Impact of hair-care products on FAEE hair concentrations in substance abuse monitoring

Abstract

Previous studies have indicated that the use of high-ethanol-content (>65%) hair-care products may elevate fatty acid ethyl ester (FAEE) concentrations in hair. In this case series, nine individuals were identified by FAEE analysis to be chronic alcohol abusers in the context of child-welfare substance abuse monitoring. Based on patient claims of moderate or no alcohol consumption, the presence of ethanol in the patients' hair-care regimens was investigated. Samples were additionally tested for the presence of ethyl glucuronide (EtG). From a total of nine patients, 12 hair samples were submitted for analysis. Patient histories were obtained as well as Material Safety Data Sheets (MSDS) listing hair-care product ethanol content. Hair samples were pre-washed to remove external contamination and analyzed for FAEE and EtG by GC-MS. According to the Society of Hair Testing consensus guidelines, FAEE levels exceeding 0.50 ng/mg and/or EtG levels exceeding 30 pg/mg indicate chronic excessive alcohol consumption. Upon initial analysis, the nine samples exhibited positive FAEE findings ranging from 0.496 to 4.984 ng/mg. MSDS review revealed the presence of ethanol from 10% to 95% by volume in at least one hair-care product used by each individual. Results of the EtG analysis ranged from 1.9 to 23.5 pg/mg. These findings indicate that regular use of products with ethanol content as low as 10% can impact FAEE results. EtG analysis should be used to confirm FAEE findings and appears to be unaffected by hair-care products, likely due to alternative mechanisms of incorporation.

Full text

ORIGINAL PAPER
Impact of hair-care products on FAEE hair concentrations
in substance abuse monitoring
Joey Gareri &Brice Appenzeller &Paula Walasek &
Gideon Koren
Received: 1 October 2010 / Revised: 15 January 2011 / Accepted: 16 January 2011 / Published online: 8 February 2011
#Springer-Verlag 2011
Abstract Previous studies have indicated that the use of
high-ethanol-content (>65%) hair-care products may ele-
vate fatty acid ethyl ester (FAEE) concentrations in hair. In
this case series, nine individuals were identified by FAEE
analysis to be chronic alcohol abusers in the context of
child-welfare substance abuse monitoring. Based on patient
claims of moderate or no alcohol consumption, the
presence of ethanol in the patients’hair-care regimens was
investigated. Samples were additionally tested for the
presence of ethyl glucuronide (EtG). From a total of nine
patients, 12 hair samples were submitted for analysis.
Patient histories were obtained as well as Material Safety
Data Sheets (MSDS) listing hair-care product ethanol
content. Hair samples were pre-washed to remove external
contamination and analyzed for FAEE and EtG by GC-MS.
According to the Society of Hair Testing consensus guide-
lines, FAEE levels exceeding 0.50 ng/mg and/or EtG levels
exceeding 30 pg/mg indicate chronic excessive alcohol
consumption. Upon initial analysis, the nine samples
exhibited positive FAEE findings ranging from 0.496 to
4.984 ng/mg. MSDS review revealed the presence of
ethanol from 10% to 95% by volume in at least one hair-
care product used by each individual. Results of the EtG
analysis ranged from 1.9 to 23.5 pg/mg. These findings
indicate that regular use of products with ethanol content as
low as 10% can impact FAEE results. EtG analysis should
be used to confirm FAEE findings and appears to be
unaffected by hair-care products, likely due to alternative
mechanisms of incorporation.
Keywords Fatty acid ethyl ester (FAEE) .Ethyl
glucuronide (EtG) .Alcohol .Ethanol .Hair
Introduction
The use of biomarkers of ethanol consumption is increasing
due to the short window of detection of ethanol in
traditional matrices such as blood and urine and the
importance of detecting alcohol abuse in the context of
driver licensing and substance abuse monitoring programs
[1–4]. Fatty acid ethyl esters (FAEE) and ethyl glucuronide
(EtG), both minor non-oxidative metabolites of ethanol,
have been established as effective biomarkers of excessive
alcohol consumption in human hair [5,6]. The Society of
Hair Testing recently established consensus guidelines for
the use of FAEE and EtG analysis in the proximal 3 cm of
hair; recommending cut-off values of 0.5 ng/mg and 30 pg/mg,
respectively, for the determination of frequent, excessive
alcohol consumption [7].
Published in the special issue Forensic Toxicology with Guest Editors
Frank T. Peters, Hans H. Maurer, and Frank Musshoff.
J. Gareri (*):P. Walasek :G. Koren
Division of Clinical Pharmacology & Toxicology,
Hospital for Sick Children,
555 University Avenue,
Toronto, Ontario M5G 1X8, Canada
e-mail: joey.gareri@sickkids.ca
B. Appenzeller
Laboratory of Analytical Human Biomonitoring,
Université du Luxembourg,
162A avenue de la Faïencerie,
1511 Luxembourg, Luxembourg
J. Gareri
Department of Pharmaceutical Sciences,
University of Toronto,
555 University Avenue,
Toronto, Ontario M5G 1X8, Canada
Anal Bioanal Chem (2011) 400:183–188
DOI 10.1007/s00216-011-4685-0

