Office of Aerospace Medicine

Washington, DC 20591

Accurate Assignment of Ethanol 

Origin in Postmortem Urine: 

A Case Study

Robert D. Johnson

Russell J. Lewis

Dennis V. Canfield

Kurt M. Dubowski

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, Oklahoma 73125

C. LeRoy Blank

Department of Chemistry and Biochemistry

University of Oklahoma

Norman, Oklahoma 73019

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, OK 73125

August 2004

Final Report

This document is available to the public

through the National Technical Information

Service, Springfield, Virginia 22161.

DOT/FAA/AM-04/13
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. 

����������������

4. Title and Subtitle 5. Report Date 

������������������������������������������������������������������������ ������������

7. Author(s) 6. Performing Organization Code 

����������������������������������������������������������������������������

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

��������������������������������������

��������������� 10. Work Unit No. (TRAIS) 

������������������������

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

13. Type of Report and Period Covered 

�����������������������������

��������������������������������

����������������������������

��������������������� 14. Sponsoring Agency Code 

15. Supplemental Notes 

��������������������������������������������������������������������

16. Abstract 

��������������������������������������������������������������������������������������������������������������

����������������������������������������������������������������������������������������������������������������

������������������������������������������������������������������������������������������������������������

�����������������������������������������������������������������������������������������������������

�������������������������������������������������������������������������������������������������������

�����������������������������������������������������������������������������������������������

������������������������������������������������������������������������������������������������������

�����������������������������������������������������������������������������������������������������������

����������������������������������������������������������������������������������������������������������

����������������������������������������������������������������������������������������������

��������������������������������������������������������������������������������������������������������

����������������������������������������������������������������������������������������������������

�����������������������������������������������������������������������������������������������������������

������������������������������������������������������������������������������������������������������������������

������������

17. Key Words 18. Distribution Statement 

�������������������������������������� ��������������������������������������������� �

���������������������������������������������� �������������������������������������������

�������������������������������������� ����������������������

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

������������� ������������� ���

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

1

ACCURATE ASSIGNMENT OF ETHANOL ORIGIN

IN POSTMORTEM URINE: A CASE STUDY

INTRODUCTION

The microbial formation of ethanol in postmortem 

specimens is by far the most likely complication encountered when examining ethanol results. The first 

report dealing with postmortem formation of ethanol 

in corpses appeared in 1936.1 That report established 

that postmortem presence of ethanol in itself could not 

be used as proof of antemortem ingestion. Today, it is 

known that many different microbes are responsible for 

postmortem formation of ethanol.2 Investigations have 

been performed to identify particular species of bacteria, 

yeast and/or fungi responsible for ethanol production 

and the mechanism by which it is formed.3-6 Candida 

albicans has been the microbe most often ascribed to 

be responsible for postmortem production of ethanol 

in humans.7, 8 This species of yeast is commonly found 

in humans in vivo.

9

 However, approximately 100 species 

of bacteria, yeast, and fungi have been shown capable 

of producing postmortem ethanol.3 Glucose is the 

most prevalent substrate in the human body utilized by 

ethanol-forming microbes to produce ethanol.10 Other 

endogenous compounds can also be utilized as substrates 

including, but not limited to, lactate, mannitol, galactose, 

maltose, sucrose, and lactose.10-13 

Possible exploitation of the metabolism of serotonin 

as a biological marker for ethanol consumption began 

to gain considerable interest in the past decade.14-16 The 

metabolism of 5-HT initially involves oxidative deamination to form the transitory intermediate, 5-hydroxyindole-3-acetaldehyde (5-HIAL). This intermediate can 

undergo either oxidation or reduction, as shown in Figure 

1. Oxidation of the aldehyde, catalyzed by aldehyde 

dehydrogenase, leads to formation of 5-hydroxyindole3-acetic acid (5-HIAA), the predominant metabolite of 

5-HT.16, 17 Reduction, catalyzed by aldehyde reductase, 

leads to formation of 5-hydroxytryptophol (5-HTOL), 

usually a relatively minor metabolite of 5-HT.18 However, 

ethanol consumption has been shown to lead to a relatively 

significant enhancement of 5-HTOL production.

