Journal of Analytical Toxicology 2013;37:227 –232
doi:10.1093/jat/bkt008 Advance Access publication March 6, 2013
Article
EtG/EtS in Urine from Sexual Assault Victims Determined by UPLC –MS-MS
Solfrid Hegstad1*, Arne Helland1, Cecilie Hagemann2,3, Lisbeth Michelsen1 and Olav Spigset1,4
1
Department of Clinical Pharmacology, St. Olav University Hospital, Trondheim, Norway, 2Department of Public Health and General
Practice, Norwegian University of Science and Technology, Trondheim, Norway, 3Department of Obstetrics and Gynecology,
St. Olav University Hospital, Trondheim, Norway and 4Department of Laboratory Medicine, Children’s and Women’s Health,
Norwegian University of Science and Technology, Trondheim, Norway
*Author to whom correspondence should be addressed. Email: [email protected]
In cases of sexual assault, victims often present too late for the detection of ethanol in biological samples. An ultra-performance
liquid chromatography –tandem mass spectrometry (UPLC–MS-MS)
method was developed and validated for the determination of ethyl
glucuronide (EtG) and ethyl sulfate (EtS) in urine. Sample preparation prior to UPLC –MS-MS analysis was a simple sample dilution.
The calibration ranges were 0.2 –20 mg/L, and between-assay relative standard deviations were in the range of 0.7– 7.0% at concentrations of 0.3, 3.0 and 7.0 mg/L. Urine samples were analyzed
from 59 female patients presenting to the Sexual Assault Centre at
St. Olav University Hospital in Trondheim, Norway between
November 2010 and October 2011. EtG and EtS results were fully
concordant, and positive in 45 of the 48 cases with self-reported
alcohol intake. In contrast, ethanol was detectable in only 20 of
these cases, corresponding to sensitivities of 94 and 42%, respectively. Of the patients reporting no alcohol intake, none had positive
EtG/EtS findings. These data show that analysis of EtG and EtS
greatly increases the detection window of alcohol ingestion in
cases of sexual assault, and may shed additional light on the involvement of ethanol in such cases. The victims’ self-reported
intake of alcohol seems to be reliable in this study, according to
the EtG/EtS findings.
Introduction
Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are nonoxidative minor metabolites of ethanol formed by the conjugation
of ethanol to glucuronic acid via uridine diphosphate
(UDP)-glucuronosyltransferases and to sulfate via sulfotransferases, respectively (1 –3). EtG and EtS are detectable in urine
for considerably longer than ethanol. The detection times for
EtG and EtS in urine range from hours up to several days, depending on the amount consumed. Therefore, urinary testing
of these metabolites can be used for the identification of
recent alcohol consumption, even after the ingested ethanol
itself has been eliminated from the body (4– 8).
Drug and/or alcohol intake is often implied in cases of
sexual assault, either voluntary or as a result of suspected
spiking of drinks. Ethanol, either alone or together with recreational/illicit drugs, has been the most common finding in previous surveys of alleged drug-facilitated sexual assault (9 –11).
Many victims hesitate to seek assistance, which often leads to a
considerable delay in obtaining urine samples for analysis. In
such cases, the measurement of EtG and EtS can represent a
valuable supplement to ethanol analysis, and contribute to a
more comprehensive evaluation of the victim’s state of intoxication at the time of the assault.
Several liquid chromatography – mass spectrometry (LC –
MS) and liquid chromatography – tandem mass spectrometry
(LC – MS-MS) methods have been described in the literature
for the determination of EtG and EtS in urine (3, 12 – 16).
Recently, two ultra-performance liquid chromatography
(UPLC) – MS-MS methods have been described for the determination of EtG (17), and EtG and EtS (18), respectively, in
urine.
The aims of the present study were to develop and validate a
fast and selective UPLC –MS-MS method for the determination
of EtG and EtS in urine, using a simple sample preparation by
means of a Tecan Freedom Evo pipetting robot platform, to
apply the method on urine samples from a group of female
patients subjected to sexual assault and to explore whether
EtG/EtS analysis can aid the investigation of possible alcohol
involvement in such cases.