Hair, as a toxicological matrix external to the body, is
subject to contamination from the environment. This aspect
of hair analysis is well-characterized and can be controlled
in many circumstances through sample decontamination
and the detection of metabolites [6]. In some situations,
such as child-welfare investigations, the detection of
environmental (i.e., passive) exposure to drugs (e.g.,
cocaine) can be considered an advantage of this meth-
odology [8]. External contamination due to alcohol is less
well-described in the literature; previous studies have
demonstrated that shampooing, multiple cosmetic treat-
ments, and the presence of detectable levels of FAEE in
hair products do not affect FAEE concentrations in hair
[9]. The direct external application of an ethanol-
containing lotion (62.5% by volume) to the scalp and in
vitro incubation of cut hair in 96% ethanol vapor for over
2 days, however, has demonstrated a capability of
producing elevated FAEE concentrations [9,10]. These
studies, while raising the issue that FAEE concentrations
in hair may be subject to considerable elevation by
ethanol-containing hair-care products, are limited to the
description of only one hair-care product and an
unrealistic in vitro paradigm of external ethanol expo-
sure. Coupled with this ambiguous effect of hair-care
products is the fact that the study populations involved in
alcohol hair biomarker development have overwhelming-
ly been male addiction treatment patients and post-
mortem subjects [5,11,12]. The effectiveness of use of
FAEE hair analysis in women (who are more likely to use
cosmetic hair products) is less well-characterized. The
objective of the following study is to determine the impact
of varying concentrations of ethanol in hair-care products
on the concentration of FAEE and EtG in hair in a
substance abuse monitoring population that is primarily
female.
Methods
FAEE analysis Hair samples were submitted for routine
toxicological analysis in the context of substance abuse
monitoring for child-welfare services or private custody
dispute. Hair sample collection was conducted by cutting
approximately 50 mg of hair from the vertex posterior of
the scalp; hair samples were cut as close to the scalp as
possible. The proximal 3 or 6 cm of hair were then
sectioned, washed, mulched, and aliquoted out for analysis
of FAEE. Prior to the recently published SOHT consensus
for FAEE in hair, published data demonstrated approxi-
mately 90% sensitivity and 90% specificity for the
determination of frequent, excessive alcohol consumption
examining the proximal 6 cm of hair [11,12]. Samples
were pre-washed with heptane to remove external
contamination. Twenty milligrams of hair (±0.5 mg)
Fig. 1 FAEE concentration vs.
EtOH content of hair-care
product vs. EtG concentration.
*Data labels below the axis
show minimum EtOH fraction.
Data labels above the bars show
maximum EtOH fraction
184 J. Gareri et al.

