Postmortem Ethanol Testing 

Procedures Available to 

Accident Investigators

Dennis V. Canfield1

Captain James D. Brink2

Robert D. Johnson1

Russell J. Lewis1

Kurt M. Dubowski1

1

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, Oklahoma 73125

2

Investigative Services Crime Laboratory

State Highway Patrol

Columbus, Ohio 43209

August 2007

Final Report

DOT/FAA/AM-07/22

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.

___________

This publication and all Office of Aerospace Medicine 

technical reports are available in full-text from the Civil 

Aerospace Medical Institute’s publications Web site: 

www.faa.gov/library/reports/medical/oamtechreports/index.cfm
i

Technical Report Documentation Page 

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

DOT/FAA/AM-07/22 

4. Title and Subtitle 5. Report Date 

Postmortem Ethanol Testing Procedures Available to Accident Investigators August 2007 

6. Performing Organization Code 

 

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

Canfield DV, Brink JD,* Johnson, RD, Lewis, RJ, Dubowski, KM 

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

FAA Civil Aerospace Medical 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 Aerospace Medicine 

Federal Aviation Administration 

800 Independence Ave., S.W. 

Washington, DC 20591 

*Investigative Services Crime Lab 

State Highway Patrol 

1583 Alum Creek Dr. 

Columbus, Ohio 43209 14. Sponsoring Agency Code 

15. Supplemental Notes 

Work was accomplished under approved task AM- B-06-TOX-204 

16. Abstract 

An ethanol positive fatal case reported as being from ingestion was ultimately determined to be from postmortem 

ethanol production using the ratio of two serotonin metabolites found in urine. This case involved a 

transportation accident that could have resulted in additional hardships for the victim’s family through loss of 

compensation and reputation. 

 

17. Key Words 18. Distribution Statement 

 

 

Forensic Science, Toxicology, Medical, Postmortem Document is available to the public through the 

Defense Technical Information Center, Ft. Belvior, VA 

22060; and the National Technical Information 

Service, Springfield, VA 22161 

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

Unclassified Unclassified 8 

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

iii

Acknowledgments

The authors thank the Montgomery County (Ohio) Coroner’s Office and the Ohio State Highway Patrol’s 

Crime Laboratory for their assistance. We thank the Federal Aviation Administration Office of Aviation 

Accident Investigation and the Office of Aerospace Medicine for sponsoring research into the identification 

of ethanol origin in transportation accidents.

1

Postmortem Ethanol Testing Procedures

Available to Accident Investigators

Case History

On a stormy night in late September 2006, an Ohio 

State Highway Patrol Trooper and a Patrol Sergeant in 

a patrol cruiser were assumed to be en route to assist 

another Trooper whose ill child was being taken to the 

hospital in an emergency situation. While en route, the 

cruiser, traveling at an estimated speed of 61 to 72 mph, 

hydroplaned on the wet highway, spun around, crossed 

the center line, and crashed into a vehicle headed in 

the opposite direction. Both vehicles were engulfed in 

flames, resulting in fatal thermal and trauma injuries 

to all three occupants. Laboratory tests on non-autopsy 

specimens collected at the scene from all victims shortly 

after death by the Ohio State Highway Patrol Crime 

Laboratory (OSPCL) revealed no ethanol in the blood 

of the victims. Autopsies were performed approximately 

34 hours after the crash at a contract autopsy facility on 2 

of the victims. To reduce the decomposition of these two 

victims, formalin powder was utilized. Toxicology results 

from the autopsy found no ethanol present in one victim, 

and the other victim had a BAC of 0.01%. However, the 

autopsy for the driver of the patrol car was delayed by 60 

hours. No form of preservation was used, so the body 

was in an advanced stage of decomposition at the time 

of autopsy. The specimens collected at autopsy from the 

Trooper driving the cruiser revealed 0.07% ethanol in 

urine and 0.08% ethanol in both heart and cavity blood. 

