Journal of Analytical Toxicology, Vol. 26, July/August 2002
Excretion Profiles of Ethyl Glucuronide in
Human Urine after Internal Dilution
Marion Goll, Georg Scbmitt, Beate Gangmann, a n d R o l f E. Aderjan*
Institute of Legal Medicine and Traffic Medicine, University of Heidelberg, Vol~strasse2, D-69115 Heidelberg, Germany
Abstract
Ethyl glucuronide (EG) is a useful marker of alcohol consumption
because its presence in urine can be detected up to five days. We
investigated the impact of diuresison the urinary excretion of FG,
a minor ethanol metabolite. Seven healthy volunteers drank
250 mL of wine (25 g ethanol) in 15 min and, 240 rain later,
ingested 1 L of water within 15 min. Urine was voided before the
drinking started and every 30-60 min for 400-550 min thereafter.
Urinary ethyl glucuronide (UEG), creatinine, and ethanol were
determined using liquid chromatography-tandem mass
spectrometry, Jaffgs method, and the enzymatic ADH method,
respectively. The maximum diuresiscoincided with the lowest
values of the UEG concentrationsof 2 mg/L and the lowest
creatinine values of 10 mg/dL 250-400 min after drinking. After
drinking the wine, the urinary creatinine decreasedslowly. After a
short period of increasing, it decreased to minimum values caused
by the water intake. After the intake of I I_water, the diuresis
increased within 60 min to its maximum. The amount of ethyl
glucuronide excreted in urine was I0 mg (SD 5 rag) corresponding
to 0.04% (SD 0.02%) of the dose administered. In successivevoids
during the elimination phase,the UEG and the diuresiswere
influenced after the subjectsdrank I t of water. Minimum UEG
values of 0.5 mg/L could still he measured. Measuring UEG
provides a reliable way to monitor recent drinking of alcohol.
However, urinary creatinine needs to be measured additionally.
Establishinga cutoff value of 25 mg/dL for urinary creatinine in
diluted samples, like for the analysisof illicit drugs, is
recommended. If the creatinine value is too low, the analyst has to
decide about the further procedure.
centrations (2). Articles that claimed it could be used as a potential relapse and abstinence marker (3,4) and that showed its
usefulness in clinical practice and for treatment of alcohol
disease appeared (5). According to German traffic laws and
the driving license regulations, proving one year of abstinence
from alcohol is important for drivers who have lost their
driving licenses because of excessive drinking that resulted in
a blood alcohol concentration above 1.6 g/kg of blood. A similar role of the EG determination can be seen in abstinence
testing of patients before and after liver transplantation, or
workplace testing of staff doctors by analyzing urine samples
for ethanol and its conjugate. Because ethanol is degraded
after a relatively short time, the dose-dependent and delayed excretion of the metabolite EG (for several days) (6) offers a use
as an abstinence marker, closing the gap left between shortand long-term markers such as carbohydrate deficient transferrine, aIanine aminotransferase, aspartate aminotransferase,
or mean corpuscular volume of erythrocytes. We reported that
after complete ethanol degradation in alcoholics during the
treatment of alcohol disease, EG in urine is still GC-MS detectable up to five days (6) and up to 36 h in serum samples (5).
EG is produced as long as ethanol is present in the body (5). EG
can be detected after the consumption of alcohol doses of approximately 10 g. It seems even possible to enhance the time
frame of detection of EG in the hair, if 10 g ethanol/d were consumed during a longer period (7). The aim of this study was to
investigate the influence of drinking water during the elimination phase of EG on its kinetics and the excretion profile in
relation to the associated changes of urinary creatinine, a commonly used marker for highly diluted specimens (8).
Introduction
Shortly after we first synthesized ethyl glucuronide (EG)
and introduced a gas chromatography-mass spectrometry
(GC-MS) and a liquid chromatography-mass spectrometry
(LC-MS) method (1), we reported on its serum and urine con* Author to whom correspondence should be addressed.
262
Material and Methods
Healthy male (n = 3) and female (n = 4) volunteers, all social
drinkers, with a mean age of 37 years (SD 5 years) and mean
body weight of 69 kg (SD 15 kg), participated in a controlled
Reproduction (photocopying)of editorial content of this journal is prohibited without publisher's permission.
Journal of Analytical Toxicology, Vol, 26, July/Ausust 2002
Pa~cipant A1
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Figure 1. The concentration-time profiles of urinary creatinine, UEG, and UEGloo for the time after water intake for all participants.
