A modification of the gas chromatographic procedure of Natelson and Stellate has been developed which enables analyses of serum ethanol to be performed ...
An Improved Procedure for the Determination Serum Ethanol by Gas Chromatography
of
John Savory, F. William Sunderman, Jr., Norris 0. Roszel, and Paul Mushak
A modification of the gas chromatographic procedure of Natelson and Stellate has been developed which enables analyses of serum ethanol to be performed with increased sensitivity, precision, and convenience. Serum (50 i,.l.) is dehydrated by injection onto anhydrous copper sulfate in a stoppered tube at 800 in a water-jacketed rotary mixing apparatus. After 2 mm., ethanol vapor is swept by a stream of helium into a gas chromatographic column packed with a mixture of Flexol 8N8, diisodecylphthalate, propyleneglycol 600, and firebrick C-22. Ethanol emerges from the column after a mean retention time of 6.7 mm. and is measured by a hydrogen-flame ionization detector. The coefficient of variation of replicate analyses is 2.0%. The recovery of ethanol added to serum is 98.7 ± 3.8%. The limit of sensitivity for the detection of ethanol in serum is 0.1 mg./100 ml., and assay can be made in the presence of constituents which are normally present in serum, and volatile compounds which are commonly encountered in clinical toxicology. Analyses of serum ethanol by gas chromatography provide close correlations with measurements by the alcohol dehydrogenase reaction.
A
GAS CHROMATOGRAPHIC METHOD for the quantitative determination of ethanol in blood or serum, was described by Fox (1) in 1958 and has been modified by numerous subsequent investigators (2-18). These modifications have differed primarily in the technics for handling the samples of blood or serum prior to injection onto the columns of the gas chromatographs. In several procedures, ethanol is separated from blood proteins by diffusion (18), distillation (1, ii), solvent extraction (2, 10, 16), or protein precipitation (5, 13). In most of the other proFrom the Pathology Department, University of Florida College of Medicine, Gainesville, Fla. 32601. Supported by U. S. Atomic Energy Commission Grant AT-(40.1)-3461, by American Cancer Society Grant E-374B, and by 11. S. Public Health Service Research Grant CA.08783.02 from the National Cancer Institute. Presented at the Aiinunl Meeting of the American Society of Biological Chemists, Chicago, Ill.,Apr. 21, 1967. The authors acknowledge the skillful technical assistance of Mr. Manindra. C. Jayaswal. Received for publicntioii June 12, 1907; n(’cepfe.l for publication Aug. 3, 1907. 32
Vol. 14. No. 2, 1968
133
SERUM ETHANOL
cedures, a volatile internal standard is added to the blood, and a small sample of tile mixture is injected directly onto the gas chromatographic column (3, 4, 7-9, 12, 15, 17). In 1965, Natelson and Stellate (14) reported a novel technic whereby the sample is dehydrated by addition of arihydrous copper sulfate, and the liberated ethanol is swept by a stream of carrier gas onto the gas chromatographic column. The Natelson-Stellate procedure has been employed in our laboratory during the past 2 years, and has undergone a number of modifications which have significantly improved the sensitivity, precision, and adaptability of the technic for routine use in clinical laboratories. In many respects, this modified procedure is similar to the gas chromatographic method for determination of lactic acid in blood which has been described by Savory and Kaplan (19).
Method Principle Serum is injected into a gas-tight dehydration tube which contains an excess of anhydrous copper sulfate. Water is rapidly extracted from the serum by the formation of copper sulfate pentahydrate, an exothermic reaction which simultaneously liberates serum ethanol as a vapor. The ethanol vapor is swept onto the column of a gas chromatograph, where it is separated from the other volatile constituents of serum. Ethanol is detected and measured in the effluent gas from the chromatographic column by means of a hydrogen-flame ionization detector. Reagents
Copper is dried
powder
sulfate, overnight
is dispensed
anhydrous in a heated
powder, vacuum
into dehydration
reagent desiccator
grade at
This reagent 103#{176} before the
tubes. Ethanol stock standard solution, 1% (w/v) Using a 2-mi. buret graduated at 0.01 ml., 1.26 ml. of absolute ethanol is transferred to a 100-mi. volumetric flask containing approximately 50 ml. of distilled water. The contents of the flask are diluted to tile mark with distilled water. The stock standard should be prepared at an ambient temperature of approximately 20#{176}. Otherwise, appropriate adjustments should be made in the volume of absolute ethanol in order to compensate for changes in specific gravity. The ethanol stock standard solution is stored in the refrigerator. Ethanol working standard solutions Aqueous dilutions of the ethanol stock standard solution containing 25, 50, 100, 150, 200, and 250 mg. of ethanol per 100 ml. are prepared immediately before use.