Material and Methods
Patients and sampling
Urine samples were obtained from 59 female patients 12
years who were examined at the Sexual Assault Centre at
St. Olav University Hospital in Trondheim, Norway, between
November 2010 and October 2011. Self-reported alcohol consumption in relation to the assault, in addition to the time
lapse from the end of alcohol intake to urine sampling, were
estimated by assessment of the patient files. Intake of alcohol
was converted to alcohol units and classified in the following
groups: no alcohol intake, intake of 1 –6 alcohol units, intake
of .6 alcohol units and intake of an unknown/uncertain
amount. Six alcohol units was chosen as a reasonable limit separating social drinking from excessive drinking that may cause
drunkenness. The national Norwegian definition of one alcohol
unit was used, i.e., 12.8 g of ethanol, which corresponds to approximately one standard can or bottle of 4.5% beer (330 mL),
one standard glass of 12% wine (120– 150 mL) or one standard
drink with 40% spirits (40 mL) (19).
The present study represents a sub-sample of a larger study
of 315 patients 12 years of age who presented to the Sexual
Assault Centre from July, 2003, to October, 2011, and in which
urine and/or blood were obtained for toxicological analyses.
Exclusion criteria from the study were male sex, no (suspected) sexual assault or no medical examination performed.
According to instructions from the Regional Committee for
Research Ethics, which approved the study, these patients
received a letter of information about the study. Those who
declined to participate on the basis of this letter were also
excluded.
# The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Analysis of EtG and EtS
Chemicals and reagents
EtG, EtS, EtG-d5 and EtS-d5 were obtained from Lipomed
GmbH (Weil am Rhein, Germany). Other chemicals were of
LC–MS, high-performance liquid chromatography (HPLC) or
analytical grade from various commercial sources. External
quality control samples were purchased from Equalis (Uppsala,
Sweden: http://www.equalis.se/sv/start.aspx).
Preparation of solutions
For each compound, stock solutions were prepared to a concentration of 1 mg/mL in methanol and were used for calibrator and quality control (QC) samples. The stock solutions were
fortified with methanol (1,000 mg/L) and used for calibration
and QC solutions prepared in urine with concentrations of
0.20, 0.50, 1.0, 5.0, 10.0 and 20.0 mg/L, and 0.30, 3.0, 7.0 and
15.0 mg/L, respectively. The internal standards EtG-d5 and
EtS-d5 were diluted with water to a concentration of 2.5 mg/L.
The stock solutions were stored at –208C; standards in urine
and internal standard were stored at 48C.
Sample treatment
A 100 mL aliquot of a urine specimen was mixed with 100 mL
of internal standard and diluted 10 times with water directly
into a 96 well plate by Tecan Freedom Evo 100 or 150 pipetting robots (Tecan Nordic, Mölndal, Sweden).
Instruments
UPLC was performed using a Waters Acquity System (Waters,
Manchester, UK). Separation was performed on a Waters HSS
T3 column (2.1 100 mm, 1.8 mm) using gradient elution at a
flow rate of 0.6 mL/min with the following binary solvent
system: 0.1% formic acid in water (A) and 100 % methanol (B).
The gradient was run as follows: 0 min, 99% A, 1% B; 1.0 min,
80% A, 20% B; 1.1 min 10% A, 90% B; 1.7 min, 99 % A, 1%
B. The time before the next injection of 1 min was sufficient to
equilibrate the column. The column temperature was kept constant at 508C. The injection volume was 5 mL performed by
partial loop injection using needle overfill as injection technique. A dual wash was applied to the autosampler using
600 mL methanol –isopropanol–acetonitrile –water –ammonia
(25:25:25:23:2, v/v), and 200 mL methanol–isopropanol –
acetonitrile –water–formic acid (25:25:25:23:2, v/v), denoted as
weak and strong wash.
