Robert D. Johnson

Russell J. Lewis

Dennis V. Canfield

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, OK 73125

C. LeRoy Blank

Department of Chemistry and Biochemistry

University of Oklahoma

Norman, OK 73019

November 2003

Final Report

This document is available to the public

through the National Technical Information

Service, Springfield, Virginia 22161.

Office of Aerospace Medicine

Washington, DC 20591

Ethanol Origin in Postmortem 

Urine: An LC/MS Determination 

of Serotonin Metabolites

DOT/FAA/AM-03/18
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

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1

ETHANOL ORIGIN IN POSTMORTEM URINE: AN LC/MS 

DETERMINATION OF SEROTONIN METABOLITES

INTRODUCTION

The Federal Aviation Administration’s (FAA’s) Civil 

Aerospace Medical Institute (CAMI) is responsible, under 

Department of Transportation (DOT) orders 8020.11A 

and 1100.2C, to “conduct toxicologic analysis on specimens from ... aircraft accident fatalities” and “investigate 

... general aviation and air carrier accidents and search for 

biomedical and clinical causes of the accidents, including evidence of ... chemical (use).” Therefore, following 

an aviation accident, samples are collected at autopsy 

and sent to CAMI’s Toxicology and Accident Research 

Laboratory, where toxicological analysis is conducted on 

various postmortem fluids and tissues.

Ethanol analysis is one of the most common tests performed on forensic specimens. The presence of ethanol 

in fatal aircraft accident victims constitutes an important 

part of aircraft accident investigations and litigation. An 

ethanol positive also invites legal scrutiny as to its validity. With the accuracy of today’s analytical techniques, 

there should be no doubt concerning the quantitative 

determination of ethanol found in a biological specimen. 

However, the origin of the ethanol found in a specimen is 

another, and highly pertinent, variable to consider when 

interpreting ethanol results.

The microbial formation of ethanol in postmortem 

specimens is the most significant problem encountered 

when evaluating ethanol results. The first report dealing with postmortem formation of ethanol in corpses 

appeared in 1936.1 But while a postmortem increase in 

ethanol was observed, the precise source of the increase 

was unknown. Today, it is known that many different 

microbes may be responsible for postmortem formation of 

ethanol. Investigations have been performed to identify the 

particular species of bacteria, yeast and/or fungi primarily 

responsible for ethanol production and the mechanism 

by which ethanol is formed.2, 3, 4, 5 Candida albicans has 

been identified as the microbe most often responsible for 

postmortem production of ethanol in humans.6, 7 This 

species of yeast is commonly found in humans in vivo. 

Although located ubiquitously throughout the body, the 

highest concentrations are typically in the mouth and on 

the skin.8 Glucose is the most prevalent substrate in the 

human body used by these microbes to form ethanol.9

Other endogenous compounds can also be utilized as 

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

galactose, maltose, sucrose and lactose. 9, 10, 11, 12

When working with forensic specimens, postmortem 

ethanol formation is generally avoided by storage at appropriate temperatures and the addition of preservatives 

such as sodium fluoride.13 These precautions, however, do 

not rule out the possibility of ethanol formation prior to 

sample collection and preservation.14 Furthermore, studies 

have demonstrated that if a specimen’s storage temperature is not carefully controlled, the preservative sodium 

fluoride may become ineffective in blocking postmortem 

formation of ethanol.15, 16 Although several methods are 

available to help address the postmortem ethanol issue, 

in many cases, the source of ethanol remains elusive.

Recently, exploitation of the metabolism of serotonin 

as a biological marker for ethanol consumption has begun 

to gain interest in the field of forensic science. Serotonin 

(5-hydroxytryptamine, 5-HT) is an indoleamine commonly found in nature.17 In humans, 5-HT is found 

throughout the body, with substantial concentrations 

found in the gastrointestinal tract and blood platelets.18

The metabolism of 5-HT initially involves oxidative 

deamination to form the intermediate aldehyde, 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-hydroxyindole-3-

acetic acid (5-HIAA).19 Reduction, catalyzed by aldehyde 

reductase, leads to formation of 5-hydroxytryptophol (5-

HTOL).20 Under normal circumstances 5-HIAA is the 

predominant metabolite of 5-HT.18 However, ethanol 

consumption has been shown to significantly alter the 

normal metabolism of serotonin.

