtually eliminated.

17. Key Words 18. Distribution Statement

Combustion Toxicology, Carbon Monoxide,

Carboxyhemoglobin, Methemoglobin, Aircraft Accident

Investigation

Document is available to the public through

the National Technical Information Service

Springfield, Virginia 22161

19. Security Classif. (of this report) 20. Security Classif. (of this page) 21. No. of Pages 22. Price

Unclassified Unclassified 9

Form DOT F 1700.7 (8-72) Reproduction of completed page authorized

1

AN ACCURATE METHOD FOR THE DETERMINATION OF CARBON MONOXIDE IN

POSTMORTEM BLOOD USING GC/TCD

INTRODUCTION

During the investigation of aviation accidents,

postmortem samples from accident victims are submitted to the FAA’s Civil Aerospace Medical Institute

for toxicological analysis (1). The analysis of carbon

monoxide (CO) is conducted to determine if the

accident victim was exposed to an in-flight/post crash

fire or faulty heating/exhaust system (2, 3, 4).

Carbon monoxide (CO) is a deadly, odorless, tasteless gas that is a product of the incomplete combustion of carbon-containing materials (5). While our

laboratory predominantly uses a spectrophotometric

method for the determination of carboxyhemoglobin

(COHb) (6), we consider it essential to confirm with

a second technique based on a different analytical

principle. The second technique chosen was gas chromatography (GC) with a thermal conductivity detector (TCD).

There are many different GC methods employed

for the determination of CO in blood (7-12). The

most common and rapid GC method determines

%COHb from a ratio of the CO peak area of COunsaturated blood to CO-saturated blood (13). When

working with fresh or well preserved blood samples,

this GC-ratio method is extremely accurate. It does

encounter limitations, however, when analyzing postmortem blood that may have experienced some degree

of deterioration or may have been improperly stored.

It is well known that methemoglobin (MetHb) can

be elevated in a postmortem blood sample. The oxidation of hemoglobin to MetHb (14) can result from

postmortem deterioration due to a lack of timely

preservation (11), exposure to heat (15), and as a

result of the freezing of a blood sample at and above

temperatures of –30°C (16, 17). MetHb does not

bind CO (18). Therefore, elevated levels of MetHb in

a sample will result in a loss of CO binding capacity

and will produce an erroneously high %COHb value.

Dominguez et al. (19) in 1964, when determining

%COHb in various dog tissues, incorporated a reducing agent to reduce any potential MetHb back to

hemoglobin. Dubowski et al. (20) also made reference

to the need for a reducing agent when determining

%COHb in decomposed postmortem blood when

using the GC-ratio method. Clearly, the importance

of using a reducing agent has been demonstrated in an

animal model. This paper describes an improved GC

method for the determination of COHb in human

postmortem blood using a reducing agent to eliminate any error due to substantial MetHb levels.

Materials and Methods

Reagents and Supplies

The chemicals used for these experiments were

sodium dithionite, saponin, sulfuric acid, and ammonium hydroxide, which were obtained from Fisher

Scientific (Fischer Scientific Co., Fair Lawn, NJ).

Distilled-deionized water was prepared using a

Millipore model Milli-QT bench-top purification

device (Millipore, Continental Water Systems, El

Paso, TX) and was used for all reagent preparations.

The reducing agent used for the GC procedure was

0.287 M sodium dithionite. The CO liberating agent

used was 1 M sulfuric acid with 1.5% (w/v) saponin.

The 10 mL headspace vials (MicroLiter Analytical,

Suwanee, GA) were sealed with aluminum crimp caps

fitted with a silicon septa (Supelco, Bellefonte, PA).

GC Method for CO Analysis

The analysis of blood for CO by GC was achieved

using a Varian Micro-GC model CP-2003P fitted

with a thermal conductivity detector. The inlet temperature was 110°C. The GC was equipped with a

Varian 20 m molsieve 5 Å, 0.32 mm ID column

module, which was held isothermally at 120°C. The

carrier gas used was He and the column pressure was

set at 35 psi. A sampling time of 40 sec was used,

followed by a 100 msec injection time. The TCD

sensitivity was set at high for all analyses. The total

chromatographic run time was 195 sec. The GC

system control and data analysis were accomplished

on a Dell Latitude C600 laptop computer using

Varian Star software.

Blood Controls

Blood CO controls were prepared by saturating

human CO-negative blood with 99.95% CO gas in

the following manner. Fifty mL of CO-negative human blood was placed in a 100 mL glass bottle. The

headspace was purged with CO for 5 min. The bottle

was then sealed and rocked front to back on a tube

rocker for 45 min. The bottle was removed, re-purged,

and rocked for an additional 45 min. The bottle was
2

then flushed with He for 5 min. Diluting various

amounts of the CO-saturated blood with CO-negative blood made multiple levels of CO controls, including 16, 25, 35, 50, and 67%. The controls were

stored in vials with zero-headspace to avoid any changes

in blood CO levels (11).