An increase in 5-HTOL concentration following ethanol consumption was first reported in 1967.19 Since that 

time, it has been clearly demonstrated that consumption of ethanol shifts 5-HT metabolism to promote 

formation of 5-HTOL and, some reports indicate, to 

reduce the formation of 5-HIAA.18, 20-22 Thus, ingested 

ethanol leads to an elevation in the 5-HTOL/5-HIAA 

ratio. The 5-HTOL/5-HIAA ratio has been reported 

to remain elevated for six to eight hours after ingested 

ethanol has been eliminated from the body.18, 21, 23 The 

5-HTOL/5-HIAA ratio has already been applied to 

ethanol cessation monitoring programs as a marker of 

recent ethanol ingestion.23-25 The 5-HTOL/5-HIAA ratio 

has also been briefly investigated for possible use with 

postmortem urine samples.21

Historically, concentrations of 5-HTOL and 5-HIAA 

in individual samples have been measured using two 

completely different analytical techniques. 5-HIAA 

concentrations are typically measured at sub-nanomolar 

concentrations using liquid chromatography with electrochemical detection (LC/EC).26, 27 5-HTOL can also be 

measured by LC/EC, but the detection limits are typically 

insufficient to measure this compound in most pertinent biological specimens. For that reason, 5-HTOL has 

routinely been analyzed using gas chromatography with 

mass spectrometric detection (GC/MS) following derivitization with pentafluoroproprionic acid anhydride.28

The employment of two different analytical techniques 

to obtain the 5-HTOL/5-HIAA ratio in a specimen 

obviously decreases the precision and reliability of the 

final result. Recently, our laboratory developed a single 

analytical method for the simultaneous determination of 

both 5-HTOL and 5-HIAA using liquid chromatography 

with mass spectrometric detection (LC/MS).14 

Our laboratory evaluated five ethanol-positive fatalities from separate general aviation accidents in which 

the source of the ethanol present could not conclusively 

be attributed to either antemortem ethanol consumption or postmortem microbial ethanol formation. 

Utilizing this newly developed analytical technique 

for the determination of the 5-HTOL/5-HIAA ratio, 

the origin of the ethanol present in each of these cases 

was investigated.

MATERIALS AND METHODS

Chemicals and Solutions

All aqueous solutions were prepared using double 

deionized water (DDW), which was obtained using a 

Milli-QTplus Ultra-Pure Reagent Water System (Millipore®, Continental Water Systems, El Paso, TX). All 

chemicals were purchased in the highest available purity and used without any further purification. Sodium 

chloride, sodium acetate, acetic acid, β-glucuronidase, 
2 

HO

N

H

NH2

HO

N

H

H

O

HO

N

H

HO

N

H

OH

OH O

Serotonin

Monoamine oxidase 

5-HIAL 

5-HTOL 5-HIAA 

Aldehyde reductase Aldehyde dehydrogenase 

Figure 1. The Metabolism of serotonin.

5-hydroxytryptophol, 5-hydroxyindole-3-acetic acid, and 

5-methoxy-2-methyl-3-indoleacetic acid were purchased 

from Sigma Chemical Company (St. Louis, MO). Methanol, acetonitrile, ammonium hydroxide, hydrochloric 

acid, ethyl acetate, and nitric acid were purchased from 

Fisher Scientific (Pittsburgh, PA). Formic acid (97%) was 

purchased from ICN (ICN Biomedicals, Inc., Irvine, CA). 

N,O-bis[trimethylsilyl]trifluoroacetamide with 1% trimethylchlorosilane (BSTFA/1%-TMCS) was purchased 

from Pierce Chemicals (Rockford, IL). All acetate buffers 

described below were prepared by first making stock solutions of the desired final concentration of both sodium 

acetate and acetic acid. One of these was then titrated 

with the other until the desired pH was achieved. The 

pH of all solutions was measured using a Corning model 

430 pH meter (Corning Life Sciences, Acton, MA) with 

a Corning 3-in-1 model pH electrode.

Two separate 10 mL stock solutions of both 5-HTOL 

and 5-HIAA were prepared independently at 1.00 mg/mL 

(5.64 mM 5-HTOL; 5.23 mM 5-HIAA) in methanol. 

Each of these stock solutions was derived from a unique 

lot of dry chemical obtained from the manufacturer. 

These two stock solutions were subsequently identified 

as calibrators and controls. 5-Methoxy-2-methyl-3-indoleacetic acid (5-MMIA), a compound not known to 

occur in vivo, was used as the internal standard for these 

experiments and was prepared at a concentration of 100 

μg/mL (0.450 mM) in 10 mL of methanol. These indolic 

compounds are light sensitive, so care was taken to use 

volumetric flasks wrapped in aluminum foil to prevent 

photodegradation. Once prepared, the solutions were 

transferred to 20 mL amber glass bottles, capped, and 

placed in freezer storage at -20°C. While these solutions 

are stable for at least 180 days,14 for maximum assurance 

of the quality of data, we used no stock solutions that 

were over 30 days old. 

