Journal of Analytical Toxicology,Vol.
21, March/April 1997
An Accurate,Automated,Simultaneous
Gas ChromatographicHeadspaceMeasurementof
Whole BloodEthanoland Acetaldehydefor
Human In Vivo Studies
D. Gail McCarver-May and Lubica Durisin
Department of Pediatrics and Pharmacology, Wayne State University, School of Medicine and Division of Clinical
Pharmacology, Children's Hospital of Michigan, Detroit, Michigan 48201
Abstract
Simultaneous assessmentof ethanol and acetaldehyde
concentrations is necessary to address hypotheses in alcohol
research. Accurate measurement of in vivo acetaldehyde following
ethanol exposure is problematic because acetaldehyde is present in
blank blood, is volatile, and is formed enzymatically and
nonenzymatically in blood containing ethanol. Because
acetaldehyde is carried in red blood cells, previously reported
plasma methods may not reflect total body acetaldehyde. We
developed an accurate, sensitive, automated gas chromatographic
whole blood method using headspace injection and flame
ionization detection. Sensitivity was 5.4 pmol/L and 1.13 pmol/L
for ethanol and acetaldehyde, respectively. Linearity (r z > 0.99,
both) and reproducibility (coefficients of variation = 1.6-7.7%)
were acceptable. Because a whole blood method completely
inhibiting in vitro oxidation of ethanol has not been reported, we
evaluated multiple reported sample processing methods. The
optimum method, which uses saturated sodium nitrite as the
inhibitor, resulted in a 30% in vitro increase in acetaldehyde in
blood containing 21.7 mmol/L (0.1 g/dL) ethanol, in contrast to the
9-40-folcl increase observed with other inhibitors (p = 0.001).
Using the described technique, the median acetaldehyde and
ethanol peak concentrations in six African-American women
following a 0.5 g/kg oral ethanol dose were 6.1 pM and 17.1mM,
respectively.
Introduction
The accurate measurement of acetaldehydeconcentrations in
blood containing ethanol is difficult as acetaldehyde is formed
in vitro both enzymatically and nonenzymatically. In addition,
acetaldehyde is volatile and spontaneous and enzymatic oxida-
tion of acetaldehyde occurs rapidly. The choice of method is
controversial and is dependent on the needs of a particular
investigation and on the degree of accuracy and validity of the
assay technique. Currently employed methods use breath ana-
lysis (1), high-performance liquid chromatography (HPLC)
(2,3), and gas chromatography (4,5). Breath acetaldehyde sam-
pling escapes the issue of in vitro formation of acetaldehyde.
134 Reproduction(photocopying)of editorialcontentof thisjournal is prohibitedwithoutpublisher'spermission.
However, even with elevated, easily measurable levels of
acetaldehyde, breath acetaldehyde concentrations do not cor-
relate with blood concentrations (6). Breath acetaldehyde may
be increased by the formation of acetaldehyde in the mouth, in
the airway, or by lung microsomes (7,8).
Common HPLC methods require extraction and derivatiza-
tion techniques that are labor intensive and require the syn-
thesis of several fresh reagents. In addition, the stability of the
adducts formed, typically hydrazines (2,9) or cyclohexadiones
(10,11), has not been reported. Moreover,without immediate
deproteinization, derivatized acetaldehyde continues to be
formed until the enzymes present in the blood are removed by
centrifugation or by deproteinization. Thus, methods that
employ derivatization without deproteinization eliminate the
loss of acetaldehyde by vaporization and oxidation but cannot
eliminate artifactual in vitro formation from ethanol. Two
HPLC methods combine deproteinization and adduct formation
with fluorescent markers using either methanolic dinitro-
phenylhydrazine (DNPH) (9,12) or organic diphenylacetylin-
dandione-hydrazone (DIH) (3,13). The organic portion of these
reagents acts as a deproteinizer while the DNPH or DIH forms
a fluorescent adduct to acetaldehyde. Another method uses
perchloric acid deproteinization and centrifugation followed
by derivatization to dinitrophenylhydrazine (14). These
methods are labor intensive and also have the technical
problem of a lack of a feasible internal standard. Furthermore,
a separate analytical method would be required for ethanol
measurement in in vivo studies of ethanol metabolism. Depro-
teinization of whole blood has been criticized based on the
belief that it causes the formation of spurious acetaldehyde by
the oxidation of ethanol by red cells (15). Recognizing that
increased oxidation of acetaldehyde and increased in vitro for-
mation of acetaldehyde from ethanol were associated with red
blood cells (16,17), some investigators preferred to measure
plasma acetaldehyde (5,6,18,19). By quick separation of plasma
from red cells, much, but not all, of the in vitro formation of
acetaldehyde from ethanol can be eliminated.
A major disadvantage of plasma methods is that the majority
of acetaldehyde is circulated in red blood cells (20,21). After
ethanol exposure, acetaldehyde metabolized from ethanol is
Journal of Analytical Toxicology, Vol. 21, March/April 1997
converted into Schiffbases bound to lysine and valine residues
in proteins (22). These linkages appear to occur with hemo-
globin, red cell membranes (23), and plasma proteins (24).
This binding tends to occur rapidly after acute ethanol exposure
(25). It is likely that deproteinization releases the more unstable
forms of this bound acetaldehyde. Thus, an alternative explan-
ation to spurious acetaldehyde exists for the large increase in
acetaldehyde seen with deproteinization techniques. Rather
than discrediting whole blood techniques, higher concentra-