A Xevo TQ-S tandem-quadrupole mass spectrometer (Waters)
was used, equipped with a Z-spray electrospray interface.
Negative ionization was performed in the multiple reaction
monitoring (MRM) mode. The capillary voltage was set to
1.0 kV, the source block temperature was 1208C and the desolvation gas nitrogen was heated to 6008C and delivered at a
flow rate of 1,000 L/h. The m/z 221.0 . 85.1 and 221.0 . 75.1
transitions (cone voltages: 40 V; collision energy: 15 eV) were
monitored for EtG, m/z 125.0 . 97.1 transition (cone voltages:
50 V; collision energy: 15 eV) was monitored for EtS, m/z
226.0 . 85.1 transition (cone voltages: 40 V; collision energy:
15 eV) was monitored for EtG-d5 and m/z 130.0 . 97.8 transition (cone voltages: 50 V; collision energy: 15 eV) was monitored for EtS-d5. System operation and data acquisition were
228 Hegstad et al.
controlled using Mass Lynx 4.1 software (Waters). All data were
processed with the Target Lynx quantification program
(Waters). The analytes were identified by comparing the retention time of the corresponding calibrator and QC samples. The
ratio between the two MRM transitions (EtG) was also compared with those of the corresponding calibrator and QC
samples and should not deviate more than 20%.
Method validation
The six-point calibration curves (five replicates of each standard) were based on peak area ratios of the analyte relative to
the internal standard using a weighted (1/x) linear line, which
included the origin. Within-assay precision was estimated by
the analysis of 10 separate replicates of QC samples at four
concentrations in a single assay. Between-assay precision and
accuracy were determined by the analysis of one replicate at
four QC concentrations on 10 different days. Matrix effects
(MEs) were evaluated by the method proposed by Matuszewski
et al. (20). The analyte signal in the spiked water was compared with the analyte signal in the matrix, and the ME was
defined as ME (%) ¼ (matrix area/water area) 100. Five replicates of urine sample extracts (from five different individuals)
were analyzed. The concentrations were 0.30 and 7.0 mg/L.
Negative urine samples supplemented with various concentrations of analytes (0.01– 0.10 mg/L) were analyzed to determine
the limit of quantification (LOQ). Samples of 0.10 mg/L of EtG
and 0.080 mg/L of EtS were run in one replicate on six different days, and the concentrations were calculated using a calibration curve in the ranges of 0.10 –5.0 mg/L (EtG) and 0.080 –
5.0 mg/L (EtS). The signal-to-noise (S/N) criteria for the LOQ
samples were 10 and precision of calculated concentrations
were within + 20%. The limit of detection (LOD) was determined by dilution and evaluation of S/N (3).
Analysis of ethanol and creatinine
Ethanol and creatinine were determined by the test kits
EthanolGen2 (ETOH2) and Creatinine Jaffé Gen.2 (CRJ2U)
on a Cobas Intergra 400þ multianalyzer (Roche Diagnostics
Norway AS, Oslo, Norway). The analytical cutoff for ethanol
was 10 mg/dL.
Calculations and statistics
The results from the analyses of ethanol, EtG and EtS were
compared with the victims’ self-reported ethanol intake to determine the reliability of the method. The analytical efficiency
is reported in terms of sensitivity, specificity, positive predictive value, negative predictive value and accuracy.
For EtG and EtS, the concentrations in urine are dependent
on whether urine is relatively concentrated or diluted. To
correct for this factor when assessing the time-dependent
elimination of EtG and EtS, the concentrations of EtG and EtS
were adjusted to a standard creatinine concentration of
100 mg/dL, by using the following equation: standardized concentrations of EtG or EtS ¼ (measured concentrations of EtG
or EtS/measured creatinine concentration in mg/dL) 100 mg/dL. In contrast, ethanol easily passes biologic membranes and the urinary level is rapidly equilibrated with the
plasma level; thus, the ethanol concentration in urine is not
subject to urinary dilution variances (21). Therefore, no creatinine adjustment is necessary for ethanol.
