Clinical Chemistry 43: 1003-1009, 1997;
(Clinical Chemistry. 1997;43:1003-1009.)
© 1997 American Association for Clinical Chemistry, Inc.
Highly sensitive gas chromatographic analysis of ethanol in whole blood, serum, urine, and fecal supernatants by the direct injection method
Albert Tangerman
Department of Gastroenterology and Hepatology, University Hospital Nijmegen, 6500 HB Nijmegen, The Netherlands.
Address for correspondence: Laboratory of Gastroenterology and Hepatology, University Hospital Nijmegen, 6500 HB Nijmegen, The Netherlands. Fax 0031-24-3540103; e-mail A.Tangerman{at}gastro.azn.nl
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Abstract
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A highly sensitive, reproducible, and rapid gas chromatographic method
for ethanol determination in various biological specimens (human whole
blood, serum, urine, and fecal supernatants) was developed. The method
involves direct injection of the biological specimen into the gas
chromatograph, without any pretreatment. Contamination of the gas
chromatographic column with nonvolatile material was prevented by the
use of a glass liner in the injector. This liner, which acted as a
precolumn, was partly filled with small glass beads. Injection was
performed in between the glass beads. More than 50 injections of the
various biological specimens could be done before the liner had to be
replaced by a new one. This injection technique between glass beads
allows direct injection of large sample volumes up to 10 µL without
disturbing the gas chromatographic separation. Injection of these large
sample volumes made the method very sensitive. The detection limit for
ethanol amounted to 0.1 mg/L (2 µmol/L) when using an injection
volume of 5 µL. Attention has also been paid to simultaneously
monitoring ethanol, methanol, acetaldehyde, and acetone in blood and
urine of control subjects.
Key Words: indexing terms: alcohol • acetaldehyde • acetone • methanol • propanol • butanol.
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Introduction
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Numerous methods have been described for the determination of
ethanol in whole blood, serum, and urine, the most popular being gas
chromatography (GC), chemical assays, and enzymatic assays
(1)(2)(3). GC is the most precise and reliable method for
alcohol determination in blood and other biological fluids, and has
become the gold standard in forensic toxicology. However, in clinical
chemistry, GC has often been disregarded as a technique full of
difficulties and requiring specifically trained personnel.
Concerning GC, many methods are available in the literature
(2)(3). Methods requiring solvent extraction
or distillation are time and sample consuming and should be considered
obsolete. The two major techniques used nowadays are headspace sampling
and direct specimen injection. These two techniques can also be fully
automated. The headspace technique is quite laborious, requires larger
volumes of the biological specimen than does direct injection, and is
less sensitive than the direct injection technique. Moreover, the
headspace technique might be subject to serious analytical errors due
to variations in partitioning of ethanol between the gas and liquid
phases, depending on the liquid matrix used
(4)(5). Direct injection
(3)(5)(6)(7) obviates all the sample type
discrepancies observed with headspace analysis. The only drawback of
direct injection is its possible polluting effect on the injection
port, on the precolumn, on the column, and on the injection syringe.
Many investigators have advocated preparation of protein-free filtrates
of the biological specimen or dilution before analysis
(3).
The objective of the present paper was to develop a sensitive,
reliable, easy-to-use, and rapid procedure for the determination of
ethanol in whole blood, serum, urine, and fecal supernatants by using
the direct injection GC technique.
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Materials and Methods
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reagents
Ethanol, methanol, acetone, n-propanol, isopropanol,
n-butanol, and isobutanol, all analytical grade (>97%
purity), were obtained from Merck (Darmstadt, Germany).
