SELECTION OF AN INTERNAL STANDARD
For PostMorreM ETHANOL ANALYSIS

INTRODUCTION

The Office of Aviation Medicine, Civil Aero-
medical Institute (CAMI) is required under Public
law 100-591 to help assess the role of potential
medical or drug related pilot impairment in avia-
tion accidents. This includes the identification and
quantitation of various alcohols in addition to ac-
etaldehyde and acetone found in postmortem speci-
mens. The laboratory has tested 2880 specimens
from fatal pilots over the past 8 years for the
presence of alcohols (Table 1).

The CAMI Forensic Toxicology Laboratory has
characteristically used n-propanol as an internal
standard, using headspace gas chromatography
analysis, for the identification and quantitation of
various alcohols, acetaldehydes, and ketones that
are typically found in postmortem specimens. How-
ever, various studies have reported the presence of n-

propanol in some postmortem specimens contain-
ing ethanol.®3>'® According to Fumio Moriya,
“n-propanol, which is produced with ethanol post-
mortem, can be an indicator of postmortem ethanol
production because normally it does not exist in the
living body”.'® This compound is the most com-
monly reported “other volatile associated with post-
mortem synthesis of ethanol.”'® The finding of
n-propanol in postmortem specimens logically sug-
gested a new internal standard be considered for
ethanol analysis in postmortem specimens. The
following criteria were emphasized in selecting an
improved internal standard: (1) The chemical prop-
erties of the internal standard must be similar to the
chemical properties of the compounds being quan-
titated or separated; (2) The retention time of the
internal standard should be in the middle range of

Table 1. Positive alcohol cases for pilots involved in fatal accidents

for the past 8 years.
Fatal Pilots with % of the Total Fatal
Year 40mg/dL of Total Fatalities Pilots
ethanol or more
1989 28 8.0 349
1990 29 7.9 367
1991 30 7.7 389
1992 29 7.3 400
1993 30 8.8 340
1994 24 6.9 347
1995 15 43 352
1996 28 8.3 336
Total 213 7.4 2880



DISCUSSION & CONCLUSION

One of the three candidates proposed as a new
internal standard best met the criteria established
for this study. Tert-butanol is not found in post-
mortem specimens, has a retention time and peak
area similar to ethanol, has acceptable baseline
separation from other components, and does not
degrade with time. With t-butanol as an internal
standard, the mean concentration for a 150 mg/dL
ethanol standard was found to be 149.75 mg/dL,
and the calculated standard deviation is 2.24. This
is even better than the 3.48 standard deviation
found for n-propanol.

Propionaldehyde was eliminated from consider-
ation because it did not have acceptable baseline
separation from compounds commonly found in
postmortem samples. Propionic acid methyl ester
was not found in postmortem specimens, had
baseline separation, and had a retention time and
peak area comparable to ethanol. Problems arose
when the concentration of propionic acid methyl
ester was found to decrease steadily with time.
Propionic acid methyl ester is not a suitable internal
standard for the analysis of ethanol because it
degrades over time.

In summary, since t-butanol best met our selec-
tion criteria, t-butanol will be the future routine
internal standard for use in the quantification of
ethanol in postmortem specimens.

REFERENCES:

1. Alha AR, Tamminen V (1971). Detection of
Alcohol in Aviation and Other Fatalities in Fin-
land. Aerospace Med, 42, 564-8.

2. Briglia EJ, Bidanset JH, Dal Cortivo LA (1992).
The Distribution of Ethanol in Postmortem
Blood Samples. J Forensic Sci, 37, 991-8.

3. Blackmore DJ (1968). The Bacterial Production
of Ethyl Alcohol. J Forensic Sci Soc, 8, 73-8.

4. Canfield DV, Kupiec TC, Huffine E (1993).
Postmortem Alcohol Production in Fatal Aircraft
Accidents. J Forensic Sci, 38, 914-7.

5. Caughlin JD (1994). An Unusual Source for
Postmortem Findings of Methyl Ethyl Ketone
and Methanol in Two Homicide Victims. Foren-

sic Sci Int, 67, 27-31.

12.

13.

14.

15.

16.

17.

Chikasue F, Yashiki M, Miyazaki T, Okamotoa
I, Kojima T (1988). Abnormally High Alcohol
Concentration in the Heart. Forensic Sci Int, 39,
189-95.