Statistical analyses were performed with SPSS version 18.0.
McNemar’s test was used to compare differences in the reliability of urinary ethanol, EtG and EtS for verifying self-reported
alcohol intake. Spearman’s rank correlation was used to test for
the relationship between concentrations of EtG and EtS, and
between EtG/EtS concentrations and time from alcohol intake
to urine sampling, respectively. P values , 0.05 were considered statistically significant.
Results
Method validation
Calibration curves were made for each compound in the concentration range, as shown in Table I. The correlation coefficient was above 0.999 for both compounds. In cases with a
concentration higher than 20 mg/L, the samples were diluted
and reanalyzed.
The MRM chromatograms of a real sample are shown in
Figure 1. The calibration range, LOD, LOQ, within-assay precision, between-assay precision and bias for EtG and EtS are presented in Table I. The within-assay coefficients of variation
(CVs) were 2.7 –4.9%, and the between-assay CVs were 0.7 –
7.0%. The bias was in the range of 21.0 to 4.0%. The observed
matrix effects indicated some ion suppression for EtG and an
ion enhancement for EtS (Table II). However, when corrected
to the internal standard, the observed matrix effects and CVs
were reduced significantly for both compounds. External
quality control samples, which were run with the presented
method, showed good correspondence in the low to medium
concentration range. The Z-scores were jZj 1.3 for all measurements when compared to the mean value of the other laboratories using the same methodological platform (i.e., LC–
MS-MS) as this study, thus indicating good accuracy for the
quantitative results obtained with the method.
Application
The mean age of the 59 women included in the study was 24
years (range 14– 61 years). Of the women, 48 (81%) reported
intake of alcohol in relation to the assault. The mean time from
alcohol intake to when the urine samples were obtained was
20.9 h (range 1.5 –108 h).
Ethanol was found in 20 samples, whereas EtG and EtS were
found in 45 samples. In no cases, ethanol, EtG or EtS were
found in urine from patients reporting no intake of alcohol.
The positive ethanol concentrations ranged from 37 to
280 mg/dL. The positive EtG concentrations ranged from 0.34
to 1,123 mg/L and the positive EtS concentrations ranged from
0.18 to 322 mg/L (not corrected for creatinine).
There was full concordance between the findings of EtG and
EtS, and there was also a close and highly significant relationship (r ¼ 0.981; p , 0.001) between the concentrations of EtG
and EtS (Figure 2), with a mean EtG/EtS ratio of 3.5. There
were, however, large interindividual variations in this ratio,
ranging from 1.4 to 7.4.
Using self-reported intake of alcohol as a reference, the
numbers of true and false positives and negatives, in addition
to sensitivity, specificity, positive and negative predictive value
and accuracy, are shown in Table III. The sensitivities and accuracies for EtG and EtS were significantly higher than for
ethanol ( p , 0.001). The three subjects with false negative
EtG/EtS results had the following characteristics: reported
intake of three alcohol units, time interval of 15.5 h from
alcohol intake to sampling (Case A); reported intake of one
alcohol unit, time interval of 51 h from alcohol intake to sampling (Case B); reported intake of an unknown amount of
alcohol, time interval of 65 h from alcohol intake to sampling
(Case C).
There was a significant decrease with time after alcohol
intake in creatinine-corrected EtG and EtS concentrations
(Figure 3). As shown in the figure, approximate detection
times of 2 –3 days could be anticipated.
Discussion
Method validation
Dilution of the urine sample prior to the LC–MS-MS analysis of
EtG and EtS has been reported previously (14, 17, 18).
Solid-phase extraction (SPE) with an anion-exchange column
combined with LC–MS-MS has been shown to be a more reliable method for the quantification of EtG in urine than a direct
injection LC– MS-MS method (17). EtG and EtS are highly polar
analytes and a selective SPE method is difficult to conduct simultaneously for both compounds. The anion-exchange extraction method is not suitable for EtS due to low extraction
recovery (22). Therefore, a simple and time-saving dilution
method was developed with a sufficient selectivity and S/N
ratio at the lowest calibration level for both compounds. In this
study, calibrator and QC samples were prepared from the same
vendor. An external QC system was used to assure the validity
of the method.