Since 5-HTOL in human-derived specimens is 

predominately found as the glucuronide derivative, we 

initially hydrolyzed the samples using the enzyme β-glucuronidase. A solution of β-glucuronidase was prepared 
3

by adding 2.5 mL of pH 5.00, 0.10 mM sodium acetate 

buffer to 250,000 units of the solid enzyme and mixing 

to dissolve. This yielded a final concentration of 100,000 

units/mL. This solution was stored at -20°C. Like the 

stock standards, this solution was discarded after storage 

for a maximum of 30 days. However, it typically was used 

entirely within 7 days following preparation.

The aqueous portion of the HPLC buffer was 50.0 mM 

formic acid adjusted to pH 5.00 with conc. ammonium 

hydroxide. Aqueous buffer and acetonitrile were mixed 

in a 98:2 ratio, respectively, to help prevent the growth 

of microbes, and this mixture was filtered through a 

vacuum filtering apparatus that incorporated a 0.45 μm 

GH polypro 47 mm hydrophilic, polypropylene membrane filter obtained from Pall Gelman laboratory (Pall 

Corp., East Hills, NY). The primary organic component 

of the mobile phase was HPLC grade methanol, which 

was filtered prior to use through a vacuum filter apparatus 

that incorporated the same type of membrane filter. The 

ratio of the previous aqueous mixture to methanol was 

20:80 in the final HPLC mobile phase.

Instrumentation

Analyte separation was achieved using a Hewlett 

Packard 1100 HPLC (Hewlett Packard Co., Wilmington, DE) equipped with a Security Guard C-8 guard 

column (4.0 mm x 3.0 mm i.d., 3 μm particles) from 

Phenomenex (Torrance, CA), followed immediately by 

a Supelcosil LC-18 (150 mm x 4.6 mm i.d., 3 μm particles) analytical column from Supelco (Supelco/SigmaAldrich, Bellefonte, PA). Samples were injected using 

a Hewlett Packard G1313A autosampler. Identification 

and quantitation were accomplished using a Finnigan 

model LCQ atmospheric pressure chemical ionization 

(APCI) ion trap mass spectrometer (ThermoFinnigan 

Corp., San Jose, CA), which utilized nitrogen as the 

sheath gas and helium as the reagent gas. Control of 

the HPLC system, integration of the chromatographic 

peaks, and communication with the mass spectrometer 

were accomplished using a Gateway 2000 E-4600-SE 

personal computer using Xcalibur LC/MS software 

(ThermoFinnigan Corp., San Jose, CA).

LC/MS/MS and LC/MS/MS/MS Methods

The analytical method employed for these experiments 

was described in detail by Johnson et al.14 In brief, the 

method employed was as follows. For all determinations, 

the HPLC was operated in an isocratic mode with a 

flow rate of 1.0 mL/min. The sample injection volume 

was 10 μL. Detection by the ion-trap mass spectrometer 

was divided into three unique segments, with each segment dedicated to the detection of a specific compound. 

Segment 1 was used for analysis of 5-MMIA, segment 2 

analyzed for the TMS derivative of 5-HTOL, and segment 

3 analyzed for the TMS derivative of 5-HIAA.

Specimen Preparation and Extraction

Calibration curves were prepared by dilution utilizing 

human certified negative urine as the diluent. Human 

certified negative urine, as obtained from the manufacturer 

(UTAK Laboratories Inc., Valencia, CA), is guaranteed 

to be free of any artificial pharmaceutical compounds 

and abnormal organic volatiles. Through the course of 

our initial investigations, this processed urine was also 

found to be negative for both 5-HTOL and 5-HIAA. 

The calibrators were prepared from one set of the original stock standard solutions of 5-HTOL and 5-HIAA. 

Controls were prepared in a similar manner to calibrators, using the same human certified negative urine as 

diluent, but employing the second set of original stock 

solutions. Calibrators, controls, and postmortem urine 

specimens, all simply referred to below as samples, were 

prepared and extracted in the following manner. 

Three mL aliquots of individual samples were transferred to 16 x 150 mm screw-topped culture tubes. To 

each sample, 1.00 mL of a 1000 ng/mL (4.50 μM) internal standard solution, prepared by dilution of its stock 

solution with water, was added. β-Glucuronidase solution 

(7500 units; 75 μL) followed by 1.00 mL of 0.10 mM 

pH 6.00 sodium acetate buffer was added to each sample. 

The samples were vortexed briefly and incubated at 70°C 

for 45 minutes to facilitate hydrolysis of the glucuronide 

conjugate. In our initial investigations, complete hydrolysis of the 5-HTOL-glucuronide conjugate was achieved 

after incubation with β-Glucuronidase for 30 minutes at 

70°C. However, an incubation time of 45 minutes was 

chosen to ensure that specimens with extremely elevated 

5-HTOL concentrations were also completely hydrolyzed. 