Glucose test strips were negative for glucose in the urine, 

and no vitreous fluid was available for testing. Because of 

the coroner’s report, the Trooper driving the patrol car 

was erroneously reported by the media as being drunk at 

the time of the crash. An investigation found no evidence 

that this officer had consumed ethanol or had acted in an 

intoxicated manner before or during his tour of duty. The 

OSPCL was informed of a laboratory test that could, in 

some cases, differentiate between ingested ethanol and 

ethanol produced postmortem by microbial action. It was 

decided that the specimens would be subjected to this 

test to ascertain whether the officer had indeed recently 

consumed ethanol. This novel technique was developed 

and reported in the peer-reviewed literature by the Federal Aviation Administration’s (FAA’s) Bioaeronautical 

Sciences Research Laboratory in Oklahoma City, OK 

(1). This procedure involves the simultaneous analysis 

of two serotonin metabolites whose ratio is significantly 

altered when ethanol is ingested. Urine from both officers 

was analyzed using this procedure, and in each case, was 

found to have a ratio of 5-Hydroxyindole-3-Acetic Acid 

(5-HIAA) to 5-Hydroxytryptophol (5-HTOL), indicating 

that the ethanol found at autopsy was from postmortem 

ethanol formation and not from ingestion.

Frequency of Postmortem Ethanol Formation

Postmortem ethanol production in human bodies has 

been well documented by many forensic scientists over 

the past 70 years (2-4). A recent postmortem ethanol 

article referenced 323 postmortem ethanol papers (5). 

A study reported in 1993 by the FAA laboratory found 

that postmortem ethanol occurred in 27% of all ethanolpositive aviation accidents, and that the differentiation 

of postmortem ethanol production from ingestion could 

not be determined in 43% of all ethanol positive aviation 

accidents (6). Ethanol ingestion could only be confirmed 

in 30% of all positive aviation ethanol cases.

 

Distribution of Ethanol in Biological Specimens

Knowing vitreous fluid ethanol concentrations are approximately 18% higher than blood ethanol, and urine 

ethanol concentrations are approximately 30% or more 

than the blood ethanol concentrations (assuming the 

existence of the post-absorptive or elimination phase), it 

has been suggested that the best approach to differentiate ethanol ingestion from postmortem ethanol is the 

distribution of ethanol in blood, urine, and vitreous fluid 

(7-9). Vitreous fluid is considered an excellent specimen 

for determining ingested ethanol, even though it may 

contain nutrients (glucose) needed for postmortem ethanol formation, because it is protected by the skull from 

the microorganisms responsible for postmortem ethanol 

production (10). Under normal conditions, urine lacks the 

nutrients required for postmortem ethanol production; 

however, certain medical conditions can cause elevated 

glucose concentrations in urine and increase the chances 

of postmortem ethanol production in urine. 

Trauma as a Factor in Postmortem Ethanol

Postmortem specimen contamination with ethanolproducing microorganisms increases with an increasing 

extent of trauma to the body. Therefore, postmortem 

ethanol formation is far more likely to occur in cases 

involving severe trauma from high speed crashes such 

as aviation accidents. Furthermore, if there is a fire that 

damages the protective dermal layer of the body, there is 
2

an increased opportunity for ethanol producing microorganisms to invade the body and produce postmortem 

ethanol. 

Time as a Factor in Postmortem Ethanol Formation

Time is another factor in the amount of postmortem 

ethanol produced (11); however, due to the many other 

variables that affect the production of ethanol, it is impossible to predict postmortem ethanol production solely 

from the amount of time before specimen collection. In 

one aviation fatality investigated by several of the authors, 

an accident occurred at an airport, and postmortem 

specimens were collected two hours after the accident. 

An ethanol concentration of 0.055% was found in the 

pilot’s blood, but no ethanol was found in vitreous fluid, 

brain, or muscle (Fig. 1). The variability of postmortem 

ethanol formation can also be seen in accidents involving multiple victims where one body forms postmortem 

ethanol and the other does not, even though they were 

in the same accident. 