263
Journal of Analytical Toxicology, Vol. 26, July/August 2002
experiment. In two participants (one male and one female)
the experiment was repeated twice to investigate within-subject
reproducibility (Figure 1, participants A1, A2, C1, and C2).
The ethical review committee of the University Hospital of
Heidelberg approve8 the study protocol, and the subjects gave
verbal consent.
All participants were required to abstain from drinking
alcohol for 36 h before the experiment started, and alcohol-free
status was confirmed by breath-alcohol analysis with an
Alcomat T 02-019 (Siemens, Munich, Germany). After an initial
urinary void, the subjects were required to consume 250 mL
(25 g ethanol) of wine within 15 rain corresponding to a mean
dose of 0.36 g ethanol/kg mean body weight. Specimens of
urine were collected at 60, 120, 180, and 240 rain after subjects
finished the wine. When urine was voided, the subjects were requested to empty their bladder completely. The specimen was
collected into a measuring cylinder, and the volumes were
recorded to the nearest 2 mL. At 240 min postdrinking (255
rain from start), the subjects were required to drink 1 L of
water in 15 min. Urine samples were collected again at 30, 60,
90, 120, 150, 180, 210, and 240 rain after finishing the water intake. The concentration of ethanol in urine was determined by
the enzymatic ADH method (9), which had a limit of detection
of 0.05 g/L. Urinary creatinine was determined by Jaffg's
method on a Hitachi 911 analyzer (Hitachi, Tokyo,Japan) (I0).
The EG analysis was performed on a triple quadrupole spectrometer (API 365, PE Sciex, Toronto, ON, Canada) with a
Turbo Ionspray| Interface. The interface was coupled to a
PerkinElmer series 200 pump, which was equipped with a
PerkinElmer series 200 autosampler (PE, Uberlingen, Germany). The samples were eluted isocratically from a Zorbax|
Eclipse XDB-C8 narrow bore (2.1 x 150-ram i.d., 5 pro, HP,
Waldbronn, Germany) at ambient temperature and a flow rate
of 0.25 mL/min. The mobile phase consisted of 40% eluent I
(20raM ammonium acetate, pH 6.5-7) and 60% eluent II
(methanol/acetonitrile, 1:1, v/v). The temperature of the Turbo
Ionspray was set at 450~ For detection, the mass spectrometer operated in the negative modus, and the interface was set
at --4200 V. Multiple reaction monitoring was performed using
nitrogen at a collision energy of-26 eV. Precursors to product
ion transitions were monitored for EG at m/z 221 to 75 and for
EG-d5 at m/z 226 to 75. All solvents and substances used were
HPLC grade.
For LC-MS-MS analysis the urine samples (500 pL) were extracted using Bond Elut~ SAXas resin, which was conditioned
with 1 mL methanol, 1 mL water, and I mL acetonitrile. The
samples were diluted with 500 pL acetonitrile (pH 2, hydrochloric acid) and applied to the cartridges. The cartridges were
washed with n-hexane. EG was eluted with a solution of ammonia (25%) in methanol (1:10, v/v, pH 12). Thereafter, the
samples were evaporated under nitrogen. The residues were
dissolved in the mobile phase.
The calibration curve covered a concentration range of
0.25-20 and 10-100 mg/L of the analyte in urine. The limit of
detection (0.04 rag/L) and the limit of quantitation (0.13 rag/L)
were determined from a 10-point calibration: 0, 0.05, 0.075,
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mg/L according to DIN 32 645
at a probability of 99% by using BEN| program (M. Herbold,
264
Heidelberg, Germany). Recovery was assessed by comparing
peak areas of the compound of the urine extracts with the
particular peak areas of standards injected directly into the
LC-MS-MS system. The recovery was found to be approximately 70%.
The concentration-time profiles of EG and their normalizations to creatinine values of 100 mg/dL (EG100) as well as
changes in urinary creatinine were plotted for each participant
(Figure 1).
The amount of EG excreted in urine was calculated from the
EG concentration and the volume voided at each sampling
time. The diuresis was reported as the volume of urine produced per minute.