tions of acetaldehyde would be anticipated from whole blood
measurements than from plasma measurements. Considering
the larger quantitative amount of acetaldehyde in red blood
cells compared with plasma, total whole blood acetaldehyde
rather than the plasma acetaldehyde concentration would seem
to better reflect total body acetaldehydefollowingacute ethanol
exposure. Although only free acetaldehyde is thought to cross
the blood-brainbarrier, free acetaldehydeis rapidly metabolized
by aldehydedehydrogenase.Removalof free acetaldehydewould
be expectedto leadto the release of looselybound acetaldehyde.
Thus, for exposure purposes, total circulating acetaldehyde
would appear more relevant than free acetaldehyde.
The major advantages of gas chromatography methods are
the ease of use and the simultaneous measurement of acet-
aldehyde and ethanol. The sensitivity of newer flame ionization
detectors allows adequate acetaldehyde detection for in vivo
studies. We developed an accurate, sensitive gas-liquid chro-
matography assay for simultaneous measurement of ethanol
and acetaldehyde using headspace injection and flame ioniza-
tion detection to study ethanol and acetaldehydeconcentrations
in human blood following ethanol ingestion. Because com-
plete inhibition of ethanol oxidation has not been achieved
with any of the published deproteinization methods, we com-
pared multiple sample processing methods for inhibition of
the in vitro oxidation of ethanol and for the relative recovery of
acetaldehyde.
Methods
Standards
Stock standards for ethanol (Aaper Alcohol and Chemical,
Shelbyville, KY) and acetaldehyde (Aldrich Chemical,
Milwaukee, WI) were prepared by dissolving each in double-
distilled water that was freshly boiled and analyzed for pos-
sible acetaldehyde contamination. Ethanol, acetaldehyde, and
the internal standard, 1-propanol (IS), stock standard solu-
tions were made to concentrations of 2.17 mol/L (10 g/dL),
22.7 mmol/L (100 mg/dL), and 16.6 mmol/L (100 mg/dL),
respectively.All standards were prepared in the cold (4~ using
cold glassware and pipette tips and were prepared fresh the
evening before the run day to vent the cold room of acetalde-
hyde vapors.
Evaluationof sampleprocessingmethods
Compounds selected to test their ability to inhibit the in
vitro formation of acetaldehyde from ethanol included satu-
rated sodium nitrite, sodium azide (10raM), polyethylene
glycol (20%) with sodium azide (10raM), perchloric acid
(0.6N), zinc sulfate (5%) followed by barium hydroxide (0.3N),
and semicarbazide (6mM) followed by perchloric acid (3N).
The inhibitors and the concentrations selected were chosen
based on their reported use by others (5,15,26,27). Fresh whole
blood (6 mL total) was drawn by venipuncture from ten indi-
viduals using a butterfly needle and two syringes (5 mL each).
No preservatives or anticoagulants were used. For each indi-
vidual, this whole blood sample was divided into 1-mL aliquots
that were placed into ice-cold 10-mL gas chromatography
vials. For each inhibitor, two sets of three vials each were pre-
pared. In each set, one vial was a blank control (no analyte
added). Ethanol stock solution was added to the whole blood in
the second vial of each set to yield a concentration of 21.71JM.
Acetaldehyde stock solution was added to the whole blood in
the third vial of each set to yield a concentration of 22.71JM.
The choice of the concentrations of ethanol and acetaldehyde
used were based on concentrations anticipated in clinical
studies (16,28). To one set of vials, the inhibitor was added
within 2 s of the addition of ethanol or acetaldehyde. To the
second set, saline was added for volume equivalence. Ten mi-
croliters of the internal standard stock solution was added to
each vial. After all compounds had been added to the whole
blood, the vials were gas-tight sealed and stored at 4~ until
analysis. Each sample was assayed for ethanol and acetaldehyde
by gas chromatography within 2 h of processing.
In order to compare the in vitro formation of acetaldehyde
from ethanol across techniques, we evaluated the increase in
acetaldehyde in the presence of ethanol by defining the oxida-
tion inhibition ratio as follows:
Relative inhibition ratio = AA PAI+ETOH
AA PA,
where AA PAI+ETOnis the area under the acetaldehyde chro-
matogram peak in blood containing 21.7 mmol/L ethanol and
inhibitor, and AAPAl is the area under the acetaldehyde peak in
blood containing inhibitor but no ethanol. Complete inhibition
of in vitro ethanol oxidation would generate a ratio of 1.0. We
defined the relative recovery of acetaldehyde to compare the
impact of the different methods of deproteinization on the
recovery of acetaldehyde:
Relative recovery ratio = AA PAI
AA PA0
where AAPAI is the area under the acetaldehyde chromatogram
peak in blood containing 22.7 pmol/L acetaldehydeto which the
inhibitor was added, and AA PAo is the area under the acet-
aldehyde peak in blood containing 22.7 pmol/L acetaldehyde
and 1 mL saline for volume equivalence, but no inhibitor.
Paired t-tests were used to compare the oxidation inhibition and
relative recovery ratios of inhibitors. An (~ level less than 0.05
was accepted as statistically significant.
Gas chromatographyconditions
Samples were analyzed on a Varian (Sugar Land, TX) 3500
gas chromatograph equipped with a Hewlett Packard (Wilm-
ington, DE) headspace injector. Headspaceincubation time was
16 rain at 75~ A 3-mL headspace injection loop was equili-
brated with the sample using ultrapure helium (99.999%) at a
pressurization of 1.7 bars. Headspace injection time was 30 s
135