L-Lactic acid and periodic acid came from Sigma (St. Louis,
MO). The column packing material, 10% SP-1200/1%
H3PO4 on 80/100 Chromosorb W AW, came
from Supelco (Bellefonte, PA). The glass wool (dimethylchlorosilane
treated) was from Chrompack (Middelburg, The Netherlands) and the small
glass beads with a diameter of 1 mm from Tamson (Zoetermeer, The
Netherlands).
preparation of biological specimens
Whole blood, urine, and fecal material were obtained from healthy
volunteers who were recruited from the laboratory personnel. In the
experiments with whole blood, heparinized whole blood was used. Fecal
samples were homogenized with a blender and ultracentrifuged for 2
h at 4 °C and 30 000g. The supernatant (fecal water) was
carefully removed and stored at -20 °C until analysis. For a more
convenient procedure, one might dilute the feces 3 to 5 times with
distilled water. After vortex-mixing, 1 mL of the homogeneous
suspension was transferred into a conical polypropylene micro sample
tube (Eppendorf, Hamburg, Germany; 1 mL) and centrifuged for 1 min at
10 000g in an Eppendorf centrifuge. The clear dark brown
fecal supernatants were used for the gas chromatographic experiments.
For concentration measurements, one should take into account the
dilution factor.
The procedures followed were approved by the local Medical Ethical
Review Committee.
gas chromatography
The gas chromatograph used was a Chrompack Model CP 9001, equipped
with a flame ionization detector, and a CP-9010 automatic liquid
sampler (Chrompack). Data handling was done with the Maestro
chromatography data system (Chrompack). The injection port of the
chromatograph was installed with a hand-made glass liner (length 8 cm,
o.d. 6 mm, i.d. 3 mm) (Fig. 1
). This liner, which acted as a precolumn to prevent
contamination of the gas chromatographic column with nonvolatile
material from blood, urine, and fecal supernatants, was stoppered with
a dimethylchlorosilane-treated glass wool plug and partly filled with
small glass beads with a diameter of 1 mm. Injection of whole blood,
serum, urine, and fecal supernatants was performed by means of a
50-µL Hamilton syringe (Model 1705, Chrompack) with a removable
needle (needle gauge 22S), penetrating the glass beads by at least 1.5
cm. Injection by <1.5 cm beneath the surface of the glass beads mostly
resulted in a broad tailing peak for ethanol. The plunger of the
syringe had a Teflon tip to provide an inert leak-tight seal. For
routine analyses, 2-µL injections were performed. For more sensitive
determinations, injection volumes up to 10–20 µL might be used. The
liner was replaced within seconds by a new one after some 50 2-µL
injections or 10 10-µL injections of whole blood, serum, urine, or
fecal supernatants. By then, the glass beads in the vicinity of the
injection area had a brown-black color, due to contamination with
nonvolatile material.
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Figure 1. GC injection port.
1, Carrier gas flow; 2, injection port head
retainer; 3, injection port head; 4, septum
retaining cap; 5, septum; 6, O-ring;
7, glass liner, stoppered with a glass wool plug and partly
filled with small glass beads; 8, Hamilton injection syringe
(25 or 50 µL), penetrating the glass balls inside the liner by at
least 1.5 cm during injection.
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The conditions were as follows: Column: 2 m x 2 mm i.d., glass,
packed with 10% SP 1200/1% H3PO4 on
80/100 Chromosorb W AW. Column temperature: 120 °C or 60 °C;
injection port temperature: 200 °C; detector temperature: 180 °C.
Detector output attenuation: 27. Carrier gas:
N2, 20 mL/min; H2, 30 mL/min; air, 300 mL/min.
Freshly packed columns were conditioned overnight at 190 °C with a
flow of nitrogen carrier gas, before being connected to the detector. A
few 1-µL injections of 10% formic acid were made to clear the column
of unknown impurities. When using a new liner, two 2-µL injections of
distilled water were made to clear the new glass beads inside the liner
of some unknown impurities that might disturb the gas
chromatographic separation. The time to replace the liner, stabilize
the system, and to decontaminate the new liner took ~3 min.
Acetaldehyde and ethanol separated at a column temperature of
120 °C. Acetaldehyde was generated inside the gas chromatographic
liner by oxidation of lactic acid with periodic acid (8).