Following hydrolysis, samples were allowed to cool to 

room temperature. Then 2.00 mL of a 0.10 M sodium 

acetate buffer, pH 6.00, and 0.50 mL of a saturated sodium chloride solution were added to each sample, and 

the tubes were briefly vortexed. Ethyl acetate (9.00 mL) 

was added to each tube, and the tube was tightly capped. 

The mixture was then placed on a rotary mixing wheel 

and gently mixed for 20 minutes by simple rotation of 

the wheel at 6 rpm. Following mixing, the samples were 

centrifuged at 820xg for 5 minutes. The organic (upper) 

layer of each sample was transferred using a disposable 

pipette to a clean 10 mL conical tube and dried in a water 

bath at 40°C under a constant stream of nitrogen. Once 

dryness was achieved, the samples were removed from the 

evaporator. Both ethyl acetate (50 μL) and BSTFA/1%-

TMCS (50 μL) were added to each sample. The tubes were 
4 

capped, vortexed briefly, and placed in a heating block at 

80°C for 20 minutes. Following derivitization, the tubes 

were removed from the heating block, allowed to cool 

to room temperature, and subsequently evaporated to 

dryness in a water bath at 40°C under a constant stream 

of nitrogen. The samples were reconstituted in 50 μL of 

methanol, vortexed briefly, and transferred using a 50 μL 

pipette to micro-vials for LC/MS analysis. 

CASE EXAMINATION

Case #1

A civil aviation accident occurred in the Atlantic 

Ocean. The wreckage from the accident was located approximately 9 hours after last contact with the victim. 

The victim, however, was not discovered until reappearing 

on shore approximately 1.5 months after the accident 

had occurred. Specimens examined from the victim for 

volatile organic compounds (VOCs) included blood, 

urine, skeletal muscle, and brain. Ethanol was present 

in every tissue and fluid examined from this case. The 

concentrations of ethanol found in these specimens 

were 38 mg/dL, 25 mg/dL, 12 mg/hg, and 39 mg/hg, 

respectively for the four samples mentioned above. The 

blood and tissue samples were noted as putrefied by 

visual inspection, and the urine was bloody. Also present in this case were numerous other VOCs, including 

n-propanol, n-butanol, and acetaldehyde at substantial 

concentrations. These other VOCs, like ethanol, were 

present in each of the specimens analyzed. The distribution of ethanol in this case was questionable because: 1) 

the highest concentration of ethanol detected was in the 

brain, which has a lower water content than both blood 

and urine and, therefore, would normally be expected 

to contain lower concentrations of ethanol, and 2) the 

blood ethanol concentration considerably exceeded the 

urine ethanol concentration, which is possible, but is not 

common in aviation fatalities. The presence of numerous other VOCs also supported the possibility that the 

ethanol present in this case resulted from postmortem 

microbial formation and not antemortem consumption. 

By LC/MS analysis, the urinary 5-HTOL/5-HIAA ratio 

for this case was determined to be 2.1 pmol/nmol. This 

value is well below the cutoff of 15 pmol/nmol established 

for antemortem ethanol consumption14, 23, 29, supporting 

the conclusion that recent ethanol consumption had not 

occurred prior to the accident.

Case #2

A civil aviation accident occurred in the Pacific Ocean 

near Alaska. The body of the victim was recovered 17 days 

after the accident. The specimens initially examined for 

ethanol and other VOCs were blood and urine. These 

specimens contained ethanol at concentrations of 54 

mg/dL and 9 mg/dL respectively. The distribution of 

ethanol in this case raises immediate questions as to its 

source. However, the low urinary ethanol concentration, 

when compared to the blood value, may be the result 

of dilution of the urine due to the length of time the 

victim was submerged in the ocean. Analysis of other 

VOCs present in the victim was limited to only acetaldehyde, which was present at 12 mg/dL in the blood. This 

concentration of acetaldehyde result could reasonably 

be considered to support either antemortem consumption or postmortem microbial formation. To provide a 

more definitive answer to the ethanol origin question, 

we examined the 5-HTOL/5-HIAA ratio for this case. 

The ratio was determined to be 3.2 pmol/nmol in urine. 

This value is well below the cutoff established for recent 

antemortem ethanol consumption, leading to the conclusion that microbes were responsible for the formation of 

the ethanol present in the blood of this victim.