and occurred via the Varian split-splitless injector at a helium
flow rate of 7.9 mL/min with a measured split ratio of 10. Sep-
aration utilized a Carbowax megabore capillary column (All-
tech, 30 m, 0.54 ram, 1.2-1Jm film thickness). The makeup gas
was nitrogen (99.998%) with a total detector gas flow of 30
mL/min. Detection was by flame ionization (FID) with a 10-fold
change in detector range programmed at 2.5 min. Data acqui-
sition used the Varian Star integrator.
Results
The retention times of acetaldehyde, ethanol, and 1-propanol
were 1.44, 3.13 and 3.81 rain, respectively (Figure 1). The limits
of assay sensitivity were 5.4 iJmol/L and 1.13 IJmol/L for ethanol
and acetaldehyde, respectively.
Saturated sodium nitrite was best at preventing the in vitro
oxidation of ethanol to acetaldehyde (Table I, p < 0.001, all
comparisons). However, the presence of 21.7 mmol/L ethanol
increased the amount of acetaldehyde present by 30%. Sodium
azide (10raM) was second best, but was much less effective
than sodium nitrite (p = 0.001) with an oxidation inhibition
ratio indicating more than a ninefold increase in acetaldehyde
in blood containing ethanol (Table I). Perchloric acid, alone and
when combined with semicarbazide, and the combination of
zinc sulfate with barium hydroxide were much less effective
than sodium nitrite or sodium azide at inhibiting in vitro for-
mation, and, in fact, were no better than saline (Table I). The ox-
idation inhibition ratios of these three methods indicated an in
vitro increase in acetaldehyde of 20-40-fold in the presence of
ethanol (Table I).
In relative recovery of acetaldehyde, sodium nitrite was sig-
nificantly better than azide with relative recoveries of 3.8 and
2.7, respectively (Table I, p --- 0.001). The relative recovery of
acetaldehyde was greatest with perchloric acid and with the
combination of zinc sulfate and barium hydroxide (Table I).
Table I. Sample Processing Techniques and Inhibition of In Vitro Oxidation of
Ethanol and Relative Recovery of Acetaldehyde*
Oxidationinhibition
ratio+
(mean_+s.d.)
Saturatedsodium nitrite 1.3 + 0.1
10mM Sodium azide 9.2 + 3.9w
20% Polyethyleneglycol + 10mM sodium azide 21.1 + 7.6w
Normal saline 44.2 + 14.6w
0.6N Perchloricacid 19.3 + 10.7w
5% Zinc sulfate + 0.3N barium hydroxide 26.8+6.5w
6mM Semicarbazide+ 3N perchloric acid 39.8 + 38w
* N= 10, each inhibitor
* Oxidation inhibition ratio = acetaldehyde peak area in blood with 21.7 mmol/L ethanol + inhibitor
acetaldehyde peak area in blank blood + inhibitor
~: Recovery ratio = acetaldehyde peak area in blood with 22.7 pmol/L acetaldehyde + inhibitor
acetaldehyde peak area in blood with 22.7 pmol/L acetaldehyde (no inhibitor)
w Comparison with satured sodium nitrite: p < 0.001.
# Comparison with satured sodium nitrite: p < 0.05.
136
Journal of Analytical Toxicology, Vol. 21, March/April 1997
Additional Studies with Sodium Nitrite
Effect of sodium nitrite concentration
Fresh whole blood from five individuals was drawn into ice-
cold syringes and placed into two sets of ice-cold gas chro-
matography vials (1 mL each) to evaluate whether inhibition of
in vitro ethanol oxidation could be improved by altering sodium
nitrite concentration. One milliliter of cold sodium nitrite
ranging from ] to 75% (w/v) (saturated) was added immediately
to one set of vials. Ethanol (21.71Jmol) was added to the second
set of vials. Within 2 s, I mL of sodium nitrite over the same
concentration range was added. Deproteinization ability was
determined by the oxidation inhibition ratio. Results were com-
pared using paired t-testing with an or value of less than 0.05
accepted as significant.
Sodium nitrite at every concentration significantly reduced
the in vitro formation of acetaldehyde (Table II, p < 0.05, all
comparisons with blank). However, 60 and 75% (saturated)