An increase in 5-HTOL concentration following ethanol consumption was first reported in 1967.21 Since that 

time it has been clearly demonstrated that the consumption of ethanol shifts serotonin metabolism to favor formation of 5-HTOL and reduces the formation of 5-HIAA.20, 

22, 23, 24 Two primary factors contribute to this increase in 

the 5-HTOL/5-HIAA ratio. The first factor involves the 

oxidation of ethanol into acetaldehyde. As acetaldehyde is 

produced, it is rapidly oxidized by aldehyde dehydrogenase. The oxidation of acetaldehyde results in competitive 

inhibition of aldehyde dehydrogenase and prevents its 

contribution to 5-HIAA formation.25 The second factor 

capable of increasing the 5-HTOL/5-HIAA ratio is the 

higher than normal concentrations of the reduced form 

of nicotinamide adenine dinucleotide (NADH), resulting 
2 

as a byproduct from the oxidation of both ethanol and 

acetaldehyde.26 The oxidation of these two compounds 

consumes NAD+, a necessary cofactor for aldehyde dehydrogenase activity, and creates an excess of NADH, a 

cofactor for aldehyde reductase, thereby promoting the 

formation of 5-HTOL. These two factors combine to dramatically increase the 5-HTOL/5-HIAA ratio following 

ethanol ingestion.25 The 5-HTOL/5-HIAA ratio remains 

elevated for hours after ethanol has been eliminated from 

the body.23 As such, the 5-HTOL/5-HIAA ratio has been 

applied, in a limited manner, to ethanol cessation monitoring programs as a marker of recent ethanol ingestion 

in urine specimens. The 5-HTOL/5-HIAA ratio has also 

been briefly examined in postmortem urine samples. This 

ratio may serve as a powerful tool in forensic science for 

differentiating between the postmortem formation and 

ingestion of ethanol.

Historically, levels of 5-HTOL and 5-HIAA have been 

examined using two considerably unrelated analytical 

techniques. 5-HIAA concentrations are easily measured at 

sub-nanomolar levels using liquid chromatography with 

electrochemical detection (LC/EC).27, 28 5-HTOL may 

also be detected with LC/EC, but the detection limits are 

typically insufficient to monitor this compound in most 

pertinent biological specimens. For this reason, 5-HTOL 

has typically been analyzed using gas chromatography with 

mass spectrometric detection (GC/MS).29 The employment of two different analytical techniques to obtain a 

5-HTOL/5-HIAA ratio in a specimen obviously increases 

the probability of substantial errors. To date this has effectively prevented the widespread use of this ratio as a 

marker for ethanol ingestion. In this study we describe 

a method for the rapid and simultaneous determination 

of 5-HTOL and 5-HIAA using liquid chromatography 

with mass spectrometric detection (LC/MS). Following 

validation of the method, we investigated the 5-HTOL/5-

HIAA ratios in both ethanol-positive and ethanol-negative 

postmortem urine samples.

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 

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

chloride, sodium acetate, β-glucuronidase, 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 (Pierce Chemicals, Rockford, IL).

Stock solutions of 5-HTOL and 5-HIAA were prepared 

at 1.00 mg/mL (5.64 mM 5-HTOL; 5.23 mM 5-HIAA) 

in methanol. 5-Methoxy-2-methyl-3-indoleacetic acid 

(5-MMIA) was used as the internal standard for these 

experiments and was prepared at a concentration of 100 

μg/mL (0.450 mM) in methanol. These 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 the freezer for 

storage at -20°C. These solutions were used for 1 month 

and then discarded. β-glucuronidase solution was prepared 

by adding 2.5 mL 0.1 mM sodium acetate buffer pH 5.00 

to 250,000 units of enzyme, yielding a final concentration of 100,000 units/mL. This solution was stored in 

the freezer at -20°C and expired after 1 month.