Table I
Calibration Range, Correlation Coefficient, LOD, LOQ, Within-Assay and Between-Assay Precisions and Bias for EtG and EtS in Urine
Analyte
Calibration range
(mg/L)
Correlation coefficient (r value)
(n ¼ 5)
LOD
(mg/L)
LOQ
(mg/L)
Theoretical concentration
(mg/L)
Within-assay CV (%)
(n ¼ 10)
Between-assay CV (%)
(n ¼ 10)
Bias (%)
(n ¼ 10)
EtG
0.20– 20
0.999
0.030
0.10
EtS
0.20– 20
0.999
0.020
0.080
0.30
3.0
7.0
15.0
0.30
3.0
7.0
15.0
4.7
4.1
3.3
3.9
4.9
3.6
2.7
3.6
7.0
3.8
2.4
0.7
5.8
4.5
2.8
1.7
2.4
4.0
2.8
–0.9
2.5
3.1
3.4
–1.0
EtG/EtS in Urine from Sexual Assault Victims Determined by UPLC– MS-MS 229
Figure 1. MRM chromatograms of an authentic sample: EtG (A) (the peaks at 0.93 and 1.1 min are due to unknown interfering compounds observed to be present in some
samples) EtS (B). Determined concentrations: EtG ¼ 1.9 mg/L, EtS ¼ 0.81 mg/L.
Table II
Evaluation of MEs for EtG and EtS in Urine
Analyte
EtG
EtS
Table III
Results from the Analyses of Ethanol, EtG and EtS Compared to Self-Reported Intake of Ethanol
in 59 Victims of Sexual Assault*
Theoretical
concentration
(mg/L)
ME
(%)
Relative
ME, CV
(%)
ME corrected
with internal
standard (%)
Relative ME to
internal standard,
CV (%)
0.30
7.0
0.30
7.0
82.9
82.2
159.7
138.9
11.7
11.1
12.7
15.0
98.2
94.3
107.4
99.8
5.1
3.2
2.4
2.6
Variable
Ethanol
EtG
EtS
True positives
True negatives
False positives
False negatives
Sensitivity
Specificity
Positive predictive value
Negative predictive value
Accuracy
20
11
0
28
20/48 ¼ 0.42
11/11 ¼ 1.0
20/20 ¼ 1.0
11/39 ¼ 0.28
31/59 ¼ 0.53
45
11
0
3
45/48 ¼ 0.94†
11/11 ¼ 1.0
45/45 ¼ 1.0
11/14 ¼ 0.79
55/59 ¼ 0.95†
45
11
0
3
45/48 ¼ 0.94†
11/11 ¼ 1.0
45/45 ¼ 1.0
11/14 ¼ 0.79
55/59 ¼ 0.95†
*Note: there was a full concordance between the results of EtG and of EtS.
†
p , 0.001 versus ethanol.
Figure 2. Scatterplot showing the relationship between EtG and EtS concentrations
in 45 positive urine samples. The correlation is highly significant (r ¼ 0.981,
p , 0.001); both axes are logarithmic.
Application
The results demonstrate that the analysis of EtG and EtS
improves the sensitivity and detection times for ethanol in
cases of sexual assault. EtG and EtS were detected in 45 out of
48 cases with self-reported intake of alcohol, whereas ethanol
230 Hegstad et al.
was detected in only 20 cases, corresponding to sensitivities of
94 and 42%, respectively. This is in accordance with previous
studies, which have shown that urinary EtG and EtS remain
positive considerably longer than urinary ethanol (6, 7). The
improved detection times and sensitivity for EtG and EtS may
provide a more complete assessment of toxicological involvement in the investigation of cases of sexual assault, because the
delay in reporting is particularly common in cases of drug
facilitated sexual assault. The detection of EtG and EtS may corroborate the victims’ self-reported alcohol intake, and may lend
credence to possible claims by victims of being assaulted while
intoxicated.