Case #3

A civil aviation accident occurred in the Pacific Ocean 

near Southern California. The pilot’s body was recovered 

from the ocean at a depth of approximately 70 feet, 16 

days following the accident. Specimens initially analyzed 

for volatile organic compounds in this case were blood, 

urine, brain, and skeletal muscle. The blood and urine 

both contained ethanol at concentrations of 22 and 18 

mg/dL, respectively. Brain and skeletal muscle were 

found to contain ethanol at concentrations of 27 and 9 

mg/hg, respectively. The distribution of ethanol in these 

fluids and tissues, although not completely consistent 

with ethanol ingestion, does not exclude a conclusion 

of antemortem ethanol consumption. Various volatiles 

including acetaldehyde, n-propanol, n-butanol, and 

acetone were also present in these specimens, which 

indicates postmortem microbial activity. This case represents another classic example of inconclusive ethanol 

origin. Without further investigation it would be difficult 

to conclusively determine whether the pilot had recently 

consumed ethanol. Utilizing our newly validated method, 

we found the 5-HTOL/5-HIAA ratio to be 0.41 pmol/

nmol in urine. This value is considerably below the 15 

pmol/nmol cutoff, thus strongly indicating the absence 

of recent antemortem ethanol ingestion. The presence of 

ethanol in this case was thus reported as resulting from 

postmortem microbial formation.

Case #4

A civil aviation accident occurred in a rural region 

of Northern Michigan. The accident was witnessed by 

numerous people, which resulted in a quick response 

by emergency personnel and the prompt recovery of 

the victim. Specimens initially analyzed for VOCs were 
5

blood, urine, vitreous humor, brain, and skeletal muscle. 

The blood, urine, and vitreous humor contained ethanol 

at concentrations of 17, 25, and 25 mg/dL, respectively. 

Brain and skeletal muscle were found to contain ethanol 

at concentrations of 146 and 170 mg/hg, respectively. 

The distribution of ethanol in the fluids initially examined from this case is fully consistent with antemortem 

ethanol consumption. The tissues examined, however, 

contained ethanol completely inconsistent with the 

fluid results. Three other VOCs were detected in these 

specimens including n-propanol, acetaldehyde, and 

n-butanol. The concentrations of the three VOCs were 

significantly higher in brain and muscle than those in the 

fluids examined. For example, n-propanol was detected 

at 14 mg/hg in the brain but only 1 mg/dL in the blood 

and urine; and n-butanol was detected at 19 mg/hg in 

the muscle but only 1 mg/dL in the blood and the urine. 

Upon specimen collection by the medical examiner in 

aviation fatalities, blood specimens are obtained and stored 

in sealed tubes that contain a preservative, while urine 

and vitreous humor are collected in sealed tubes that 

contain no preservative.30 Additionally, tissue specimens 

are placed in plastic zip-top bags, also without addition 

of preservative. When our laboratory received this case 

it was noted that the temperature of the container carrying the specimens in questions was 10°C, which is 

above the desired transport temperature of 4°C. It was 

also noted by visual inspection that the tissue specimens 

were putrefied, and they had a foul odor. It is probable 

that the tissue specimens in this case were contaminated 

with microbes either before or during collection and that 

these microbes had sufficient time to produce substantial 

quantities of ethanol.

This case represents one of the most difficult ethanol 

origin scenarios possible. It appears upon initial examination that ethanol was consumed antemortem. Then 

as a result of trauma experienced during the accident, 

microbes contaminated both brain and muscle specimens. 

Ethanol formation subsequently occurred while these tissue specimens were in transit. We applied our LC/MS 

method to the urine available from this case and found 

the 5-HTOL/5-HIAA ratio to be 67 pmol/nmol. This 

value is well above the established 15 pmol/nmol cutoff, 

thus confirming the recent consumption of ethanol prior 

to death as suggested by the fluid ethanol distributions.

Unlike the interpretation of a 5-HTOL/5-HIAA ratio 

below 15 pmol/nmol, which conclusively eliminates the 

possibility of recent ethanol consumption, caution must 

be used when interpreting a 5-HTOL/5-HIAA ratio above 

the 15 pmol/nmol cutoff. In antemortem experiments, it 

has been demonstrated that the 5-HTOL/5-HIAA ratio 

may remain elevated above the 15 pmol/nmol cutoff value 

for some 2 - 8 hrs after all consumed ethanol has been 

eliminated from the body.18, 23 Therefore, there remains 

a possibility that all of the ethanol present in this case 

was produced by postmortem microbial formation, and 

the elevated 5-HTOL/5-HIAA ratio was due to ethanol 

that had been consumed within the previous 8 - 12 hrs 

but completely eliminated from the body prior to the 

fatal accident.

Case #5

A civil aviation accident occurred in a rural region of 

California. The accident site was not discovered until 7 

days following the crash, and the victim was found submerged in a river. Specimens initially analyzed for VOCs in 

this case included blood, urine, skeletal muscle, and brain. 