For this, the syringe was filled with 0.3 µL of 150 g/L periodic acid
and 1 µL of a lactic acid solution (25 mmol/L). Injection inside the
glass beads of the liner resulted in a sharp single peak for
acetaldehyde. Ethanol, methanol, and acetone did not separate at a
column temperature of 120 °C. However, in ethanol-intoxicated
patients the methanol and acetone concentrations are usually <1% of
that of ethanol and therefore do not interfere with the ethanol
determination in these patients. When concerned with simultaneously
monitoring ethanol, methanol, acetaldehyde, and acetone, a lower column
temperature of 60 °C must be used, resulting in an almost baseline
separation between all the mentioned volatiles.
calibration and recovery studies
An aqueous stock calibrator of ethanol was prepared with a
concentration of 500 g/L.1 This solution was stored at 4 °C. To 1-mL
samples of water, whole blood, serum, urine, and fecal supernatants
were added 1, 2, 4, 6, 8, and 10 µL of this stock calibrator,
resulting in solutions of 0.5, 1, 2, 3, 4, and 5 g/L, respectively. The
most diluted calibrator (0.5 g/L) was further diluted to afford
solutions with ethanol concentrations ranging from 0.25 to 10 mg/L. The
aqueous calibrators were used for daily calibration.
The intraassay reproducibility was determined for three calibration
solutions (0.01, 0.5, and 5 g/L) in water, whole blood, serum, urine,
and fecal supernatant, by analyzing each sample six times on the same
day. The interassay reproducibility was determined by analyzing the
same samples on six different days during a 3-month period. In between,
the samples were stored at -20 °C.
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Results
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gas chromatographic separation
Figure 2
a–e shows gas chromatograms of five calibrator solutions of
ethanol (1, 2, 3, 4, and 5 g/L) in whole blood. Injection within glass
beads gave a sharp peak for ethanol with a retention time of 0.43 min
(oven: 120 °C). The same sharp peaks were obtained for injections
with water, serum, urine, and fecal supernatants, supplemented with
ethanol. Some broadening of the ethanol peak was sometimes observed
after 50–100 injections of the biological specimen, due to
contamination of the glass beads with nonvolatile material. Injection
of large sample volumes (10 µL or more) also sometimes resulted in
peak broadening. Peak broadening resulted in lower peak heights but the
peak area was not influenced by peak broadening. Because peak area was
used for daily calibration, peak broadening did not influence the
outcome of the analysis. Nevertheless, the liner was routinely replaced
by a new one after 50 2-µL injections of biological specimen.
Injection within the glass beads must be performed at a distance of at
least 1.5 cm beneath the surface of the glass beads inside the liner.
Injection at a distance of <1.5 cm beneath the surface gave a broad
tailing peak for ethanol (Fig. 2f
). Injection in the gas phase of an
empty liner without glass beads often gave broad or double peaks for
ethanol.
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Figure 2. (a–e) Gas chromatograms of five calibrator
solutions of ethanol in whole blood (1, 2, 3, 4, and 5 g/L,
respectively).
Column temperature: 120 °C; injection volume: 2.0 µL; injection
depth: at least 1.5 cm beneath the surface of the glass beads in the
liner. (f) Gas chromatogram of the same solution as under
(e), but at an injection depth of 1 cm beneath the surface
of the glass beads. Recorder output: 0.2 V.
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Many reported methods on analysis of ethanol are concerned with
simultaneously monitoring acetaldehyde, methanol, or acetone
concentrations (5)(9)(10)(11). The peak of
acetaldehyde (retention time: 0.36 min) was clearly separated from that
of ethanol (retention time: 0.43 min) at a column temperature of
120 °C. Ethanol, methanol, and acetone coincided at 120 °C. An
almost baseline separation between acetaldehyde, methanol, acetone, and
ethanol was obtained at a column temperature of 60 °C (Fig. 3
). Some forensic important congeners (12) of
ethanol (isopropanol, n-propanol, isobutanol,
n-butanol) are also included in Fig. 3
and showed baseline
separation at 60 °C. No carryover problems were seen for ethanol,
nor for any of the other volatiles studied.
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Figure 3. Gas chromatogram of a mixture of acetaldehyde
(1), methanol (2), acetone (3),
ethanol (4), isopropanol (5),
n-propanol (6), isobutanol (7), and
n-butanol (8).
Column temperature: 60 °C; injection volume: 1.0 µL; concentration
of all volatiles: 0.8 g/L; recorder output: 60 mV.