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. The 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 organic portion 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 as above.

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/Sigma-Aldrich, 

Bellefonte, PA). Samples were injected using a Hewlett 

Packard G1313A autosampler (Hewlett Packard Co., 

Wilmington, DE). Identification and quantitation were 

accomplished using a Finnigan model LCQ atmospheric 

pressure chemical ionization (APCI) ion trap mass spectrometer (ThermoFinnigan Corp., San Jose, CA). 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 Method

Working with standards of 5-HIAA and 5-HTOL, we 

began the initial investigation by looking into the response 

received from these 2 compounds when injected directly 

into the LCQ. As described in detail below, 5-HTOL and 

5-HIAA were derivatized with TMS to form 5-HTOLTMS and 5-HIAA-TMS derivatives. Initial ionization 

evaluation of these derivatives indicated that positive 

chemical ionization (PCI), creating the [M+H]+ ions, was 

more effective than negative chemical ionization (NCI), 

which formed the [M-H]- ions. APCI-PCI-MS conditions were optimized by infusing the desired compound 

at approximately 10 μg/mL into the LCQ at 1 mL/min. 

Tuning the MS for the desired ions was accomplished 

using the autotune feature of the Xcalibur software. As a 

result of preliminary APCI-PCI-MS investigations, each 

sample analysis was subsequently split into 3 unique data 

collection segments.

The operating conditions for segment 1, which 

analyzed for 5-MMIA, were as follows: APCI capillary 

temperature, 150°C; APCI vaporizer temperature, 450°C; 

source voltage, 10.0 kV; source current, 5.0 μA; capillary voltage, 8.0 V; tube lens offset, 25.0 V; octapole 1 

offset, -1.75 V; octapole 2 offset, -6.5 V; interoctapole 

lens voltage, -16.0 V; ion trap DC offset, -10 V; multiplier voltage, 0.0 V; micro-scan injection time, 200 

msec. Additionally, segment 1 was split into 2 separate 

scan events. Scan event 1 collected the [M+H]+ parent 

ion at m/z 220.1, and scan event 2 performed MS/MS 

on this parent ion using a collision energy of 42% and 

collected the daughter ion at m/z 174.1.

The operating conditions for segment 2, which 

analyzed for 5-HTOL, were as follows: APCI capillary 

temperature, 150°C; APCI vaporizer temperature, 450°C; 

source voltage, 10.0 kV; source current, 5.0 μA; capillary 

voltage, 17.0 V; tube lens offset, 25.0 V; octapole 1 offset, -3.25 V; octapole 2 offset, -7.0 V; interoctapole lens 

voltage, -16.0 V; ion trap DC offset, -10 V; multiplier 

voltage, 0.0 V; micro-scan injection time, 200 msec. 

Additionally, segment 2 was split into 3 separate scan 

events. Scan event 1 collected the [M+H]+ parent ion 

at m/z 250.1. Scan event 2 performed MS/MS on this 

parent ion, using a collision energy of 38%, and collected 

the daughter ion at m/z 232.1. Scan event 3 performed 
4 

MS/MS/MS on the daughter ion at m/z 232.1, using a 

collision energy of 48%, and collected the granddaughter 

ion at m/z 216.1.

The operating conditions for segment 3, which analyzed for 5-HIAA, were as follows: APCI capillary temperature, 150°C; APCI vaporizer temperature, 450°C; 

source voltage, 10.0 kV; source current, 5.0 μA; capillary 

voltage, 3.0 V; tube lens offset, 10.0 V; octapole 1 offset, -4.25 V; octapole 2 offset, -7.0 V; interoctapole lens 

voltage, -22.0 V; ion trap DC offset, -10 V; multiplier 

voltage, 0.0 V; micro-scan injection time, 200 msec. Segment 3 was split into 3 separate scan events. Scan event 

1 collected the [M+H]+ parent ion at m/z 264.1. Scan 

event 2 performed MS/MS on this parent ion, using a 

collision energy of 36%, and collected the daughter ion 

at m/z 218.1. Scan event 3 performed MS/MS/MS on 

the daughter ion at m/z 218.1, using a collision energy 

of 48%, and collected the granddaughter ions at m/z

146.1, 191.1 and 202.1.