These specimens each contained ethanol at concentrations of 31 mg/dL, 48 mg/dL, 49 mg/hg, and 57 mg/hg, 

respectively. The distribution of ethanol in the fluids and 

tissues examined from this case, although not considered 

normal, does not immediately rule out the possibility 

of antemortem ethanol ingestion.31 Other VOCs were 

detected in these specimens as well. The volatiles found 

were n-propanol in each specimen, acetaldehyde in blood 

and urine, n-butanol in each specimen and isopropanol in 

blood, urine, and skeletal muscle. As previously discussed, 

the detection of other VOCs in the specimen of interest 

indicates postmortem microbial activity and, therefore, 

the possibility of postmortem ethanol formation.

This case represents another difficult question of ethanol origin. The classic ethanol origin indicators could 

not conclusively rule out the possibility of antemortem 

ethanol ingestion. We applied the LC/MS method to the 

urine available from this case and found the 5-HTOL/

5-HIAA ratio to be 0.08 pmol/nmol. This value is well 

below the 15 pmol/nmol cutoff, thus confirming no 

recent antemortem ethanol ingestion had occurred, but 

instead, indicated the postmortem microbial formation 

of ethanol in this case.

DISCUSSION

Two classic indicators are commonly employed by 

forensic laboratories to discern the origin of ethanol in 

postmortem cases. These indicators are: (1) the distribution of ethanol between various bodily fluids and tissues 

from a single victim and (2) the presence of additional 

VOCs in the bodily fluids and tissues of the victim. During life, ethanol distributes fairly rapidly throughout the 

body according to the water content of the various fluids 

and tissues.31, 32 Postmortem microbial ethanol formation 

alters the ethanol concentration in the examined fluids 

and tissues and consequently disrupts the usual distribution pattern of ethanol. By comparing ethanol results 

obtained from two or more fluids and tissues from one 
6 

case, one can make an informed assumption concerning 

the origin of any ethanol present. The presence of other 

volatiles such as acetaldehyde, acetone, sec-butanol, 

isopropanol, n-propanol, methanol, and n-butanol in 

an ethanol-positive specimen suggests microbial action 

and, therefore, possible postmortem ethanol formation. 

The lack of other commonly analyzed volatiles suggests the absence of microbial postmortem activity but 

does not exclude the possibility of postmortem ethanol 

formation.3, 8, 33-38

Furthermore, urine was once considered immune to 

microbial ethanol formation because it is sealed in the 

bladder and not normally exposed to microbes and/or a 

substrate such as glucose for the microbes to consume. 

However, this view of urine has been recently questioned.4, 

12, 39-41 It is now known that postmortem microbial production of ethanol in urine is quite feasible and, indeed, 

under the circumstances of a fatal aviation accident may 

be likely. After careful application of the above-mentioned 

ethanol origin indicators to the five cases in question, the 

precise source of ethanol origination was still unclear. 

Therefore, application of the newly validated LC/MS 

method14 for determination of the serotonin metabolite 

ratio was undertaken. 

A 5-HTOL/5-HIAA ratio below 15 pmol/nmol clearly 

eliminates the possibility of recent ethanol consumption 

prior to death, while a 5-HTOL/5-HIAA ratio above 15 

pmol/nmol indicates recent ethanol ingestion. It must be 

stressed, however, that in an ethanol positive case with 

a 5-HTOL/5-HIAA ratio above 15 pmol/nmol, there 

remains a possibility that the ethanol present in the specimen originated from postmortem microbial formation, 

and not consumption. This possibility arises from the 

time lag between complete ethanol elimination from the 

body and the return of the 5-HTOL/5-HIAA ratio to 

normal levels. In fact, the 5-HTOL/5-HIAA ratio can 

remain elevated above normal for 2-8 hours after ingested 

ethanol has been completely eliminated from the body.21, 

42 Further, chemically identical ethanol resulting from 

postmortem microbial formation can be superimposed 

upon any residual ethanol from recent consumption.

The cases represented in this paper exemplify the 

usefulness of employing the 5-HTOL/5-HIAA ratio 

to determine if recent ethanol consumption had occurred. We suggest that the relative simplicity of this 

procedure should make the 5-HTOL/5-HIAA ratio 

methodology more readily applicable to the routine assessment of antemortem versus postmortem origin of 

ethanol found in postmortem fluid and tissue samples. 

The application of this method is also notably not 

limited to postmortem samples. Indeed, contested 

ethanol results from routine screenings in various 

drug programs, and criminal cases could readily be 

evaluated through the use of this procedure. 