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calibration, recovery, and precision
Daily calibration was performed with the aqueous ethanol
calibration solutions (concentration: 1–5 g/L). A quite good linear
correlation was obtained between peak area and concentration (Fig. 4
). No significant differences were observed between the
calibration line of ethanol in water and those of ethanol in whole
blood, serum, urine, and fecal supernatant. The biological matrix did
not influence the gas chromatographic analysis. Although not shown, the
calibration curves were also linear in the low concentration range
(0.0001–0.5 g/L).
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Figure 4. The calibration curves for ethanol in water
(y = 50 157x + 5929, r =
0.9996), whole blood (y = 50 728x -
1036, r = 0.9991), serum (y =
48 676x + 2518, r = 0.9999), urine
(y = 49 929x + 4012, r =
0.9998), and fecal water (y = 51 059x +
3225, r = 0.9997).
Column temperature: 120 °C; injection volume: 2.0 µL.
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The percentage recovery of ethanol from water, whole blood, serum,
urine, and fecal supernatant was excellent, with low intra- and
interassay CVs (Table 1
). The interassay reproducibility was determined by analyzing
the same samples on six different days during a 3-month period. In
between, the samples were stored at -20 °C.
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Table 1. Mean percentage recovery of ethanol from water, whole
blood, serum, urine, and fecal supernatant, and the intraassay and
interassay variation, as measured for three different
concentrations.
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The detection limit, corresponding to a peak area equal to four times
the background noise, was 0.25 mg/L, when using 2-µL injections and a
column temperature of 60 °C. This limit could be lowered to 0.10
mg/L by injecting larger amounts (5 µL) of biological sample.
Injection between glass beads allows injection of at least 5–10 µL
of biological sample without resulting in peak broadening or other
disturbances. Injection of larger volumes sometimes resulted in peak
broadening without disturbing peak area. For a given concentration, a
linear correlation was obtained between the volume injected and the
peak area, at least for injections up to 10 µL (y =
24 293x + 1035, r = 0.9993, Fig. 5
). Above this volume, the response was not linear anymore. When
applying these large volumes, the liner should be replaced by a new one
after some 10 injections. Although not studied in detail, the detection
limits for acetaldehyde, methanol, and acetone lie in the same low
range as that for ethanol. Injection of 1 µL of an aqueous calibrator
solution (Fig. 3
) gave comparable peak areas for all the volatiles
studied.
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Figure 5. The peak area response of ethanol vs injection
volume.
Injected sample: whole blood with an ethanol concentration of 1 g/L;
column temperature: 120 °C.
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Injection of 5 µL of a blank urine sample (Fig. 6
a), serum sample (not shown), or blank whole-blood sample (Fig. 6d
) from a healthy volunteer who had no alcoholic drinks during the
last 3 days before the sampling gave no detectable ethanol peaks (<0.1
mg/L). This was repeated for two other volunteers with the same
results. No interferences were observed with other constituents from
whole blood, serum, urine, or fecal supernatant, allowing detection of
very small amounts of ethanol. Fig. 6b
and 6e
show the spectra after
addition of 5 mg/L ethanol to the corresponding blanks (see Fig. 6a
and 6d
). Fig. 6c
shows the spectrum of the first morning urine of the same
volunteer who drank one glass of wine the evening before. The ethanol
concentration in this sample amounted to 3.4 mg/L. The ethanol
concentration in the serum of a normal social drinker amounted to 1.2
mg/L (Fig. 6f
). The control urine, whole-blood, and serum samples
studied here all contained small amounts of acetone (0.3–2.0 mg/L). In
the control samples containing no ethanol, methanol was not detected
either (<0.2 mg/L). Methanol was present in urine and blood samples of
volunteers who had several alcoholic drinks and consequently high
ethanol concentrations. The methanol concentration in a urine sample
containing 0.8 g/L ethanol amounted to 5.2 mg/L and in a serum sample
containing 0.8 g/L ethanol to 3.7 mg/L. The acetone concentrations in
these two samples amounted to 5.2 and 1.3 mg/L, respectively. The first
two small peaks in the spectra of Fig. 6
were also present after
injection of 5 µL of distilled water and were designated as injection
peaks. The latter had the same retention time as acetaldehyde. The area
of this injection peak corresponded to 0.5–0.8 mg/L acetaldehyde. When
measuring acetaldehyde, the concentration has to be corrected for by
this amount. Moreover, one should use a new liner because after ~10
injections a contaminated liner resulted in two liner peaks with the
same retention time as acetaldehyde and isopropanol.