sodium nitrite were better than the lower concentrations
(1-40%) (Table II, p < 0.05, all comparisons).
Effect of saturated sodium nitrite volume.
To evaluate the possibility that increasing the volume of
saturated sodium nitrite would improve oxidation inhibition,
blood from a single individual (10 mL), was aliquoted into ten
ice-cold gas chromatography vials (1 mL each). Five vials served
as blanks. To the other five vials, 21.71Jmol ethanol was added.
Saturated sodium nitrite was added to both the blank and
ethanol vials. The volume of saturated sodium nitrite was either
1, 2, 3, 4, or 5 mL. The ability to inhibit ethanol oxidation
was expressed as the oxidation ratio. With increasing volumes
of sodium nitrite, the oxidation inhibition ratio increased
sequentially from 1.43 to only 1.85, suggesting that increasing
the volume of sodium nitrite offered no significant improve-
ment in oxidation inhibition.
Confirmation of inhibition without suppression of recovery
A second series of experiments was performed to evaluate the
effectiveness of sodium nitrite as an inhibitor
of in vitro oxidation. Blood (6 mL) was drawn
from ten individuals with ice-cold syringes.
For each individual, the sample was split into
two sets of three 1-mL vials. In each set, one
vial without additives was considered a blank.
Recovery ratio*
(mean + s.d.) To the second and third vials in each set,
21.71Jmol ethanol and 22.71Jmol acetaldehyde
3.8 _+ 1 were added, respectively.One set, without the
2.7 _+1w addition of sodium nitrite, served as controls.
1.3 _+0.4w To the other set, 1 mL saturated sodium ni-
l.Ow trite was added within 2 s after acetaldehyde
13.8 -+5.2w or ethanol. All vials were immediately gas-
13.3 _+15.6w tight sealed and stored at 4~ until analysis
5.7 + 2.7~ was performed (within 2 h). The acetalde-
hyde peak area obtained in the presence of
21.7 mmol/L ethanol was compared with the
acetaldehyde peak area in the blank samples
to verify the effectiveness of nitrite as an in-
hibitor. The acetaldehyde areas in blank blood
with and without nitrite were compared, and
Journal of Analytical Toxicology, Vol. 21, March/April 1997
the acetaldehyde areas in blood containing 22.7 IJmol/L
acetaldehyde with and without nitrite were compared in order
to verifya lack of suppression of sodium nitrite on acetaldehyde
recovery.All comparisons used Student's paired t-test with an (x
value less than 0.05 accepted as significant.
Sodium nitrite significantly reduced the acetaldehyde peak
area in blood containing 21.7 mmol/L ethanol (Figure 2, right
bars, p < 0.001) such that the acetaldehydepeak area in the pres-
ence of sodium nitrite was not different from the acetaldehyde
observedin blank blood (Figure 2, left and fight bars). In contrast,
the addition of sodium nitrite had no impact on the acetaldehyde
peak area in blank blood (Figure 2, left bars) or in blood con-
taining 22.7 lJmol/Lof acetaldehyde (Figure 2, center bars).
Standard curves
Assay linearity and intersubject variation in the in vitro
formation of acetaldehyde were evaluated by performing stan-
dard curves for ethanol and acetaldehyde in blood drawn from
seven individuals. Fresh whole blood was drawn into ice-cold
syringes and placed into ice-cold GC vials. Vials were prepared
one at a time. The internal standard and ethanol standard (0-50
IJL) were added to one set of vials to yield final ethanol con-
centrations of 0 to 32.5 mmol/L. The internal standard and ac-
etaldehyde standard (0-50 I~L)were added to a second set of
vials to yield acetaldehyde concentrations of 0 to 113.5 ]Jmol/L.
For all vials, 1 mL of cold saturated sodium nitrite was added
within 2 s of the addition of ethanol or acetaldehyde. All vials
were immediately gas-tight sealed and stored at 4~ until assay,
which was performed within 2 h.
The ethanol assay was linear (r2 = 0.999, Figure 3A) over the