CONCLUSIONS

A newly developed LC/MS procedure for the analysis 

of 5-HTOL and 5-HIAA and the associated 5-HTOL/

5-HIAA ratio was applied to five ethanol-positive civil 

aviation fatalities where ethanol origin could not previously be determined. The application of this method 

has demonstrated the utility of this approach in assessing ethanol origin in postmortem specimens. One of 

the most important aspects of this novel method is the 

simultaneous analysis of both compounds using a single 

extraction method and a single analytical technique. This 

greatly increases the precision in the methodology and, 

thus, the certainty of the subsequent conclusions.

REFERENCES

1. Nicloux, M. Neoformation d’alcool ethylique dans 

le cadavre humain en voie de putrefaction. Compte 

Rendu des Seances de la Societe de Biologie, Paris. 

121: 975-8 (1936).

2. Corry, J.E. 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).

3. O’Neal, C.L. and Poklis, A. Postmortem production 

of ethanol and factors that influence interpretation: 

a critical review. Am J Forensic Med Pathol. 17: 8-20 

(1996).

4. Saady, J.J., Poklis, A., and Dalton, H.P. Production 

of urinary ethanol after sample collection. J Forensic 

Sci. 38: 1467-71 (1993).

5. Bogusz, M., Guminska, M., and Markiewicz, J. 

Studies on the formation of ethanol and of pyruvate as its precursor from some di- and tricarbonic 

compounds in putrefying blood in vitro. Forensic 

Sci. 1: 229-37 (1972).

6. Clark, M.A. and Jones, J.W. Studies on putrefactive 

ethanol production. I: Lack of spontaneous ethanol 

production in intact human bodies. J Forensic Sci. 

27: 366-71 (1982).

7. Chang, J. and Kollman, S.E. The effect of temperature on the formation of ethanol by Candida albicans in blood. J Forensic Sci. 34: 105-9 (1989).
7

8. Davis, G.L., Leffert, R.L., and Rantanen, N.W. 

Putrefactive ethanol sources in postmortem tissues 

of conventional and germ-free mice. Arch Pathol. 

94: 71-4 (1972).

9. Kupfer, D.M., Chaturvedi, A.K., Canfield, D.V., 

and Roe, B.A. PCR-based identification of postmortem microbial contaminants--a preliminary 

study. J Forensic Sci. 44: 592-6 (1999).

10. Bogusz, M., Guminska, M., and Markiewicz, J. 

Studies on the formation of endogenous ethanol 

in blood putrefying in vitro. J Forensic Med. 17:

156-68 (1970).

11. Jones, A.W., Andersson, R., Sakshaug, J., and Morland, J. Possible formation of ethanol in postmortem 

blood specimens after antemortem treatment with 

mannitol. J Anal Toxicol. 15: 157-8 (1991).

12. Sulkowski, H.A., Wu, A.H., and McCarter, Y.S. 

In-vitro production of ethanol in urine by fermentation. J Forensic Sci. 40: 990-3 (1995).

13. Blackmore, D.J. The bacterial production of ethyl 

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

14. Johnson, R.D., Lewis, R.J., Canfield, D.V., and 

Blank, C.L. 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).

15. Minegishi, A. and Ishizaki, T. Rapid and simple 

method for the simultaneous determination of 3,4-

dihydroxyphenylacetic acid, 5-hydroxyindole-3-

acetic acid and 4-hydroxy-3-methoxyphenylacetic 

acid in human plasma by high-performance liquid 

chromatography with electrochemical detection. J 

Chromatogr. 308: 55-63 (1984).

16. Kema, I.P., de Vries, E.G., and Muskiet, F.A. Clinical 

chemistry of serotonin and metabolites. J Chromatogr B Biomed Sci Appl. 747: 33-48 (2000).

17. Falkowski, A.J. and Wei, R. Optimized isocratic 

conditions for the simultaneous determination of 

serotonin precursors and metabolites by reversedphase high-pressure liquid chromatography with 

electrochemical detection. Anal Biochem. 115:

311-7 (1981).

18. Helander, A., Beck, O., Jacobsson, G., Lowenmo, 

C., and Wikstrom, T. Time course of ethanol-induced changes in serotonin metabolism. Life Sci. 

53: 847-55 (1993).

19. Davis, V.E., Brown, H., Huff, J.A., and Cashaw, 

J.L. The alteration of serotonin metabolism to 5-

hydroxytryptophol by ethanol ingestion in man. J 

Lab Clin Med. 69: 132-40 (1967).

20. Svensson, S., Some, M., Lundsjo, A., Helander, A., 

Cronholm, T., and Hoog, J.O. Activities of human 

alcohol dehydrogenases in the metabolic pathways 

of ethanol and serotonin. Eur J Biochem. 262: 324-9 

(1999).

21. Helander, A., Beck, O., and Jones, A.W. Distinguishing ingested ethanol from microbial formation 

by analysis of urinary 5-hydroxytryptophol and 5-

hydroxyindoleacetic acid. J Forensic Sci. 40: 95-8 

(1995).