Nutrients as an Indicator of Postmortem Ethanol 

Production

Urine does not normally contain the nutrients necessary for microbial ethanol production. Tests are routinely 

performed on urine specimens to check for the presence 

of glucose. The presence of glucose may suggest postmortem ethanol, but the absence of glucose does not 

exclude postmortem ethanol because ethanol-producing 

microorganisms can produce ethanol from substrates other 

than glucose (sucrose, mannose, lactose, etc). Further, the 

possibility exists that glucose originally present in the urine 

was consumed by microorganisms prior to the specimen 

being collected or analyzed. In individuals who have 

recently eaten a meal, the nutrients available in the body 

for postmortem ethanol production are increased.

Serotonin Metabolite Ratios as an Indicator of 

Postmortem Ethanol Production

Due to the high rate of postmortem ethanol formation 

in fatal aviation accidents and the frequency at which no 

determination could be made regarding ethanol origin, it 

was found to be advantageous to develop a new analytical 

procedure to determine the origin of ethanol found in 

pilots who had died in an aviation accident. In 1967, it 

was reported that the ratio of two serotonin metabolites, 

5-HIAA and 5-HTOL, were altered by the ingestion of 

ethanol and remain altered for up to 16 hours after the 

individual stopped consuming ethanol (12). Researchers 

discovered that this ratio of serotonin metabolites in urine 

could be used to determine whether a person had recently 

consumed ethanol (13). This ratio was initially used in 

ethanol cessation programs to monitor patients diagnosed 

with alcoholism. Under normal circumstances, 5-HIAA 

is found at concentrations up to 100 times as great as 

5-HTOL. When ethanol is consumed, the production 

of 5-HTOL is favored, and the ratio shifts from 100:1 

(5-HIAA/5-HTOL) up to 60:40 (5-HIAA/5-HTOL). 

The determination of this ratio has been used successfully in aviation accident investigations to differentiate 

between ingested and postmortem ethanol (14). A ratio 

of 5-HTOL/5-HIAA below 15 has proven to be evidence 

that any ethanol found in the urine specimen is from 

postmortem ethanol production and not from ingestion. 

Ratios of 5-HTOL/5-HIAA above 15 usually indicate that 

the person consumed ethanol within 8-12 hours prior to 

death. An elevated ratio does not exclude the possibility 

that the ethanol found in the specimen is from a combination of ethanol ingestion and postmortem ethanol 

production, but a ratio below 15 does exclude ethanol 

ingestion as a source of ethanol in a specimen.

Identification of Ethanol Producing Microorganisms

Procedures have been developed at the FAA Civil 

Aerospace Medical Institute (CAMI) to identify the 

ethanol-producing microorganisms present in a specimen 

containing ethanol. This procedure uses a set of DNA 

probes developed to identify DNA sequences associated 

with microorganisms capable of ethanol production. The 

procedure has been used in aviation fatalities to show 

that ethanol-producing microorganisms were absent or 

present in the specimen (15,16). 

Other Volatiles as Indicators of Postmortem Ethanol 

Formation

It has been proposed that the presence of volatiles other 

than ethanol indicates postmortem ethanol formation 

(17). Research has shown the presence of other volatiles 

to be an indication of specimen putrefaction but not 

necessarily a conclusive indicator of postmortem ethanol 

production (6,18).

Ethanol Concentration as an Indicator of Postmortem Ethanol

Some investigators believe that concentrations of 

ethanol above a certain concentration (0.04%) indicate 

the ingestion of ethanol (19). However postmortem 

ethanol concentrations above 0.350% have been found 

in postmortem specimens taken from aviation accidents. 

Therefore, no conclusions can be made regarding the 

absence or presence of postmortem ethanol based solely 

on the concentration of blood ethanol found. 
3

Figure 1: Postmortem alcohol distribution in a fatal aviation case.
4

Conclusion

Postmortem ethanol production can and does occur in 

fatal accidents. Therefore, care is necessary in investigating 

fatal accidents involving severe trauma to the body or in 

cases where long delays occurred prior to the collection 

of specimens for toxicological analysis. When abnormal 

ethanol distribution is found in blood, urine, and vitreous 

fluid, further testing may be warranted using serotonin 

metabolite ratios. Finding elevated blood ethanol concentrations but no ethanol in vitreous fluid and urine 

should be reported as negative for ingested ethanol and 

should not require further testing. It is important to realize that an incorrect finding of “intoxication,” when the 

person did not consume ethanol, can result in extreme 

hardship for the family of the deceased through loss of 

pension, workers compensation, life insurance, and the 

reputation of the individual.