In studies in healthy volunteers, the excretion profiles for
EtG and EtS in urine are well documented, and there is an
established dose-response relationship between the amount of
alcohol ingested and the detection time (6, 7). The current
data on the relationship between reported intake, a time from
assault to sampling and urinary concentrations of EtG and EtS
(Figure 3) should be interpreted with caution due to the
limited sample size and the uncertainties of recall in the selfreported amount of alcohol and time interval since the alcohol
intake took place. Nevertheless, these results are in accordance
with data from a previous study showing that following an
Figure 3. Scatterplots showing the relationship between time since alcohol intake and EtG and EtS urinary concentrations standardized to a creatinine concentration of
100 mg/dL. Samples from patients reporting alcohol intake: 1– 6 units (A); . 6 units (B). The correlations between time after intake and concentrations are statistically
significant (1– 6 units: r ¼ – 0.636 for EtG, r ¼ –0.704 for EtS; . 6 units: r ¼ –0.794 for EtG, r ¼ –0.786 for EtS; p , 0.01 for all); the plots are semi-logarithmic. From
extrapolation of the regression lines to the intercept with the LOQs for EtG and EtS in this method (0.10 and 0.080 mg/L, respectively), detection times of 2 –2.5 days could be
assumed for those ingesting 1– 6 alcohol units, whereas detection times of approximately 3 days could be assumed for those ingesting . 6 alcohol units. The detection times
seem to be slightly longer for EtG than for EtS.
intake of 0.50 g of ethanol per kg body weight (i.e., 35 g or approximately three alcohol units in a subject weighing 70 kg),
the maximum detection time for EtG and EtS would be approximately 48 h (6).
For the purpose of detecting alcohol intake in victims of
sexual assault, this study suggests that the lowest possible
cutoff (e.g., LOQ) should be employed to maximize the sensitivity of the method. The observed EtG concentrations were
always higher than the EtS concentrations, with a mean ratio of
3.5. In other studies, the mean EtG/EtS ratios have varied
between 2.6 and 4.0 (3, 6, 23). Thus, if the same cutoff values
are applied for both EtG and EtS, samples in the lower range
could turn out positive for EtG and negative for EtS. To avoid
difficulties in the interpretation and reporting of such samples,
a lower cutoff can be used for EtS than for EtG. Because the
ratio between the LOQs for EtG and EtS in the present study
was 1.25, there is still a risk that a sample containing both EtG
and EtS could be positive for EtG and negative for EtS by
employing this method.
From the high concordance between self-reported alcohol
intake and positive EtG and EtS findings in urine, it was concluded that the patients’ qualitative reporting of whether they
did or did not ingest alcohol in relation to the alleged assault
was highly credible in this study, although authors of studies
from other Western sexual assault centers claim the contrary
(24, 25). In none of the cases were EtG or EtS detected in
samples from patients not reporting alcohol intake. In three
cases, EtG or EtS could not be detected despite alcohol ingestion being reported by the patient. Two of these cases can
easily be explained, in Case B by a low alcohol intake and in
Case C by a long duration between intake and urine sampling.
In Case A, the self-reported intake was three standard alcohol
units and the time from assault to sampling was 15.5 h. This
would be expected to yield positive EtG and EtS findings in
most cases; however, the negative results could stem from individual peculiarities in ethanol metabolism and excretion, or
simply from inaccuracies in the reporting of the amount of
ethanol ingested and/or the time of the intake.
In conclusion, this study reported the development and validation of a fast and reliable method for the simultaneous quantification of EtG and EtS in urine. The application of this
method on a series of sexual assault cases confirms the superiority of EtG/EtS analysis over ethanol analysis for the verification of alcohol intake in conjunction with the assault, and
shows a high concordance between victims’ self-reported
ethanol intake and EtG/EtS findings.
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