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Quantitation of EtG and EtS in Urine by
Ion-Pairing LC-MS/MS
Kristine Van Natta, Marta Kozak, Thermo Fisher Scientific, San Jose, CA
Application Note 589
Key Words
EtG, EtS, ethyl glucuronide, ethyl sulfate, ion pairing, SPE, DHAA,
dihexylammonium acetate, TSQ Ultra, HyperSep, Hypercarb,
forensic toxicology
Goal
To develop an HPLC-MS/MS method for forensic toxicological analysis of
EtG and EtS in urine with limits of quantitation (LOQs) of 100 and 50 ng/mL,
respectively.
Introduction
Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are
long-term biomarkers for ethanol consumption. Although
they are minor metabolites of ethanol, their longer
half-lives make them useful for forensic detection of past
alcohol use. These compounds are highly polar; they
retain poorly on most reversed-phase HPLC columns and
elute on or near the chromatographic solvent front. The
result is poor peak shape and large matrix effects. This
application note demonstrates the use of solid-phase
extraction (SPE) sample preconcentration to remove
interferences and an ion-pairing reagent to retain these
compounds on the HPLC column long enough to move
them off the solvent front. This enables better peak shape,
less matrix interference, and baseline resolution of both
analytes for less risk of ion suppression.
Experimental
Sample Preparation
A 1 mL volume of urine, 25 µL of internal standard
solution (50 and 5 ng/mL of EtG–
d
5
and EtS–
d
5
,
respectively), and 50 µL of formic acid were mixed.
The 200 mg Thermo Scientific
™
HyperSep
™
Hypercarb
™
SPE column (P/N 60106-301) was conditioned with 2 mL
of 1% formic acid in water. The sample was loaded at a
rate of 1–2 mL/min. Next, the column was washed with
2 mL of water and dried under nitrogen at 10–15 mm Hg
for 10 min. The sample was eluted with 2 mL of 1%
formic acid in methanol. The eluent was evaporated to
dryness under nitrogen at 37 °C and reconstituted in
0.2 mL of water.
1
Finally, 20 µL was injected onto the
HPLC-MS/MS.
Liquid Chromatography
Chromatographic separations were performed under
gradient conditions using a Thermo Scientific
™
Accela
™
1250 pump and Accela Open autosampler. The analytical
column was a Thermo Scientific Hypersil GOLD
™
column
(50 x 3 mm, 5 μm particle size). The column was
maintained at room temperature. The injection volume
was 20 μL. The flow rate was 1 mL/min, and the total run
time was 5 min. Other size columns can be used for this
application with the appropriate adjustment in injection
volumes and flow rates (as in AN488b
2
). Mobile phases
A and B consisted of 5 mM dihexylammoniumacetate
(TCI America) ion pairing reagent in water and
acetonitrile (Fisher Chemical), respectively. Mobile
phase C was acetonitrile/1-propanol/acetone (45:45:10).
Mass Spectrometry
MS analysis was carried out on a Thermo Scientific
TSQ Quantum Ultra
™
triple-stage quadrupole mass
spectrometer equipped with a heated electrospray
ionization (HESI-II) probe. Two selected-reaction
monitoring (SRM) transitions each were monitored for
EtG, EtS, and their deuterated internal standards to
provide ion ratio confirmations (IRC). Two scan
segments, one for EtG and its internal standard and one
for EtS and its internal standard, were used to maximize
the time the mass spectrometer spent scanning each
compound.
Results and Discussion
EtG demonstrated linear response from 100 to
100,000 ng/mL with an LOQ of 100 ng/mL. EtS
demonstrated linear response from 25 to 50,000 ng/mL
with an LOQ of 25 ng/mL. Figure 1 shows representative
calibration curves for both compounds. Figure 2 shows
representative chromatograms for EtG and EtS and their
respective LOQs.