For this analysis, the HPLC was operated in an isocratic 

mode with a flow rate of 1 mL/min. The mobile phase 

composition was 80:20, methanol:50 mM formate buffer 

mixture. The sample injection volume was 10 μL. The 

HPLC column was routinely equilibrated for 2 hours 

prior to use. Following use, the column was washed and 

stored in 50:50, methanol:H2O.

Specimen Preparation and Extraction

Calibration curves were prepared by serial dilution

utilizing human certified negative urine as the sample 

matrix. Controls were prepared using separate drug standards from those used to create the calibration curve. 

Calibrators, controls and postmortem urine specimens 

were prepared and extracted in the following manner.

To facilitate the excretion of 5-HTOL from the body, 

this compound is extensively conjugated in vivo in humans 

as a glucuronide adduct. Therefore, an initial hydrolysis 

step was performed on all specimens. Three mL aliquots 

of calibrators, controls and specimens were individually 

transferred to 15 mL screw-topped culture tubes. To each 

sample, 1.00 mL of a 1000 ng/mL (4.50 μM) internal 

standard solution was added. β-Glucuronidase solution 

(7500 units) followed by 1.00 mL of 0.1 mM pH 5.0 

sodium acetate buffer was added to each calibrator, control 

and specimen aliquot. The samples were vortexed briefly 

and incubated at 70°C for 45 min. Following hydrolysis, 

samples were allowed to cool to room temperature, then 

3.00 mL of a 0.100 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 mixer and gently mixed for 20 min. 

Following mixing, the samples were centrifuged at 780xg 

for 5 min. The organic (upper) layer of each sample was 

transferred using a clean, 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, 

and both ethyl acetate (50 μL) and BSTFA/1%-TMCS 

(50 μL) were added to each. The tubes were capped, 

vortexed briefly and placed in a heating block at 80°C 

for 20 min. 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 subsequently reconstituted in 50 μL 

of methanol, vortexed briefly and transferred to vials for 

LC/MS analysis.

Extraction Efficiency

The recovery of each analyte was determined using 

the following procedure.30 Two groups of negative urine 

samples, X and Y, were extracted and prepared in the 

same manner as described above, including the addition 

of internal standard. Group X was spiked with a known 

concentration of 5-HTOL and 5-HIAA prior to extraction, and group Y was spiked with the same solution 

following the liquid/liquid extraction step. Upon analysis, 

the average response factor obtained from group X was 

divided by the average response factor obtained from 

group Y to yield the percent recovery value (100 * X/Y 

= % recovery) for each of the compounds.

Postmortem Urine Samples

Postmortem urine specimens received by our laboratory are stored at -20°C. Specimens are retained in our 

storage facility for up to 5 years following collection. The 

Toxicology and Accident Research Laboratory database 

was used to identify both positive and negative ethanol 

urine samples for this study. Our laboratory administratively defines an ethanol negative specimen as any sample 

containing ethanol at a concentration <10 mg/dL. This 10 

mg/dL cutoff is also required by the College of American 

Pathologists (CAP) in their “whole blood alcohol/volatiles 

survey” and for CAP laboratory accreditation.

RESULTS AND DISCUSSION

Method Validation

The procedure described in this paper provides a rapid, 

accurate and reproducible method for the simultaneous 

extraction and quantitation of 5-HTOL and 5-HIAA. A 

liquid-liquid extraction procedure in combination with 
5

HPLC/APCI-PCI-MS provides both superior separation 

of these two compounds and detection limits that are 

well below concentrations expected in most biological 

specimens.

Derivitization of 5-HTOL and 5-HIAA with TMS 

was found to be essential for this method. Derivatization of these compounds achieved two necessary objectives. First, the underivitized compounds provided poor 

chromatographic resolution under mobile phase conditions amenable to LC/MS. And, optimizing the mobile 

phase to increase APCI-PCI ionization of one compound 

dramatically decreased the ionization efficiency for the 

other. This was not unexpected since one compound is 

an acid and the other a primary alcohol. By producing 

a TMS derivative of each compound, complete chromatographic resolution was achieved, and the undesirable effects of mobile phase on APCI-PCI ionization was 

eliminated. Secondly, derivatization increased the mass 

of the compounds, which practically allowed for MS/MS 

and MS/MS/MS and enhanced the associated detection 

limits by approximately 1000 times compared with the 

underivitized forms.