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Figure 6. Gas chromatograms of a blank urine sample from a healthy
volunteer (a), the same urine sample supplemented with 5
mg/L ethanol (b), a morning urine sample of the same
volunteer after consumption of one glass of wine the evening before
(c), a blank whole-blood sample from a healthy volunteer
(d), the same blood sample supplemented with 5 mg/L ethanol
(e), and a serum sample from a healthy volunteer (social
drinker) (f).
Peak 1, acetaldehyde; peak 3, acetone; peak
4, ethanol. Column temperature: 60 °C; injection volume: 5.0
µL; recorder output: 4 mV.
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Discussion
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With the technique described in the present study, whole blood,
serum, urine, and fecal supernatants can be analyzed by GC for the
presence of ethanol without any pretreatment. Direct injection inside a
glass liner filled with glass beads, which acted as a precolumn,
protects the GC column against serious contamination with nonvolatile
material. This injection technique was also recently applied for the
determination of fecal short-chain fatty acids (13). The
glass beads inside the liner ensure that injection of the sample always
takes place against hot glass, providing an immediate evaporation of
the sample. Injection against hot glass appears to be very important
for obtaining sharp peaks. Broad peaks for ethanol were often seen
after injection in the gas phase of an empty liner. This might be
explained by a temperature difference. The glass beads have the same
temperature as the injector (200 °C), whereas the temperature in the
gas phase of an empty liner is surely lower, mainly because of cooling
by the N2 carrier gas stream through the liner. Injection
in the gas phase might therefore result in a slower evaporation of the
sample and, as a consequence, peak broadening. The gas chromatograph
used (Chrompack CP 9001) is ideal, because this chromatograph is
provided with an injection port containing the required liner. The same
gas chromatographic column has been in use now for >3 years. No
deterioration of the column has been observed after >10 000
injections of blood, serum, urine, and fecal supernatants. Detection of
low physiological concentrations of ethanol, methanol, and acetone
should be performed at a column temperature of 60 °C. The column
should be replaced by a new one when separation between these volatiles
becomes insufficient.
internal calibrator
Most authors applying the direct injection technique do use an
internal calibrator
(6)(11)(14)(15), also
because injection of small sample volumes (0.5 µL or less) is
subjected to unacceptable large sample errors. However, my technique
uses larger volumes (1 µL or more), eliminating these large sample
errors and consequently the need for an internal calibrator, as was
apparent from the very small intra- and interassay variations. An
aqueous external calibrator of ethanol meets all the requirements. The
calibration graphs for ethanol in water did not differ from those in
whole blood, serum, urine, and fecal supernatant. Two calibrations
during the day, one at the beginning and one at the end, were
sufficient. The intra- and interassay CVs in peak area, when injecting
1 µL of an external ethanol calibrator of 1 g/L, were <5%.
Nevertheless, when one is uncomfortable with the approach of external
calibration, one might easily use one of the higher alcohols
(n-propanol, isobutanol, n-butanol, see Fig. 3
)
as internal calibrator.