Christmore D, Kelly RC, Doshier L (1984).
Improved Recovery and Stability of Ethanol in
Automated Headspace Analysis. J Forensic Sci,
29, 1038-44.

Davis G, Leffert R, Cpt. Rantanen N (1972).
Putrefactive Ethanol Sources in Postmortem Tis-
sues of Conventional and Germ-Free Mice. Arch
Pathol, 94, 71-4.

Felby S, Nielsen E (1993). Postmortem Blood
Alcohol Concentrations. Blutalkohol, 30,
244-50.

Gilliland MGF, Bost RO (1993). Alcohol in
Decomposed Bodies: Postmortem Synthesis and
Distribution. J Forensic Sci, 38, 1266-74.

Glendening B, Harvey R (1969). A Simple
Method Using Head-Space Gas for Determina-
tion of Blood Alcohol by Gas Chromatography.
J Forensic Sci, 14, 136-45.

Kuhlman JJ, Levine B, Smith ML, Hordinsky JR
(1991). Toxicological Findings in Federal Avia-
tion Administration General Aviation Accidents.
J Forensic Sci, 36, 1121-8.

Moriya F, Ishizu H (1994). Can Microorgan-
isms Produce Alcohol in Body Cavities of a Living
Person?: A Case Report. J Forensic Sci, 39, 883-8.

Nanikawa R, Kotoku S (1974). Medicolegal
Observations on a Dead Body Drawn Up From
the Sea Bed, with Special Reference to Ethanol
and Diatoms. Forensic Sci, 3, 225-32.

Nanikawa R, Ameno K, Hashimoto Y, Hamada
K (1982). Medicolegal Studies on Alcohol De-
tected in Dead Bodies - Alcohol Levels in Skeletal
Muscle. Forensic Sci Int, 20, 133-40.

O’Neal C, Poklis A (1996). Postmortem Pro-
duction of Ethanol and Factors that Influence
Interpretation. Am J Forensic Med Pathol, 17, 8-20.

Parker K, Fontan C, Yee J, Kirk P (1962). Gas
Chromatographic Determination of Ethyl Alco-
hol in Blood for Medicolegal Purposes. Anal
Chem, 34, 1234-6.


* RUN #3 APR 16> 1996 10:185:33
START
| 6.688 ACETALDEHYDE
8.988
Cr apn vet. PROPIONALDEHYDE

———
——— 2.489 ETHANOL

1.845 ACETONE

3.675 ISOPROPANOL

NIC ACID METHYL, ESTER

8.28 SEC-BUTANOL

9.258 ISOBUTANOL

on
Oo
an

sToP

9.930 N-BUTANOL

Figure 1. Gas Chromatograph of a standard test mix with 3 internal standards
present. t-butanol was left out of test mix because it elutes at the same time as

propionic acid methyl ester.

had a decreased peak area over time. This decrease
in peak area for the internal standard resulted in
what appeared to be an increasing concentration of
ethanol with time for the same 150mg/dL control.

The t-butanol internal standard has a retention
time of 4.86 minutes (Figure 2). It had acceptable
baseline separation from the other components of
the test mix. Twenty runs were made with a known
concentration of ethanol of 150 mg/dL and the t-
butanol (Table 2). The mean for the 150mg/dL
concentration of ethanol was 149.75 mg/dL with a
standard deviation of 2.24.

The n-propanol internal standard has a retention
time of 5.73 minutes (Figure 2), which is not in the
mid range of the retention times for the compounds

of interest. N-propanol had acceptable baseline
separation from the other components of the test
mix. Twenty runs were made with a known concen-
tration of ethanol of 150 mg/dL and the n-propanol
(Table 2). The mean for the 150mg/dL concentra-
tion of ethanol was 149.9 mg/dL with a standard
deviation of 3.48.

Although n-propanol has baseline separation from
other compounds found in postmortem specimens,
naturally occurring n-propanol found in postmor-
tem specimens would interfere with the use of n-
propanol as an internal standard. In contrast, no
t-butanol was found in an examination of 2880 fatal
pilots analyzed by the laboratory over the past 8
years (Table 1).


the retention times of the compounds being sepa-
rated; (3) The internal standard must have baseline
separation from all components of the mixture.