were weighed out for extraction and analysis. Extraction
and analysis of four species of FAEE (ethyl oleate, ethyl
myristate, ethyl palmitate, ethyl stearate) was conducted
by liquid–liquid extraction with a mixture of heptane and
dimethyl sulfoxide followed by automated headspace
solid-phase microextraction and GC-MS/EI analysis as
previously reported [13]. FAEE were quantified using
deuterated internal standards.
Subject recruitment A total of nine subjects contacted our
laboratory contesting positive FAEE findings (≥0.50 ng/mg)
and claiming moderate or no alcohol consumption.
Alcohol use histories were obtained from the hair donor
themselves or their social workers. Material Safety Data
Sheets (MSDS) listing hair-care product chemical content
were submitted to the laboratory for review by hair
donors or their representatives. MSDS were reviewed for
the presence and content (by volume) of ethanol in the
respective product.
EtG analysis Sixteen hair samples were forwarded for EtG
analysis to confirm initial FAEE findings over the same
length of hair. Four samples were sent as controls; three
FAEE-positive with self-reported binge drinking histories
or positive urine alcohol screens, and one FAEE-negative
claiming 2 months of abstinence from alcohol.
Three- or six-centimeter hair samples were pre-washed with
water and acetone to remove external contamination. Hair
was then pulverized and aliquoted out for extraction and
analysis. Samples weighed between 16.7–25.24 mg, with
the exception of one sample (Sample “F”) that was 7 mg.
EtG was extracted from hair samples with water followed
by solid-phase extraction and analysis by GC-MS/NCI as
previously reported [14]. EtG was quantified using a
deuterated internal standard.
Results
Upon initial analysis, the 12 FAEE-positive samples ex-
hibited concentrations ranging from 0.496 to 4.984 ng/mg
(see Fig. 1). The EtG results for all FAEE-positive
samples fell below the cut-off for determining heavy
Product Percent ethanol by volume
Down Under Natural’s Fruit Kicks Bodifying Finishing Mist Maximum Hold 85–95%
Herbal Essences Dangerously Straight Pin Straight Non-Aerosol Hairspray 30–60%
Tresemme Tres spray European Sculpting Spray Super Hold 94–95%
Pantene Pro-V Style Classic In Control Mousse 0
Tresemme Shampoo 24 Hour Body 0
Finesse Shampoo BeautiFULL Volume 0
Infusium 23 Repairologie Conditioner 0
Fruiteen Extreme Styling Gel 20–60%
Down Under Natural’s Fruit Kicks Moisture and Shine
Conditioner—All Hair Types
0
Dove Hairspray (Aerosol)—All Variants 15–30%
Exact Hairspray (six varieties) 80–85%
Herbal Essences Set Me Up Aerosol Hairspray Maximum Hold 10–30%
Designline Pure Results Volume Mist 15%
Aussie Catch the Wave Aerosol Hairspray 10–30%
Aussie Instant Freeze Extreme Hold Aerosol Hairspray 30–60%
G2b Styl-tini Laminate Hair Spray 17–30%
Tresemme Tres Two Hair Spray Extra Hold <70%
Table 1 Concentration of etha-
nol in various cosmetic hair
products
Not all ethanol-negative prod-
ucts reviewed are listed
Sample [FAEE] ng/mg
(0.50 ng/mg cut-off)
[EtG] pg/mg
(30 pg/mg cut-off)
Details
C1 4.613 96.1 Recent positive urine alcohol screens and
third party reports of heavy drinking
C2 0.601 22.7
a
Self-reported 2-3 binge episodes in 3 months
C3 1.315 3.5 Recent positive urine alcohol screens
C4 0.421 3 Abstinent for 2 months, alcohol in hairspray
Table 2 Results for control
samples
a
Borderline positive EtG level;
likely consistent with heavy alco-
hol use, some studies indicate EtG
concentrations of 25 pg/mg are
sufficient to establish heavy
drinking [5]
Impact of hair-care products on substance abuse monitoring 185

Fig. 2 a Cumulative [FAEE]
vs. ethanol content by
volume. b[EtG] vs. ethanol
content by volume. c[EtG]
and [FAEE] vs. mean ethanol
content by volume
186 J. Gareri et al.