134
SAVORY
fT AL.
Clinical
Chemistry
Apparatus
Microsyringes, Dehydration
50-p.i. capacity (Hamilton tubes The dehydration
Co.) tubes are test
tubes,
75
mm.
in length and 12 mm. in diameter, containing 130 mg. of anhydrous copper sulfate powder, and sealed with a puncture type rubber bung (Vacutainer stopper, Beckton-Dickinson Co.). Batches of these dehydration tubes may be stored indefinitely at room temperature. nmixinq apparatus A metal cup, 130 mm. in length and 40 mm. in diameter (metal centrifuge shield, International Equipment Co., No. 367), is fitted with two 0.25-in. by 2-in, copper tubes for inlet and outlet of water, and a No. 8 rubber stopper with a hole in tile center to accommodate the dehydration tube. The apparatus is mounted on a “Vortex” rotary mixing apparatus (Scientific Industries Co.). The metal cup is connected to a circulating water bath and maintained at 80#{176} (Fig. 1). Vapor injection sI/stem A vapor injection system is assembled as illustrated in Fig. 2. The necessary supplies are: (1) polyvinylchloride tubing, 0.045 in. 1.1). (Technicon Co.); (2) two male Luer-Loc needle connectors (Becton-Dickinson Co.); (3) two hypodermic needles (No. 20, 1.5 in. long; and No. 21, 1, in. long); (4) three Quick-Off valves (Whitey Valve Co.). Heated
A Model 402 chromatograph (F and M Scientific Co.) is used in the authors’ laboratory, l)ut comparable gas chromatographs of other makers should also be satisfactory. The gas chromatograph is fitted with a hydrogen-flame ionization detector and with a stainless steel chromatographic column* (10 ft. long and in. I.D.) packed with a mixture of 23 gm. of stationary phase to 100 gm. of C-22 firebrick (42-60 mesh). The stationary phase is a mixture of 15 parts by volume of Flexol 8N8, 10 parts of diisodecylphthalate, and 3 parts of polyethyleneglycol 600. The column temperature is kept at 100#{176}; the flash evaporator is kept at 125#{176}, and the detector is kept at 140#{176}. Helium is the carrier gas and is maintained at a flow rate of 73 mI./min. The sensitivity adjustment knobs are set at 10 (range), and at 128 or 256 (attenuation). Gas
cli romatograph
Procedure
Prior to venipuncture, the patient’s antecubital region is cleansed with 0.1% (w/v) mercuric chloride. The use of ethanol as an antiseptic should obviously be avoided. Blood samples are placed in stoppered tubes containing 0.1 mg. of mercuric chloride per milliliter of blood. *prepared
columns
may
be purchased
from
the Beckman
Instrument
Co.
Vol. 14, No. 2, 1968
SERUM ETHANOL
135
1’
Fig. jection water
I. Gas ehiroinatograph adapted system is employed in conjunction
for measurement with water-jacketed
of
serum ethanol. Modified mixing apparatus and
vapor circulating
iii-
bath.
The serum is removed and kept in stoppered tube until the time of analysis.