The internal standard chosen for these experiments 

was 5-methoxy-2-methyl-3-indoleacetic acid (5-MMIA). 

This compound is structurally related to both analytes 

and has similar chromatographic characteristics. Furthermore, it is not endogenous in biological samples of any 

type. 5-MMIA was found to be stable in solution for an 

extended period of time.

An ion trap mass spectrometer is a collection devise, 

which allows for the “trapping” or isolation of ions from 

a target compound, followed by the subsequent formation of unique spectra from these ions. APCI is a soft 

ionization technique and, when used in the PCI mode, 

becomes an excellent source of [M+H]+ parent ions. This 

ionization technique, in combination with an ion trap, 

enabled us to perform MS/MS/MS on the 5-HTOL and 

5-HIAA derivatives, and MS/MS on 5-MMIA. 5-HTOL 

had a [M+H]+ parent ion at m/z 250.1. The parent ion 

was collected by the ion trap and subjected to MS/MS, 

resulting in a daughter ion at m/z 232.1. Collecting m/z

232.1 and performing MS on it resulted predominantly 

in the granddaughter ion at m/z 216.1. The same process 

was used on 5-HIAA, which had a parent [M+H]+ ion at 

m/z 264.1, a daughter ion at m/z 218.1 and several granddaughter ions with high-abundance, including m/z 146.1, 

191.1 and 202.1. The internal standard had a [M+H]+ at 

m/z 220.1, which, when subjected to MS/MS, resulted 

in a daughter ion at m/z 174.1. Since it is unlikely that 

a specimen matrix will produce the same product ions 

as a target compound, full scan MS/MS and MS/MS/

MS spectra for 5-HTOL and 5-HIAA, and MS/MS for 

5-MMIA provided the “fingerprints” used for analyte 

identification and confirmation. The full scan MS/MS 

and MS/MS/MS spectra are shown in Figures 2-6.

Quantitation of 5-HTOL and 5-HIAA was achieved 

via an internal standard calibration procedure. Calibration curves were prepared by plotting a linear regression 

of the analyte/internal standard response factor versus 

the analyte concentration. Response factors for both 

compounds were determined for each specimen. The 

response factor was calculated by dividing the area of the 

analyte peak by the area of the internal standard peak. 

The MS/MS/MS ion, at m/z 216.1 was used for 5-HTOL 

quantitation. Quantitation of 5-HIAA was accomplished 

using the MS/MS ion at m/z 218.1. The MS/MS ion at 

m/z 174.1 was used for 5-MMIA.

As can be seen in Figure 7, 5-MMIA, 5-HTOL and 

5-HIAA are completely resolved chromatographically. 

The peak shape is excellent for all three compounds, and 

the noise was found to be negligible for most injections 

throughout the analysis. These compounds experienced 

no interference from endogenous sample matrix components. Typical retention times were 1.59, 2.34 and 3.30 

min for 5-MMIA, 5-HTOL and 5-HIAA, respectively. 

The number of theoretical plates calculated for each 

compound ranged from 2000 to 5000.

The extraction efficiency of 5-HTOL and 5-HIAA 

from postmortem urine samples, as described in the 

experimental section, was determined at 1, 10, 50 and 

800 ng/mL. The individual recovery values are presented 

in Table 1. The recovery of 5-HTOL and 5-HIAA across 

this broad concentration range averaged 82% and 80%, 

respectively. These values exceeded our initial expectations, 

considering the simplicity of the extraction.