alcohol use (30 pg/mg); EtG concentration ranged from
1.9to23.5pg/mg.
Review of the MSDS sheets for numerous hair-care
products revealed the presence of ethanol in concentrations
between 10% and 95% by volume in at least one hair-care
product used by each individual. See Table 1for a full
listing of the products and their corresponding ethanol
concentrations. The presence of ethanol in these products
was primarily confined to hairsprays and ‘mists’. Ingredient
listings on the hair products themselves listed ethanol as
“SD Alcohol 40”,“Denatured Alcohol”,“Alcohol”, and
“Ethyl Alcohol”.
Three positive control samples were analyzed for EtG as
well (C1–C3). Analysis of the control samples for EtG
confirmed a recent history of heavy alcohol use in two
individuals as evidenced initially by positive FAEE find-
ings, but returned a negative result for the third control
sample (see Table 2). One negative control was tested as
well, and confirmed to be negative for heavy alcohol use by
EtG analysis.
FAEE, EtG, and hair product ethanol-content data were
compared to assess the possible presence of an association
between these variables. Minimum, maximum, and mean
ethanol concentrations in the hair-care products did not
correlate with the concentration of FAEE or EtG in hair
samples (see Fig. 2a–c).
Discussion
The significant impact of common hair-care products on
FAEE levels indicates that confirmation of FAEE results
by EtG is recommended, particularly when testing a
female population. The ability of ethanol content as low
as 10% by volume possibly affecting FAEE levels
suggests further studies in low-level ethanol exposure,
for example individuals working as hair dressers, bar-
tenders, or in distilleries.
When monitoring individuals for heavy alcohol use via
FAEE analysis, the hair donor should be advised to
discontinue use of any ethanol-containing hair-care products
for a minimum of 1 month prior to analysis. Consecutive hair
samples were collected from four of the subjects in this study
(see Fig. 3); subject 1962F submitted her second sample
3 weeks after discontinuation of hairspray use and demon-
strated a corresponding 80% reduction in FAEE concentra-
tion. Exact schedules of hair-care product application or
discontinuation amongst the four subjects were not available.
Ethanol was primarily present in hairsprays; none of the
shampoos and conditioners reviewed contained any listed
ethanol, therefore routine hair hygiene evidently does not
have the potential to affect hair FAEE concentrations. It is
evident that the false elevation of FAEE levels is a result of
localized FAEE production due to the local presence of
ethanol on the scalp; other typical hygienic ethanol exposures,
such as ethanol-containing mouthwashes, because they are not
applied directly to the scalp and will exhibit limited ethanol
absorption through the oral mucosa, have no theoretical basis
upon which to expect an impact on hair FAEE levels.
The data herein also demonstrate that the use of EtG to
confirm total abstinence may be considerably affected by
hair-care products. A cut-off value of 7 pg/mg has
demonstrated some efficacy as a guideline to differentiate
abstinence from any alcohol consumption in primarily male
Fig. 3 Consecutive samples
from four individuals
Impact of hair-care products on substance abuse monitoring 187

subjects [5,6,15]. Five of 12 samples negative for heavy
drinking (<30 pg/mg EtG) in this study had EtG levels
above 7 pg/mg. Amongst these five subjects, reported
history in addition to the hair-care products was not
available for three of them. Both of the other subjects had
alcohol abstinence histories that included both self-report of
abstinence and third party report (addiction worker and
social worker) of good compliance with abstinence require-
ments. One subject, 1948F (see Fig. 3), tested above 7 pg/
mg on her first sample in spite of a third-party-supported
history of over 1 year of abstinence from alcohol. A clear
pattern of hair-product ethanol impact on EtG levels in the
moderate drinking range (7–30 pg/mg) does not emerge
based on the data herein; however, this particular case
report, coupled with the fact that over 40% of the samples
(5/12) exhibited EtG levels above 7 pg/mg suggest a
possible effect that warrants further study.
Conclusions
These findings suggest that the use of EtG in hair to
establish a history of excessive alcohol consumption is
unaffected by ethanol-containing hair-care products. Alter-
natively, a cautionary approach is warranted to the use of
EtG hair analysis in providing a history of any alcohol
consumption until further studies are conducted to clarify
the possible impact of hair-care products in raising hair EtG
levels into the moderate drinking range (7–30 pg/mg).
Regular use of products with ethanol content as low as
10% can potentially elevate FAEE results above the
recommended cut-off to determine heavy drinking behavior
(0.50 ng/mg). No correlation is evident between FAEE or
EtG concentrations and % volume of ethanol in hair-care
products (one individual (C4) showed negative results with
ethanol-positive product use).
The risk for false-positive results appears high when
monitoring a female population, individuals under mon-
itoring should be advised to discontinue use of any
ethanol-containing hair-care products for a minimum of
1 month prior to analysis. EtG analysis should be used to
confirm FAEE findings and appears to be unaffected by
hair-care products, likely due to alternative mechanisms
of incorporation into the hair shaft.
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