REFERENCES

1. R.D. Johnson, R.J. Lewis, D.V. Canfield, et al. 

Accurate assignment of ethanol origin in postmortem urine: Liquid chromatographic-mass spectrometric determination of serotonin metabolites. J 

Chromatogr B Analyt Technol Biomed Life Sci 

805:223-34 (2004).

2. D.J. Blackmore. The bacterial production of ethyl 

alcohol. J Forensic Sci Soc 8:73-8 (1968).

3. M. Nicloux. Neoformation d’alcool ethylique 

dans le cadavre humain en voie de putrefaction. 

Compte Rendu des Seances de la Societe de Biologie 

121:975-8 (1936).

4. J.E. Corry. A review. Possible sources of ethanol 

ante- and post-mortem: its relationship to the 

biochemistry and microbiology of decomposition. 

J Appl Bacteriol 44:1-56 (1978).

5. F.C. Kugelberg and A.W. Jones. Interpreting results 

of ethanol analysis in postmortem specimens: A 

review of the literature. Forensic Sci Int 165:10-29 

(2007).

6. D.V. Canfield, T. Kupiec, and E. Huffine. Postmortem alcohol production in fatal aircraft accidents. 

J Forensic Sci 38:914-7 (1993).

7. A.W. Jones and P. Holmgren. Uncertainty in estimating blood ethanol concentrations by analysis 

of vitreous humour. J Clin Pathol 54:699-702 

(2001).

8. B.E. Stone and P.A. Rooney. A study using body 

fluids to determine blood alcohol. J Anal Toxicol 

8:95-6 (1984).

9. A.W. Jones. Urine as a biological specimen for 

forensic analysis of alcohol and variability in the 

urine-to-blood relationship. Toxicol Rev 25:15-35 

(2006).

10. Y.H. Caplan and B. Levine. Vitreous humor in the 

evaluation of postmortem blood ethanol concentrations. J Anal Toxicol 14:305-7 (1990).

11. R J. Lewis, R.D. Johnson, M.K. Angier, et al. 

Ethanol formation in unadulterated postmortem 

tissues. Forensic Sci Int 146:17-24 (2004).

12. V.E. Davis, H. Brown, J.A. Huff, et al. The alteration 

of serotonin metabolism to 5-hydroxytryptophol by 

ethanol ingestion in man. J Lab Clin Med 69:132-

40 (1967).

13. A. Helander, O. Beck, and A.W. Jones. Urinary 

5HTOL/5HIAA as biochemical marker of postmortem ethanol synthesis. Lancet 340:1159 

(1992).

14. R.D. Johnson, R.J. Lewis, D.V. Canfield, et al. 

Utilizing the urinary 5-HTOL/5-HIAA ratio to 

determine ethanol origin in civil aviation accident 

victims. J Forensic Sci 50:670-5 (2005).

15. D.M. Kupfer, A.K. Chaturvedi, D.V. Canfield, et al. 

PCR-based identification of postmortem microbial 

contaminants--a preliminary study. J Forensic Sci 

44:592-6 (1999).

16. N.T. Vu, A.K. Chaturvedi, D.V. Canfield, et 

al.DNA-based detection of ethanol producing 

microorganisms in postmortem blood tissues by 

polymerase chain reaction. Federal Aviation Administration. Report No. DOT/FAA/AM-00/16, 

2000.

17. K. Ziavrou, V.A. Boumba, and T.G. Vougiouklakis. 

Insights into the origin of postmortem ethanol. Int 

J Toxicol 24:69-77 (2005).

18. D. Yajima, H. Motani, K. Kamei, et al. Ethanol 

production by Candida albicans in postmortem 

human blood samples: Effects of blood glucose 

level and dilution. Forensic Sci Int 164:116-21 

(2006).

19. B. Levine, M.L. Smith, J.E. Smialek, et al. Interpretation of low postmortem concentrations of 

ethanol. J Forensic Sci 38:663-7 (1993).