The limit of detection (LOD), limit of quantitation 

(LOQ) and linear dynamic range (LDR) for each analyte 

are listed in Table 2. The LOD was defined as the lowest 

concentration of analyte having a minimum signal-tonoise ratio (S/N) of 5, in addition to meeting a MS/MS 

and MS/MS/MS spectral “fingerprint“ confirmation and 

±5% retention time criteria. The LOQ was defined as 

meeting all LOD criteria plus having a S/N of 10 and 

having an experimentally determined value within ± 20% 

of its prepared concentration. The LOD was found to 

be 0.100 ng/mL for both 5-HTOL and 5-HIAA. The 

LOQ was found to be 0.390 ng/mL and 0.780 ng/mL for 

5-HTOL and 5-HIAA, respectively. The LDR for these 

two compounds was 0.390 - 800 ng/mL for 5-HTOL 

and 0.780 - 12800 ng/mL for 5-HIAA. The correlation 

coefficients for both of the LDR curves exceeded 0.99. 

Non-linearity was observed with 5-HTOL at concentrations greater than 800 ng/mL, while concentrations above 

12,800 ng/mL for 5-HIAA were not evaluated.
6 

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Carryover from one sample to the next was not found 

to be a problem. It was, however, monitored by the use of 

solvent injections. A methanol blank injected following 

the highest calibrator showed no carryover contamination. 

Blanks were also used randomly throughout the sample 

sequence to verify that no sample-to-sample contamination occurred.

Intra-day (within day) and inter-day (between days) 

accuracy and precision were examined for this extraction. 

The accuracy was measured as the relative error between 

the experimentally determined and prepared concentrations of a sample. The precision was measured as the 

relative standard deviation (RSD) in the experimentally 

determined concentrations. Urine controls at 1 and 10 

ng/mL were prepared in pools on day 1 and stored in the 

refrigerator at 4°C until extracted. A calibration curve 

was extracted along with 5 replicates of each control concentration on day 1 of the experiment. The intra-day 

relative error and RSD for 5-HTOL were +9% and 2% 

at 1 ng/mL and -4% and 8% at 10 ng/mL, respectively. 

The intra-day relative error and RSD for 5-HIAA were 

0% and 8% at 1 ng/mL and -8% and 2% at 10 ng/mL, 

respectively. These data are summarized in Table 3. Interday accuracy and precision were evaluated by extracting 5 

replicates of each of two control concentrations on days 

2, 4 and 8 and basing the quantitation on the original 

calibration curve. The inter-day relative error and RSD 

for 5-HTOL were +1% and 6% at 1 ng/mL and -9% 

and 4% at 10 ng/mL, respectively. The inter-day relative error and RSD for 5-HIAA were -3% and 6% at 1 

ng/mL and -13% and 2% at 10 ng/mL, respectively. The 

inter-day accuracy and precision are shown in Table 4. 

The short-term stability of these two compounds at 4°C 

was evaluated by looking at the data from day 8 alone, as 

shown in Table 5. After storage for 8 days, the 1 ng/mL 

control was found to have a 5-HTOL concentration of 

0.99 ± 0.04 ng/mL and a 5-HIAA concentration of 0.91 

± 0.07 ng/mL. The 10 ng/mL control was found to have 

a 5-HTOL concentration of 8.7 ± 0.1 ng/mL and a 5-

HIAA concentration of 8.3 ± 0.1 ng/mL. The decrease in 

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concentrations at 10 ng/mL was not unexpected, however, 

due to the ease of autoxidation of these compounds. This 

decrease may be explained by the use of the original calibration curve for the quantitation of controls extracted up 

to 8 days later. These results were found to be acceptable 

for general use and agree well with previously reported 

short-term stability studies.27 As a good laboratory practice 

and in an effort to maintain a high degree of accuracy, 

we would recommend preparing new calibration curves 

at the beginning of each new analysis.

Since actual postmortem urine samples are stored for 

extended periods of time at -20°C, and not 4°C, the 

long-term stability of 5-HTOL and 5-HIAA at -20°C 

was investigated. Controls were separated into 4 mL 

aliquots and stored at -20°C. Five control replicates 

were then analyzed on days 1, 14, 30, 60, 90 and 180. 