Considering these criteria, propionaldehyde, pro-
pionic acid methyl ester, and t-butanol were se-
lected as potential replacements for the n-propanol
internal standard. Propionaldehyde and propionic
acid methyl ester were favorable choices because
their retention times are close to the retention time
of ethanol. The t-butanol was a favorable choice
because it had alcohol chemical properties and a
retention time in the middle range of those found in
the test mix.

MATERIALS & METHODS

The gas chromatograph (GC) was an HP 5890
series II gas chromatograph with an FID detector,
equipped with HP 19395A Headspace Sampler.
The GC column was a 60/80 Carbopack B, 5%
Carbowax 20, 6 foot X %-inch OD glass-packed
column. The GC oven temperature was initially
65°C for 6.5 minutes, ramping at 20°C/min. to a
final temperature of 140°C and held for 2 minutes
at this temperature. The GC had an injection
temperature of 150°C and a detector temperature of

170°C.

Preparation of “Working Test Mix”
The following test mix was prepared from a stock
solution:

Volatile Component Concentration (mg/dL)

Acetaldehyde 31.33 mg/dL
Methanol . 158.28 mg/dL
Acetone... 31.59 mg/dL
Ethanol...... - 157.46 mg/dL
Isopropanol - 157.10 mg/dL
n-Propanol - 241.25 mg/dL
sec-Butanol 80.00 mg/dL
Isobutanol . 32.76 mg/dL
n-Butanol . 80.95 mg/dL

Preparation of Internal Standards

A 5.04 mg/dL solution of propionaldehyde in-
ternal standard was prepared. This reagent was
stored at 2 to 8°C. A 17 mg/dL propionic acid
methyl ester internal standard was prepared. This

reagent was stored at 2 to 8°C. A 39.43 mg/dL t-
butanol internal standard was prepared and stored
at 2 to 8°C.

Preparation of Samples

Five hundred mL of the 17 mg/dL propionic acid
methyl ester or t-butanol internal standard solution
and 500 mL of the ethanol standard (150 mg/dL) or
Working Test Mix were pipetted into an appropri-
ately labeled 10 mL glass reaction vial. The vial was
sealed immediately after the addition of the Work-
ing Test Mix or ethanol standard. This was repeated
for all remaining vials. All specimens were vortexed
to ensure all materials were well mixed. The
propionaldehyde was rejected as an internal stan-
dard because it did not have baseline separation
from compounds commonly found in postmortem
specimens.

After calibration of the instrument with an inter-
nal standard, the analysis was run with known
ethanol concentration of 150 mg/dL. This format
was used for each run:

Well Position Sample Type

Wells 1-3 Internal Standard & Working Test
Mix

Wells 4-24 — Internal Standard & 150 mg/dL

Ethanol Standard

Analysis of Prior Year Case Reports

The results of 2880 cases, analyzed over the past 8
years, were examined for the presence of specific
compounds detected in postmortem specimens, with
particular emphasis on t-butanol.

RESULTS

Propionaldehyde had a retention time of 1.63
min. (Figure 1) and did not have baseline resolution
from methanol and acetone. It eluted at the same
retention time as an unidentified peak commonly
seen in postmortem specimens. It had a short
retention time relative to the compounds of interest.

Propionic acid methyl ester, with a retention
time of 4.49 minutes, did resolve from the peaks of
interest and had a retention time in the mid range
of the compounds of interest (Figure 1). However,
this internal standard was found to be unstable and


Table 2. Comparisons of n-Propanol and t-Butanol internal
standards.

Sample n-Propanol t-Butanol | n-Propanol

1 144 146 Mean 149.9
2 150 146 sD 3.48
3 152 148 N 20
4 150 147 cv 2.32
5 152 147 VAR 12.09
6 149 151
7 147 151 t-Butanol
8 148 153 Mean 149.75
9 156 152 sD 2.24
10 150 151 N 20
1 149 152 CV 1.50
12 154 150 VAR 5.04
13 154 151
14 153 151
15 145 150
16 148 150
17 148 148
18 146 147
19 156 152
20 147 152

SUM 2998 2995



DOT/FAA/AM-98/5

Office of Aviation Medicine
Washington, D.C. 20591

Selection of an Internal

Standard for Postmortem
Ethanol Analysis

Dennis V. Canfield
Moraine D. Smith
Heather J. Adam

Eric R. Houston

Civil Aeromedical Institute

Federal Aviation Administration
Oklahoma City, OK 73125

February 1998

Final Report

This document is available to the public
through the National Technical Information
Service, Springfield, Virginia 22161.