concentration range of 2.17 to 32.5 mmol/L. The concentration
of acetaldehyde in blank blood was 1.13 _+0.9 I~mol/L,and the
acetaldehyde assay was linear over the concentration range of
0.22 to 113.5 pmol/L (Figure 3B). The interassay coefficients of
variation were 1.9 and 7.7% for ethanol and acetaldehyde,
respectively, and the intra-assay coefficients of variation were
2.0 and 1.6% for ethanol and acetaldehyde, respectively.
In the presence of sodium nitrite with ethanol concentra-
Table II. Sodium Nitrite Concentrations and Inhibition of In Vitro Ethanol
Oxidation*
% Sodiumnitrite Oxidation InhibitionRatiot
(w/v) (mean • s.d.)
0 37.2 _+15'
1 17.9 + 5*
5 11.2 + 11
10 5.7+8
20 2.4 + 0.7w
30 2.3 + 0.6w
40 2,1 + 0.4w
60 1.7 + 0.1
75 (saturated) 1.3 + 0.1
* N = 5 at each concentration.
-t Oxidation inhibition ratio = acetaldehyde peak area in blood with 21.7 lJmol/L ethanol + inhibitor
acetaldehyde peak area in blank blood + inhibitor
* Comparison with satured sodium nitrite: p < 0.01.
w Comparison with satured sodium nitrite: p < 0.05.
tions between 4.3 and 21.7 mmol/L, the spontaneous forma-
tion of acetaldehyde displayed intersubject variability with
coefficients of variation between 31--46%. Within an indi-
vidual, the in vitro formation of acetaldehyde was linearly
related to ethanol concentration (r2 > 0.99). In the individual
with the largest amount of in vitro formation, the increase in
acetaldehyde concentration at an ethanol concentration of
10.8 mmol/L was 3 IJmol/L.
Effect of storage
In order to evaluate the effect of storage, whole blood samples
(1 mL each) were spiked with ethanol to final concentrations
from 0.22 to 13.0 mmol/L (N = 5, each concentration). A second
series of whole blood samples (1 mL each) were spiked with
acetaldehyde to final concentrations from 0.22 to 113.5 lamol/L
(N = 5, each concentration). The internal standard and I mL
saturated sodium nitrate were added, and the samples were
gas tight sealed. One ethanol and one acetaldehyde sample
were analyzed immediately, the remainder were stored over
liquid nitrogen at-195~ A storage temperature of-195~ was
chosen rather than the commonly used --80~ based on the
freezing point of acetaldehyde (-123.5~ and our experience
with poor recovery of acetaldehyde when stored at-80~ Sam-
ples were thawed and assayed at 1, 2, 3, and 5 days. The amount
of variance was described by coefficients of variation using the
initial sample analysis as baseline. Coefficients of variation in
excess of 10% were considered unacceptable.
Ethanol samples remained stable with storage at-195~ for
5 days with coefficients of variation between 0.3 and 8% (Figure
4A). In contrast, acetaldehyde was stable at -195~ for only 2
days. At 2 days, all the coefficients of variation for acetaldehyde
measurement except one were between 5-10% (Figure 4B).
When acetaldehyde assays were performed at either day 3 or day
5, significant decreases in the amount of acetaldehyde present
were observed, and an increase in variation was noted with all
coefficients of variation between 12 and 28%.
Application of Method to In
Vivo Human Studies
Healthy, nonpregnant women were re-
cruited for ethanol and acetaldehyde
metabolism studies. Following a 0.5 g/kg oral
dose of ethanol, multiple 1-mL blood sam-
ples were drawn from an indwelling catheter
for 8 h. Within 2 s of being drawn, the whole
blood samples (1 mL) were added to cold GC
vials containing I mL saturated sodium ni-
trite. The internal standard, 1-propanol, was
added and the vials gas-tight sealed. Vials
were stored at 4~ to be analyzed within 2 h.
A subject-specific standard curve was pre-
pared by the addition of the individual's
blank blood to iced GC vials. The appropriate
amount of ethanol or acetaldehyde stock so-
lution and the internal standard (10-50 IJL)
137