Fresh calibration curves were prepared and analyzed on 

each day of analysis. There was no statistical decrease 

in concentration for any of the controls for all times 

investigated. Therefore, the degradation of 5-HTOL and 

5-HIAA over time in properly stored specimens should 

not be of significant concern.

Forensic Urine Analysis

Several studies have been conducted on live human 

subjects examining the shift in the urinary 5-HTOL/5-

HIAA ratio following consumption of ethanol.20, 23, 24, 

27, 31 These reports have clearly demonstrated elevated 

5-HTOL/5-HIAA ratios above those of urine baseline 

levels following ethanol ingestion. They also found that 

the more ethanol an individual consumed, the higher the 

5-HTOL/5-HIAA ratios were. Furthermore, 5-HTOL/5-

HIAA ratios remained elevated above baseline levels for 

hours after ethanol could no longer be detected in the 

body. In one study, both men and women were dosed 

with 0.8 g/kg ethanol, resulting in an average peak urinary 

alcohol concentration of 87.4 mg/dL.20 They found that 

while ethanol could no longer be detected in the body 

after 10 hours, the 5-HTOL/5-HIAA ratio remained 

significantly elevated for up to 16 hours. In a separate 

study, Hagan et al. dosed subjects at 0.6 g/kg, and found 

that the 5-HTOL/5-HIAA ratio remained elevated for 

approximately 7 hours after ethanol was eliminated from 

the body.24 Helander et al. have established a cutoff value 

for the 5-HTOL/5-HIAA ratio of 15 pmol/nmol, below 

which ethanol has not been recently consumed.32, 33, 34

Helander et al. also reported a cursory examination of 

postmortem urine samples with respect to their 5-HTOL/

5-HIAA ratio and, while finding substantial individual 

variations, demonstrated a good correlation between 

ethanol levels and 5-HTOL/5-HIAA ratios.23

We examined 44 urine specimens obtained from fatal 

aviation accident victims. Of the 44 specimens analyzed, 

21 specimens were previously reported negative for ethanol 

by our laboratory, while 23 specimens were previously 

reported positive (ethanol > 10 mg/dL). The initial focus 

of this study was to determine if the previously established 

15 pmol/nmol cutoff was appropriate for postmortem 

urine samples.

The data collected from all 44 specimens are illustrated in Figure 8a. The results show a general trend of 

increasing 5-HTOL/5-HIAA ratio with increasing urine 

ethanol concentration. There is, however, substantial 

inter-individual variation in 5-HTOL/5-HIAA ratios at 

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ethanol concentrations below approximately 150 mg/dL. 

This area has been expanded and can be seen in Figure 8b. 

These variations are not unexpected since the specimens 

utilized in this study are from postmortem sources with 

unknown dietary histories and relatively unknown medical 

and alcohol consumption histories. These results may be 

better represented by averaging groups of individual data 

points and creating a linear plot of 5-HTOL/5-HIAA ratio 

vs. ethanol concentration. This approach is illustrated in 

Figure 9 and clearly demonstrates a relationship between 

5-HTOL/5-HIAA ratio and the amount of ethanol detected 

in a specimen.

5-HTOL/5-HIAA ratios for the 21 ethanol-negative 

postmortem urine specimens ranged from 0.010 - 9.38 

pmol/nmol, with an average of 2.52 ± 2.94 pmol/nmol 

(mean ± S.D.). 5-HTOL concentrations in these specimens 

ranged from 5.22 nM - 1.67 μM (0.930 - 296 ng/mL) 

with an average of 220 nM (39.5 ng/mL). 5-HIAA concentrations ranged from 5.92 μM - 39.1 mM (1.13 - 117 

μg/mL) with an average of 142 μM (27.3 μg/mL). All of 

the negative specimen ratios fell well below the previously 

established 15 pmol/nmol cutoff. In fact, our average 5-

HTOL/5-HIAA ratio is more than 4 standard deviations 

below the 15 pmol/nmol cutoff (2.52 + (2.94 * 4) = 14.3 

pmol/nmol). Therefore, based on our average and S.D., the 

probability of having a 5-HTOL/5-HIAA ratio above the 

previously established 15 pmol/nmol cutoff from a person 

who has not consumed alcohol is less than 1 in 10,000.