Results
The complete concentration-time profiles of EG during the
course of the experiment, EG100in urine and the changes in
urinary creatinine are shown in Figure 2 for one female participant (B). The relevant time frame is marked with a circle. In
Figure 1 this time frame is plotted for all participants. As a few
(A1, A2, and E) of the participants drank up to 300 mL tea, the
starting conditions were different. Therefore, their initial creatinine concentrations were lower than those of the other participants. After 180 to 250 min, the experimental conditions for
all participants were comparable: they drank the same amount
of alcohol and were required to abstain from drinking liquids
for 4 h until the water intake started. The different starting
conditions reflect the diuretic action of ethanol caused by inhibition of the secretion of the antidiuretic hormone
vasopressin from the posterior pituitary gland (11). The effect
can be seen in Figure 2: the creatinine concentrations decreased immediately after the wine intake. The participants
who had consumed liquids before the experiment had lower
creatinine concentrations with a mean peak of 29 mg/dL (SD
12 mg/dL); the diuretic effect was low and the creatinine
concentrations declined less 22 mg/dL (SD 6 mg/dL). The
creatinine concentrations of all other participants decreased
from a mean peak of 112 mg/dL (SD 35 mg/dL) to 45 mg/dL
(SD 28 mg/dL). The creatinine concentrations were stabilized
B
Participant
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Figure2. Thewholecourseof the concentration-timeprofileof creatinine,
UEG,and UEGloofor one femaleparticipant(B)duringthe courseof the experiment.
Journal of Analytical Toxicology, Vol. 26, July/August 2002
to 119 mg/dL (SD 39 mg/dL) 230 rain after ethanol intake.
After consuming 1 L of water, values of less than 10 mg/dL (SD
2.5 mg/dL) were reached within 60 rain. Thereafter, within
60-130 rain, the creatinine concentrations rose to values of 94
mg/dL (SD 36 mg/dL) except of the participants A2, D, and G,
who reached their individual average creatinine concentrations 360 rain after water intake.
According to Figure 1, the UEG and the urinary creatinine
concentrations, show a parallel course. The UEG concentrations peaked to 34 mg/L (SD 18 rag/L) 180-230 rain after
alcohol intake. Afterwater intake, they declined parallel to the
creatinine concentrations reaching their minimum of 2 mg/L
(SD 0.5 rag/L) within 60 rain. Along with decreasing urine
production, the UEG concentrations increased to 11 mg/L (SD
6.5 mg/L). Exceptions are the smaller EG values of participants D and G. Cminand Cm~ of UEG concentrations coincided with Cma~and Cmi n of the diuresis. After 60-130 rain, the
maximum of the diuresis was reached for all participants. This
coincides with the minimum of the UEG concentrations and
the urinary creatinine with the exception of participants A2, D,
and G, who reached their diuretic maximum 150 min later.
From the administered dose of 25 g ethanol, 10 mg (SD 5
rag) corresponding to 0.04% (SD 0.02%) was eliminated in
urine as EG (12,13). This calculation is possible because EG
proved to be a very stable analyte (14).
Urinary creatinine and the UEG concentration decreased
exponentially as the diuresis increased (Figure 3). As consequence ofthe water intake the urinary creatinine content of the
80.
70, i t
60 9
specimens was less than 20 mg/dL in 23% of all urine samples.
Discussion
Unlike urinary excretion of ethanol, in which the concentration is not diminished by water intake and dilution (15), the
elimination profile of EG can be highly influenced by drinking
large amounts of water. The forced diuresis influences the
UEG concentrations parallel to the urinary creatinine values.
This can be shown by the normalization of the EG concentrations to an average creatinine value of 100 mg/dL (SD 20
mg/dL) normally observed in healthy persons (16-18). As
shown in Figures I and 4, the dilution by water intake can be
compensated by normalization of the EG content to a creatinine value of 100 mg/dL (EG100). This allows to compare the
EG concentrations of individuals.
Lowering the UEG concentration is possible by drinking
water shortly before urine will be voided. In our low-dose experiment using 25 g ethanol, 1 L of water ingested I h before
voiding urine did not reduce the EG concentration below the
limit of detection of 0.04 mg/L. However,by drinking higher
amounts of water or other beverages, an ongoing excretion of
EG during the terminal elimination phase could be hidden in
order to pretend ethanol abstinence. To recognize such conditions or manipulations, it is important to determine the urinary creatinine and to check it for values below 25 mg/dL. To
exclude potential internal adulteration and urine dilution,
such a creatinine cutoff of 25 mg/dL was established for the
analysis of illicit drugs in urine (8).
so I
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References
2010, t?=~lldPl~.m.
O,
0
- d~ll
1
2
9
3
4
w 9
5
6 7 8 9 10 11
Diuresis (mL/min)
12 13 14 15
Figure3.Associationbetween UEG-concentrationand the diuresisfor all
participants in the courseof the experiment.
50 -,
45 4
~ 30
25
2O
15
1
0
100
200
"~me(min)
300
400
Figure4.Theconcentration-time profile of UEG10o asaveragecurvefor all
participants.
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