were added to each vial, and 1 mL of cold, saturated sodium
nitrite was added immediately.Followingincubation at 75~ for
16 rain, the headspace sample (3 mL vapors) was injected onto
the Carbowax column, and concentrations were determined
by flame ionization detection.
The ethanol and acetaldehyde areas under the concentra-
tion time curves were determined using the trapezoidal rule.
The apparent ethanol Vmaxand Kmwere determined by fitting
the ethanol concentration time data using the Michaelis-
Menten equation and the computer software program Minsq
(Micromath, Salt Lake City, UT). The acetaldehyde concentra-
tion time data were fit to an exponential equation using the
U application must take into consideration what portion of total
M
I---
Figure 1. Gas chromatograms from whole blood containing 22.7FIM
acetaldehyde (A), 2.17mM ethanol (B), and 0.83 mM 1-propanol (internal
standard, [C]).
138
Journal of Analytical Toxicology, Vol. 21, March/April 1997
program Rstrip (Micromath), and the terminal exponent was
used to determine the rate constant of dimination and its
associated terminal half life. For both data sets, a model selec-
tion criteria greater than 3.0 and r 2 > 0.9 were the criteria for
acceptable fit.
Six healthy African-American women (age range, 22-35
years) gave informed consent. Four women were smokers; two
women were nonsmokers. In the two weeks before the study,
three women consumed greater than seven drinks per week,
two consumed 4-6 drinks per week,and one woman abstained.
All women abstained from alcohol in the 12 h immediately be-
fore the study. Peak ethanol and acetaldehyde concentrations
occurred within one half hour after ethanol ingestion (repre-
sentative data, Figure 5). The median peak acetaldehyde con-
centration was 6.11JM (range, 2-80pM), whereas the median
ethanol peak concentration was 17.7mM (range, 14-20mM).
The areas under the concentration time curve for acetalde-
hyde and ethanol ranged from 3.8 to 13.4 pmol/Uh and from 32
to 80 mmol/L/h, respectively.The median terminal half life for
acetaldehyde elimination was 46 rain (range, 7-105 rain). The
apparent Kmfor ethanol was from 1.1 to 12.9pM; the apparent
Vmaxwas from 38.4 to 114.7 pmol/h/kg.
Discussion
The decision regarding which method of ethanol and acet-
aldehyde determination should be chosen for a given analytical
body acetaldehyde is measured, if simultaneous measurement
of ethanol is desirable, and the accuracy of the sample pro-
cessing and assay technique. Technical documentation should
include adequate sensitivity and recovery, linearity over the
100000 t
90000
80000
10000
Z
.ooo
"-~ 6000
~9 4000
2000
0 r i i
Blank AA ETOH
Figure 2. Gas chromatographic peak area acetaldehyde measurements in
blank whole blood samples, whole blood samples spiked with acetaldehyde
(AA, 21.7tiM), and whole blood spiked with ethanol (ETOH, 22.7mM) in
the presence (hatched bars) and absence (open bars)of saturated sodium ni-
trite. The addition of 22.7mM ethanol to whole blood without inhibitor
(open bar, far right) results in a significant increase in the amount of ac-
etaldehyde compared with blank blood (open bar, far left) (*p < 0.001). In
contrast, the addition of sodium nitrite to blood containing 22.7mM ethanol
(hatched bar, far right) results in acetaldehyde peak areas that are similar to
blank blood (hatched bar, far left).
Journal of Analytical Toxicology, Vol. 21, March/April 1997