GC Analysis of CO

To determine the amount of CO in a sample, 0.5

mL of unsaturated blood was placed in a 10 mL

headspace vial followed by 0.5 mL of reducing agent,

and the vial was sealed. A different aliquot from the

same sample was used to prepare a CO-saturated

blood by placing 1 mL blood and 1 mL reducing agent

in a 10x75 mm test tube. The headspace was purged

with CO gas for 30 sec; the tube was capped and

placed on a rocker for 30 min. The tube was again

purged with CO for 30 sec and rocked for an additional 30 min. The headspace was then purged with

He for 10 sec. One mL of this saturated blood mixture

was transferred to a 10 mL headspace vial and sealed.

CO was liberated from both saturated and unsaturated blood samples by adding 1 mL of liberating

agent. Following the addition of liberating agent, the

samples were agitated at room temperature on a shaker

(Glas-Col, Terre Haute, In) for 40 min. The samples

were then analyzed using the GC.

Following the analysis, the COHb concentration was

calculated by the ratio of the chromatographic peak areas

from the unsaturated sample and the CO-saturated

sample. The CO peak area of the unsaturated specimen

(CO) was divided by the peak area of the CO-saturated

specimen (SatCO) and then multiplied by 100 to get the

percent concentration of COHb (%COHb=[CO/

SatCO]∗100).

Spectrophotometric Analysis of Carboxyhemoglobin

A detailed description of the spectrophotometric

method used has been previously published (6, 21).

Briefly, the absorption of the blood sample was determined following its dilution with sodium dithionite and

ammonium hydroxide. A UV spectrum of the sample

was collected from 450 to 700 nm. Carboxyhemoglobin

has an absorbance maximum at 540 nm. There is an

isosbestic point at 579 nm through which multiple

species of hemoglobin pass (9). A ratio of the absorbance

at 540 and 579 nm was used to determine the % COHb.

A Hewlett Packard 8453 UV spectrometer was used for

the above analysis.

RESULTS AND DISCUSSION

The GC/TCD method described here is a modified

version of that described by Van Dam et al. (13). The

method provides an excellent separation of CO from

all other common gases, as can be seen in Figure 1.

The thermal conductivity detector, described extensively by Goldbaum et al. (7) and Van Dam et al. (13),

provided superb sensitivity. CO eluted at approximately 2.5 min, thus a total run time of 3.25 min was

used. The CO liberating agent used was 1 M sulfuric

acid with 1.5% (w/v) saponin. Saponin was included

to ensure complete breakdown of all red blood cells in

the sample. Using a wide range of COHb blood

controls, it was determined that a 40 min liberation

time was sufficient for complete CO release. This data

supports what Van Dam et al. (13) have previously

reported.

A reducing agent is commonly employed in the

spectrophotometric analysis of COHb to reduce any

oxyhemoglobin and MetHb present in the blood,

thereby eliminating their potential interference with

the absorbance of COHb (6, 11, 21, 22). Reduction

of blood has also been reported for use with a COoximeter (23). However, to the best of our knowledge,

no one has yet reported the determination of %COHb

in human postmortem blood using the GC-ratio

procedure in conjunction with the use of a reducing

agent. This reduction is extremely important in postmortem specimens, which have the potential to contain high levels of MetHb. This is important because

the commonly employed GC-ratio method determines %COHb from a ratio of CO liberated from

unsaturated blood to CO-saturated blood. Since

MetHb does not bind CO, significant levels of MetHb

in a blood sample will result in a reduction of total CO

binding capacity. This less than complete hemoglobin CO saturation produces an erroneously high

%COHb value. By treating a sample with a reducing

agent, MetHb is converted back to hemoglobin, thus

restoring the total CO binding capacity of the sample.

The appropriate amount of sodium dithionite

needed to reduce any potential MetHb present in

postmortem blood was investigated. Various concentrations of sodium dithionite, ranging from 0.0287 M

to 1.0 M, were used to reduce human blood with a

MetHb level of approximately 40%. These samples

were analyzed using a CO-oximeter to determine the
3

MetHb concentrations (24). The optimum sodium

dithionite concentration was determined to be 0.287

M. Concentrations less than this value did not supply

enough reduction capacity, while concentrations substantially greater than this were found to interfere

with the binding of CO during saturation. Sodium

dithionite at 0.287 M had no effect on existing levels

of COHb. To determine if sodium dithionite had any

effect on the release of CO from a sample, a wide range

of COHb blood controls, both with and without

reducing agent added, were analyzed using the GC.