sensitivity
The effects of alcohol intoxication lie in the concentration range
0.2–6 g/L (3). In this study, a column temperature of
120 °C was used for this concentration range. No separation was
obtained between ethanol, methanol, and acetone at 120 °C. However,
the concentrations of methanol and acetone in alcohol-intoxicated
patients are usually <1% that of ethanol
(16)(17) and therefore do not interfere with
the ethanol determination. Similar low values for methanol and acetone
(~1–5 mg/L) were found in this study for a blood and urine sample
containing 0.8 g/L ethanol. Conventional gas chromatographic methods
for ethanol determination lack sensitivity at <10 mg/L (0.2 mmol/L)
(6)(7)(18)(19) and are
not suited for the determination of normal physiological concentrations
of ethanol in the blood or urine of control subjects because these
concentrations lie below this limit. GC-mass spectrometry (MS) methods
have been applied to measure such normal concentrations
(9)(15)(18). However, MS is a
technique much more sophisticated than GC and requires highly trained
personnel. The present GC method has a detection limit (0.1 mg/L)
similar to GC-MS. Such a low limit was obtained by injecting large
sample volumes (5 µL or more) and using a column temperature of
60 °C. The presence of interfering substances, particularly methanol
and acetone, has been a concern in the forensic measurement of ethanol
in blood and urine. Almost baseline separation between ethanol,
methanol, and acetone was obtained at 60 °C. This is necessary
because normal physiological concentrations of acetone lie in the same
low range as those of ethanol (9) and would interfere with
ethanol at a column temperature of 120 °C. A mean urine ethanol
concentration of 1.4 mg/L was found for healthy social drinkers
(18). I did not detect any alcohol (<0.1 mg/L) or any
methanol (<0.2 mg/L) in the urine of three healthy volunteers when
they abstained from alcohol for at least 3 days. Literature values of
normal physiological methanol concentrations in blood and urine range
from undetectable (<0.6 mg/L) to 3.8 mg/L
(20)(21)(22).
quantification
Quantification on the basis of peak area was excellent.
Quantification on the basis of peak heights is less desirable, because
peaks may become somewhat broadened, especially when injection was
performed inside a liner already contaminated with nonvolatile material
from previous injections or when applying large sample volumes (10 µL
or more). This broadening lowered peak heights but had no influence on
peak area.
injection syringe
Plugging of the syringe appeared to be a serious problem during
direct injection of whole blood or serum, especially when using the
10-µL Hamilton syringes with a needle gauge of 26S (Hamilton code
701). In my experience, immediately washing the syringe as recommended
by some authors (6)(11) did not solve the
problem, nor did the use of Hamilton syringe cleaning wires. The use of
a cleaning wire even worsened the plugging. Plugging could be overcome
by the use of a 25-µL or 50-µL gas-tight syringe with a Teflon
plunger tip and a removable needle with a needle gauge of 22S (Hamilton
code 702 and 703, respectively). Immediately after each injection, the
plunger was removed and the syringe was washed by filling it from above
with a 9 g/L saline solution by means of a second syringe. The plunger
was then reinstalled and pressed down, thereby cleaning the needle from
protein or other nonvolatile deposits.
direct injection vs headspace technique
Direct injection and headspace GC are the two most often used GC
techniques for measuring ethanol in biological specimens. The headspace
technique is quite laborious and is subject to various analytical
errors, mostly due to sample-type discrepancies (5). The
type of biological specimen influences the partitioning of ethanol
between liquid and headspace vapor, as does the type of salt added as a
salting-out agent. Moreover, headspace techniques require larger
volumes of biological specimen than direct injection and have higher
detection limits. In my hands, the headspace technique was inferior to
direct injection.
storage
The ethanol concentrations in water, blood, serum, urine, and
fecal supernatant appeared to be stable for at least 3 months at
-20 °C. No significant changes in ethanol concentrations were
observed during storage. Similar data were found in literature for
storage of blood. During a 2-week period, no significant changes were
observed in the blood concentration of ethanol, whether stored at room
temperature, under refrigeration, or in the freezer
(14)(23).
In conclusion, the direct injection method as presented here is a
highly sensitive, rapid, and reliable gas chromatographic procedure for
measuring ethanol in various biological specimens. The direct injection
method between glass beads may be a step forward in measuring all kinds
of volatile substances in biological material. Once running, the method
is easy to perform and does not require highly and specifically trained
personnel, making this gas chromatographic method also suited to the
field of clinical chemistry.
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Acknowledgments
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I thank all the healthy volunteers participating in this study.
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Footnotes
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1 In the literature, ethanol concentration has rarely been expressed in the SI-recommended unit (mmol/L). Mostly, mg/dL or g/L has been used. In the present paper, the units g/L and mg/L have been applied. The conversion factors are: from g/L to mmol/L = 21.7 and from mg/L to mmol/L = 0.0217. 
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Abstract
Introduction