anticipated concentration range, reproducibility, and adequate
stability of the compounds measured over the time the assay is
to be performed. For practical reasons, consideration must be
given to the ease of use of the method in a given application.
The method presented is an accurate, sensitive, automated
technique for the simultaneous measurement of whole blood
ethanol and acetaldehyde for in vivo human studies of ethanol
metabolism as demonstrated in the subjects evaluated. Head-
space injection eliminates the extensive sample preparation,
which decreases technician time and helps reduce the loss of
acetaldehyde from vaporization. Automation and short run
times enhance convenience and efficiency.The assay is linear
for both ethanol and acetaldehyde over the concentration
ranges expected in human in vivo studies (16,28). The sensi-
tivity achieved for acetaldehyde in this method required maxi-
mizing the sample on column using a large sample loop size,
high carrier gas flow rates, low split ratios, and a large sta-
tionary phase (data not shown). Furthermore, high-purity
helium (99.999%) minimized background noise.
The present method uses sodium nitrite as a deproteinizing
agent. Sodium nitrite is a known oxygenscavenger that depro-
teinates and also decreases the oxidation of ethanol to acet-
aldehyde by conversion of oxyhemoglobin to methemoglobin
(29). Compared with sodium azide, which has also been advo-
cated for the inhibition of ethanol oxidation (2,5), sodium
nitrite was superior in inhibiting the in vitro oxidation of
ethanol to acetaldehyde and was superior in the recovery of
acetaldehyde. It is possible that the difference observed
between nitrite and azide is secondary to the difference in con-
centrations used, and it is possible that using high concentra-
tions of sodium azide might enhance the inhibition observed.
We chose the concentration of azide based on previous pub-
lished use (5). Also, because azide is more dangerous than
nitrite, we chose to limit our use to low concentrations. The
most worrisome property of azide is its ability to release
hydrazoic acid, which is highly volatile, toxic to humans, and
highly explosive (30). In a recent azide-induced explosion and
fire, 11 workers suffered inhalational injuries that were sodium
azide induced (31). In addition, sodium azide is a potent
mutagen (32), and multiple cases of significant toxicity fol-
lowing relatively small accidental laboratory ingestions have
been reported (32,34).
As an alternative to increasing azide concentration, we
attempted to improve oxidation inhibition by combining azide,
which inhibits by reducing hemoglobin to oxyhemoglobin,
with polyethylene glycol, which deproteinates by exclusion
(35). The concentrations chosen were similar to those used by
others (5). Surprisingly, the combination of polyethylene glycol