@

U.S. Department
of Transportation

Federal Aviation
Administration




18.

19.

Takayasu T, Ohshima T, Tanaka N, Maeda H,
Kondo T, Nishigami J, Nagano T (1995). Post-
mortem Degradation of Administered Ethanol-
d6 and Production of Endogenous Ethanol:
Experimental Studies Using Rats and Rabbits.
Forensic Sci Int, 76, 129-40.

Winek T, Winek CL, Wahba WW (1996). The
Effect of Storage at Various Temperatures on
Blood Alcohol Concentration. Forensic Sci Int,
78, 179-85.

20. Zumwalt RE, Bost RO, Sunshine I (1982).

21.

Evaluation of Ethanol Concentrations in De-

composed Bodies. J Forensic Sci, 27, 549-54,

O’Neal, CL, Wolf, CE II, Levine, B, Kunsman,
G, and Poklis, A (1996). Gas chromatographic
procedures for the determination of ethanol in
postmortem blood using t-butanol and methyl
ethyl ketone as internal standards. Forensic Sci
Int, 83, 31-8.

7  ¥US. GOVERNMENT PRINTING OFFICE: 1998 | -660-088/60058


1.460 Methanol

‘a 2.471 Ethanol

ae 4.860 tertButanol

0.895 Acetaldehyde
laa
<=

1.841
Acetone

3.640 Isopropanol

TM §.798 n-Propanot

7.975 sec-Butanol

9.224 Isobutanol

9.900 n-Butanol

11.213

11.826
STOP

Figure 2. Gas Chromatograph of a standard test mix with t-butanol and n-propanol
present.




Technical Report Documentation Page

T. Report No. 2. Government Accession No. 3. Recipient's Catalog No.
DOT/FAA/AM-98/5

4, Title and Subtitle 5. Report Date
Selection of an Internal Standard for Postmortem Ethanol Analysis February 1998

6. Performing Organization Code

7. Author(s) 8. Performing Organization Report No.

Canfield, D.V., Smith, M.D., Adams, H.J., and Houston, E.R.

9. Performing Organization Name and Address 10. Work Unit No. (TRAIS)

FAA Civil Aeromedical Institute

P.O. Box 25082, 11. Contract or Grant No.
Oklahoma City, OK 73125

12. Sponsoring Agency name and Address 13. Type of Report and Period Covered
Office of Aviation Medicine

Federal Aviation Administration 14. Sponsoring Agency Code

800 Independence Ave., S.W.
Washington, DC 20591

15. Supplemental Notes

‘Work was accomplished under approved task AM-B-96-TOX-202

16. Abstract

Introduction: One mission of the Civil Aeromedical Institute is to determine the concentrations of alcohol in
postmortem specimens related to aviation accidents. This requires the ability to identify and quantitate a wide range of
alcohols that are produced in postmortem specimens. A headspace gas chromatographic procedure utilizing n-propanol
as an internal standard had been used in the past. However, n-propanol has been found in postmortem specimens,
making n-propanol an unsuitable specimen for an internal standard in the analysis of postmortem specimens. This study
evaluated 3 potential replacement internal standards for postmortem ethanol analysis. Method: A mixture of alcohols
commonly found in postmortem specimens was prepared and tested using headspace gas chromatography. Solutions
were prepared using the test mix and the new internal standards. Data were collected on the resolution and
reproducibility of the proposed new internal standards with the test mix. Postmortem cases collected over the past 8
years were reviewed for the presence of specific volatile compounds. Results: Baseline resolution from the test mix was
not obtained with propionaldehyde, while propionic acid methyl ester exhibited degradation over time. T-butanol was
found to give baseline resolution from all volatile compounds commonly found in antimortem and postmortem
specimens. No t-butanol was found in 2880 fatal pilots analyzed over the past 8 years for the presence of volatiles.
Conclusion: t-butanol is a better internal standard for the analysis of alcohols in postmortem specimens than
propionaldehyde, n-propanol, and propionic acid methy] ester, and is not produced in postmortem specimens.

17. Key Words 18. Distribution Statement

Alcohol, analysis, quantitation, aviation accidents Document is available to the public through the
National Technical Information Service,
Springfield, Virginia 22161

19. Security Classif. (of this report) 20. Security Classi. (ofthis page) 21. No. of Pages 22. Price
Unclassified Unclassified 10

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



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