22. Hagan, R.L. and Helander, A. Urinary 5-hydroxytryptophol following acute ethanol consumption: 

clinical evaluation and potential aviation applications. Aviat Space Environ Med. 68: 30-4 (1997).

23. Helander, A., Beck, O., and Jones, A.W. Laboratory 

testing for recent alcohol consumption: comparison 

of ethanol, methanol, and 5-hydroxytryptophol. 

Clin Chem. 42: 618-24 (1996).

24. Voltaire, A., Beck, O., and Borg, S. Urinary 5-

hydroxytryptophol: A possible marker of recent 

alcohol consumption. Alcohol Clin Exp Res. 16:

281-5 (1992).

25. Carlsson, A.V., Hiltunen, A.J., Beck, O., Stibler, 

H., and Borg, S. Detection of relapses in alcoholdependent patients: comparison of carbohydratedeficient transferrin in serum, 5-hydroxytryptophol 

in urine, and self-reports. Alcohol Clin Exp Res. 17:

703-8 (1993).

26. Helander, A., Beck, O., Wennberg, M., Wikstrom, 

T., and Jacobsson, G. Determination of urinary 5-

hydroxyindole-3-acetic acid by high-performance 

liquid chromatography with electrochemical detection and direct sample injection. Anal Biochem. 

196: 170-3 (1991).

27. Ikarashi, Y., Blank, C.L., and Itoh, K. Development of a liquid chromatography/multiple electrochemical detector (LCMC) and its application 

in neuroscience. Jap. J. Pharm. 97: 51-64 (1991).

28. Jones, A.W. and Helander, A. Time course and 

reproducibility of urinary excretion profiles of 

ethanol, methanol, and the ratio of serotonin 

metabolites after intravenous infusion of ethanol. 

Alcohol Clin Exp Res. 23: 1921-6 (1999).
8

29. Helander, A., Walzer, C., Beck, O., Balant, L., Borg, 

S., and von Wartburg, J.P. Influence of genetic variation in alcohol and aldehyde dehydrogenase on serotonin metabolism. Life Sci. 55: 359-66 (1994).

30. Chaturvedi, A.K., Smith, D.R., Soper, J.W., Canfield, D.V., and Whinnery, J.E. Characteristics 

and toxicological processing of postmortem pilot 

specimens from fatal civil aviation accidents. Aviat 

Space Environ Med. 74: 252-9 (2003).

31. Ito, A., Moriya, F., and Ishizu, H. Estimating the 

time between drinking and death from tissue distribution patterns of ethanol. Acta Med Okayama. 

52: 1-8 (1998).

32. Stowell, A.R. and Stowell, L.I. Estimation of blood 

alcohol concentrations after social drinking. J Forensic Sci. 43: 14-21 (1998).

33. Amick, G.D. and Habben, K.H. Inhibition of 

ethanol production by Saccharomyces cerevisiae 

in human blood by sodium fluoride. J Forensic 

Sci. 42: 690-2 (1997).

34. de Lima, I.V. and Midio, A.F. Origin of blood 

ethanol in decomposed bodies. Forensic Sci Int. 

106: 157-62 (1999).

35. Moriya, F. and Ishizu, H. Can microorganisms produce alcohol in body cavities of a living person?: A 

case report. J Forensic Sci. 39: 883-8 (1994).

36. Wigmore, J.G. and Chow, B.L.C. Case report: Detection of neo-formation of ethanol in a postmortem 

blood sample using n-propanol and a urine sample. 

Can Soc Forensic Sci J. 33: 145-49 (2000).

37. Jacobsen, E. The metabolism of ethyl alcohol. 

Pharmacol. Rev. 4: 107-35 (1952).

38. Nanikawa, R., Ameno, K., Hashimoto, Y., and 

Hamada, K. Medicolegal studies on alcohol detected in dead bodies--alcohol levels in skeletal 

muscle. Forensic Sci Int. 20: 133-40 (1982).

39. Levine, B., Smith, M.L., Smialek, J.E., and Caplan, 

Y.H. Interpretation of low postmortem concentrations of ethanol. J Forensic Sci. 38: 663-7 (1993).

40. Ball, W. and Lichtenwalner, M. Ethanol production in infected urine. N Engl J Med. 301: 614 

(1979).

41. Alexander, W.D., Wills, P.D., and Eldred, N. Urinary ethanol and diabetes mellitus. Diabet Med. 5:

463-4 (1988).

42. Helander, A., Beck, O., and Borg, S. The use of 

5-hydroxytryptophol as an alcohol intake marker. 

Alcohol Alcohol Suppl. 2: 497-502 (1994).