35
A 1o- A
4.5X
30
8 - 2.7Z
t
25 i
o..

4X
20 6-
I
5X
15
4-
10 0.3X

2- 8X
5
X
0 ~r i I i i i i i
0 =" i f i i i i
0 S 10 15 20 25 30 35 0 2 4 6 8 10 12 14
Ethanol mM Ethanol mM

35 u 6X
B
30

m 25
8.9

N 2o

ts
14Z

.~ lO !=.
IOZ
s J.7Z ][
mr
<

0 ~ i i i i i i i i i i i i J i

0 5 10 15 20 25 30 35 20 40 eo eo ~oo ~2o
Acetaldehyde pM
Acetaldehyde IJM
Figure 4. Variance in ethanol (A) and acetaldehyde (B) whole
Figure 3. Representativestandard curves for whole blood ethanol (A, r2 = blood concentrations in samples stored at-195~ for 5 days and 2 days,
0.999) and acetaldehyde (B, r2 = 0.998) assays. respectively.

139

and azide led to a decrease in the inhibition of oxidation
achieved with azide alone and, equally importantly, ted to a
loss of recovery of acetaldehyde. Other authors have suggested
that deproteinization by polyethylene glycol alone is only 80%
effective (5); therefore, we did not evaluate it individually.
In addition to sodium nitrite and azide, the current analysis
evaluated the two other sample processing methods previously
reported: the use of zinc sulfate and barium hydroxide (36),
two salts that deproteinate by increasing ionic strength (37), and
perchloric acid, which deproteinates through pH alterations
(37). The use of perchloric acid, which achieves greater than
98% deproteinization (38), is one of the most common methods
of processing ethanol samples (19,26,28). In our analysis, the
perchloric acid method appeared as ineffective as saline in in-
hibiting the in vitro oxidation of ethanol, even when the per-
chloric acid was added within 2 s as suggested by Eriksson (26).
This lack of effectiveness is not likely secondary to inadequate
deproteinization, but may reflect the activity of perchloric acid
25 "~ A stants, must be used in large amounts (an organic to sample
20 84
151
10
0 I I I
0 2 4 6 8 reported methods while offering adequate sensitivity for human
Time (h)
6' B metabolic studies than HPLC methods that require labor
, ethanol and acetaldehyde.
=
4 '~ I
3
c~
2 This work was supported in part by the U.S. Public Health
0 ,
0 2 4 References
Time (h)
Figure 5. Representativeethanol (A) and acetaldehyde (B) concentration
time data obtained from a woman following oral ingestion of 0.5 g/kg
ethanol over 5 min.
140
Journal of Analytical Toxicology, Vol. 21, March/April 1997
as an oxidizing acid with its oxygen atoms available for contri-
bution to spontaneous nonenzymatic oxidation. We also evalu-
ated perchloric acid using a high concentration in combination
with semicarbazide, a trapping reagent, that has been used
primarily in plasma analyses to decrease the loss of acetaldehyde
during centrifugation steps (2,4,39). Although the recovery of
acetaldehyde was acceptable using perchloric acid and semi-
carbazide, the in vitro oxidation of ethanol was the worst. This
may be related to the high concentration of perchloric acid pro-
viding a large amount of oxygen for nonenzymatic oxidation, or
it may be related to the delay in adding the deproteinizer, per-
chloric acid. The latter idea would suggest that methods using
trapping or derivatizing steps may resu]t in spuriously high
concentrations of acetaldehyde if deproteinization is not carried
out simultaneously.
Deproteinization methods not analyzed in the present study
included the use of organic solvents, heat denaturation, pre-
column stationary columns, and ultrafiltration. Organic sol-
vents, which deproteinate by decreasing the dielectric con-
ratio of 2:1) to achieve adequate deproteinization (37). For gas
chromatography using headspace analysis, this large volume of
organic solvent may lead to interference with the peaks of
ethanol and acetaldehyde. Heat denaturation requires prior
derivatization to prevent loss from volatility, and, even with
derivatization, it would enhance the spontaneous oxidation of
alcohol to acetaldehyde (40). Precolumn stationary columns
and ultrafiltration techniques would also require prior deriva-
tization to minimize vaporization loss.
The method presented is an accurate, sensitive, and practical
method to measure ethanol and acetaldehyde simultaneously in
whole blood. Although no method totally eliminates the in
vitro formation of acetaldehyde from ethanol (41,42), the pre-
sented deproteinization method appears superior to previously
in vivo studies. Further, the convenience of the method sug-
gests it is a more desirable approach for in vivo ethanol
intensive sample preparation and two separate assays for
Acknowledgments
Service National [nstitutes of Health grant AA07606 and by
the Research Endowment Fund, Children's Hospital of
Michigan.
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Manuscript received April 16, 1996;
revision received August 8, 1996.
141