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The 23 ethanol-positive specimens had ethanol concentrations ranging from 11 to 520 mg/dL. The corresponding 5-HTOL/5-HIAA ratios ranged from 19.1 

to 551 pmol/nmol. All of the positive specimens ratios 

fell well above the previously established 15 pmol/nmol 

cutoff. Figure 10 graphically shows the difference between the 5-HTOL/5-HIAA ratios of ethanol-positive 

and ethanol-negative specimens. Our data indicate that 

the previously established 15 pmol/nmol cutoff is highly 

appropriate for postmortem urine specimens, and we 

recommend it be employed as the ethanol positive cutoff 

ratio in such studies.

We applied this novel LC/MS method to urine samples 

from 2 cases suspected of experiencing postmortem ethanol formation. Case #1 was obtained from a fatal aviation 

accident that occurred in remote, mountainous terrain. 

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The victim was not recovered from the accident scene 

for more than 24 hours. This case had a urine ethanol 

concentration of 31 mg/dL. The case also had a blood 

ethanol value of 93 mg/dL and a vitreous humor of 3 

mg/dL. These specimens were also notably missing other 

volatiles. The abnormal nature of the ethanol distribution 

in these 3 biological matrices suggests postmortem ethanol formation. Conversely, the lack of other commonly 

analyzed volatiles may suggest the absence of postmortem 

ethanol production.16, 35, 36, 37, 38, 39, 40, 41 However, the use 

of other volatiles as a marker for postmortem ethanol 

formation can be misleading and, therefore, caution 

must be exercised.2 To conclusively determine if recent 

ethanol ingestion occurred, we investigated the 5-HTOL/

5-HIAA ratio. We found the 5-HTOL/5-HIAA ratio to 

be 1.62 pmol/nmol, which was substantially below the 

established 15 pmol/nmol cutoff. This result indicates 

an absence of recent ethanol ingestion and supports the 

conclusion that the ethanol present in this case was due 

to postmortem formation.

Case #2 was an aviation fatality that was recovered 

from water approximately 2.5 months after the accident 

occurred. The urine and blood ethanol concentrations 

were 31 mg/dL and 16 mg/dL, respectively. Vitreous 

humor, heart and skeletal muscle were also analyzed and 

were found to have ethanol concentrations of 17 mg/dL, 

8 mg/hg and 12 mg/hg, respectively. Various volatiles, 

including large amounts of acetaldehyde, n-propanol and 

n-butanol were also present in these samples. The blood 

and tissue samples were putrefied and the urine was red 

and bloody. While the distribution of ethanol between 

fluid and tissues was reasonably consistent with ethanol 

consumption, the visual confirmation of sample putrefaction and the presence of various volatiles suggests that 

the ethanol found in this case may be due to postmortem 

formation. We found the 5-HTOL/5-HIAA ratio to be 

1.95 pmol/nmol. This value is below 15 pmol/nmol, 

thus indicating the absence of recent ethanol ingestion. 

The presence of ethanol in this case was reported as 

postmortem formation.

CONCLUSION

This study shows the effectiveness of LC/MS in the 

analysis of 5-HTOL and 5-HIAA. It also demonstrates 

the utility of the 5-HTOL/5-HIAA ratio in determining 

ethanol origin in postmortem urine specimens. One of 

the most important aspects of this novel method is the 

simultaneous analysis of both compounds using one 

extraction method and one analytical technique. This 

greatly increases the precision in the methodology and, 

thus, the certainty of subsequent conclusions. The relative 

simplicity of this procedure should make the 5-HTOL/5-

HIAA ratio methodology more readily applicable to the 

field of forensic toxicology. The application of this easy, 

rapid and accurate method is not limited to postmortem 

samples. Contested antemortem ethanol results could also 

readily be evaluated through the use of this procedure. 

The postmortem formation of ethanol remains one of the 

biggest concerns in the forensic toxicology community; 

however, the utilization of this method, as it applies to 

postmortem urine samples, may offer a substantial improvement in such investigations.

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15

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