Comparing the CO area counts between the reduced

and unreduced samples yielded relative errors of less

than 1%. This demonstrates that 0.287 M sodium

dithionite had no effect on the release of CO from a

sample. Additionally, the analysis of a combination of

sodium dithionite and liberating agent alone revealed

no unique peaks.

The new GC method was tested for its accuracy and

precision. The results obtained from the GC were

compared with results obtained from the UV spectrophotometric method we commonly employ in our

laboratory. As seen in Table 1, the GC and UV results

were found to be virtually identical, demonstrating

the accuracy of the GC method. Additionally, the

coefficient of variation for each control level was less

than 10%, indicating good precision.

After showing that 0.287 M sodium dithionite had

no effect on an existing concentration of COHb or the

release of CO from a sample and proving that the

method was both accurate and precise, the GC method

was applied to real postmortem blood samples. Blood

samples from various fatal aviation accident victims

that had previously tested positive for CO were analyzed. The amount of MetHb present in the samples

was determined using a CO-oximeter. The samples

were found to have MetHb values ranging from 5% to

64%. The %COHb in the specimens was then determined using our existing spectrophotometric method

and the GC method with and without reducing agent.

We anticipated that, as the amount of MetHb increased, so would the %COHb error associated with

Figure 1. A representative GC chromatogram showing the separation of CO from

oxygen and nitrogen.

1.0 2.0 3.0

Minutes

0.0

0.5

1.0

1.5

2.0

2.5

Volts

CO (2.486)
4

the GC method not utilizing reducing agent. The

results listed in Table 2 indicate that our hypothesis

was correct. As shown in the table, the GC method

without sodium dithionite experienced errors up to

and exceeding 50% when compared with the spectrophotometric method. However, by incorporating sodium dithionite into the GC method, virtually all

errors between the GC method and the spectrophotometric method were eliminated. This illustrates that

the discrepancy between the %COHb values determined using the GC method without reduction and

the %COHb values determined using the GC method

with reducing agent is directly attributable to the

MetHb present in a sample.

To further illustrate that MetHb accounts for the

%COHb error found between unreduced and reduced samples, we mathematically predicted the unreduced %COHb values using the spectrophotometric

%COHb results and MetHb values. The equation for

predicting %COHb from unreduced samples is: Predicted %COHb = [((UV %COHb)/(100 −

%MetHb))∗100]. As MetHb levels increase, the CO

binding capacity, i.e., the denominator, decreases.

Since %COHb is calculated as a ratio of peak areas of

the unsaturated and saturated samples, if the denominator decreases, the %COHb value increases. For

example, sample number 7 from Table 2 had 22%

COHb, as determined by our spectrophotometric

method. Additionally, the sample contained 52%

MetHb, thus leaving only 48% of the total hemoglobin available for CO saturation. With these values, we

would predict that the unreduced GC method would

yield 47% COHb. The unreduced method actually

gave us 45% COHb. Not only does this show that

MetHb accounts for the error between reduced and

unreduced samples, it exemplifies the potentially significant error caused by not incorporating a reducing

agent when determining %COHb.

Table 1. Accuracy and precision of the GC method compared with the spectrophotometric method.

Method used for Target %COHb

%COHb ± s.d. Neg 16% 25% 35% 50% 67%

GC* 0.9 ± 0.1 16 ± 1 25 ± 2 38 ± 3 54 ± 2 69 ± 5

UV** 1.1 ± 0.4 16.0 ± 0.4 23.7 ± 0.3 32.6 ± 0.4 49.6 ± 0.5 66.7 ± 0.4

* n=5 for both saturated and unsaturated samples.

** n=4

Table 2. Postmortem blood results from aviation accident victims.

Sample

number

UV

%COHb

GC with

reduction

%COHb

GC without

reduction

%COHb

Predicted

unreduced

%COHb

%MetHb

1 27 27 30 29 5

2 37 36 47 44 15

3 7 7 9 9 26

4 17 16 27 26 35

5 29 30 46 47 38

6 41 38 74 73 44

7 22 20 45 47 52

8 7 7 16 18 64
5

CONCLUSION

This study is concerned with the interpretation and

the significance of CO findings in cases where human

postmortem specimens are analyzed. Through the

analysis of blood samples with varying amounts of

MetHb, this GC method has proven to be accurate,

precise, and reliable for the determination of %COHb

in postmortem and antimortem specimens. Admittedly, the GC procedure without reduction can be

used for CO detection; however, it is clear that as

MetHb levels increase in postmortem samples so does

the error introduced in the determination of %COHb.

Therefore, when analyzing postmortem blood using

the GC-ratio method, the inclusion of a reducing

agent is essential in obtaining meaningful and reliable

COHb values.

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