the
refrigerator
in
a
tightly
Using microsyrmges, SO-p.]. samples of serum, ethanol working standard solutions, and water (‘‘blank’’ sample) are injected onto anhydrous copper sulfate in stoppered dehydration tubes. Tile contents of each tube are mixed by tapping tile tube oii the laboratory bench and by shaking tile tube vigorously. A ‘‘bypass’’ of the injection system of the gas chromatograph is estal)lished by closing valves A and B and by opening valve C (Fig. 2). The inlet needle is inserted as far as the needle hub througil tile Vacutaiiier stopper of one of tile dehydration tubes. Tile ti1) of the outiet needle is positioned ill the 110110w cavity beneath tile stopper. Tile dehydration tUl)e is placed inside tile waterjacketed mixing apparatus alld is heated at 80#{176} for 2 fun. The Vortex mixer is turned on (luring tile last 30 sec. of tile heating period. After the mixer has l)een stopped, valves A and B are opened, and valve C is then closed. helium flow is thereby directed through the dehydration tube and onto the column. The dehydration tube is purged with helium carried gas for 90 sec. \alve C is then opened and valves A and ii are closed. As illustrated in Fig. 3, etllallol emerges from the chromatographic column as a symmetrical peak after a retention time of approximately 6.7 mm. The height of tile ethanol peak is measured and used in the
fT AL.
SAVORY
136
The
computations.
conceiitratioii
of
Clinkal
serum
ethanol
Chemistry
is calculated
as
follows: serum Ethanol
(mg./l00
ml.)
=
peak
standard
peak
(em.) (cm.)
X
colic,
of standard
Before the next sample is introduced into the gas chromatograph, empty tube is attached to the vapor-injection system and placed
an in the
to
(()ltJIIlfl flow
meter valves
from bet lit in
hvpoderm
ia
Ic needle
dehvtl rat ion tube water jacket
cus04
(80#{176}C)
Fig.
2. Diagram
of vapor-injection
system
for
gas
chroinatographic
nicasurenients
of serum
ethnnol.
mixing apparatus. Valves A and B are opened, and valve C is then closed. The entire chromatographic system is purged with heliuun carrier gas for at least 10 miii, to eliminate any residual volatile constituents of serum. water-jacketed
Results StandardCurve A calibration chart of peak in Fig. 4 to demonstrate the ables. Because of convenience, computations instead of peak exists between the ethanol chromatographic peaks.
height vs. ethanol concentration is given linear relationship between the two varipeak height is used routinely in the area. However, a linear relationship also concentrations and the areas under the
Precision
The precision of the method was evaluated by 22 replicate analyses of a single sample of serum. The mean concentration of ethanol in the
Vol. 14, No. 2, 1968
137
SERUM ETHANOL
::.JiH
L
TL
..
-
T:
.
t
±LL
T t
L
.L
I
iL..
±H4H
1
‘
fH± ELI
1
-
.
-
H1
. .
Li:
#{149}
1
--
[ -
L
-i
-
i T
I .
.
.
.
-
= IEL LJ__I Fig.
3.
produced ethnnol Tracing
Typical by
aqueous
.
-
gas
chromatogram ethanol
100 ml. Peak D obtained with
per
-
IIT EIELILIT I :T1_ J__
jiiiJ1I -
I
t
C obtained serum
obtained
standard from
solutions
with
serum
a control
by
the
described
containing,
from
patient
person
who
procedure: respectively,
with had
not
acute
Peaks A and B 100 and 15(1 lug.
alcoholic
ingested
intoxication.
ethanol.
sample was 104.5 mg./100 ml. with a standard deviation of ± 2.1 mg./ 100 ml., and a coefficient of variation of 2.0%. During preliminary studies of the technic, an air-jacketed heating assembly similar to that described by Natelson and Stellate (14) was used instead of the waterjacketed heating assembly. Using the air-jacketed apparatus, 25 replicate analyses of a single sample of serum gave a mean concentration of
138
SAVORY
ethanol of 83.4 mg./100 100 ml., and a coefficient
fT AL.
Clinical
nIl., with a standard deviation of variation of 4.6%.
of
Chemistry
3.9 mg./
±
Optimum Conditions for Vaporization of Ethanol
Analyses
of a single
pet’ 100 ml. were
specimen
performed
of serum
while
tile
containing
150 mg. of ethanol
water-jacketed
heating
assembly
20
IS
10-
Fig. for
4. Standard
measurement
by
nol
gas
curve of
etha-
chroinatog-
raphiy.
5-
0
0
50
bOO Ethanol
50
200
250
(mg per lOOmI)
was mailitailled at 60, 70, 80, and 900. The mean iieak heights which were obtained at these temperatures of vaporization were 10.9, 11.6, 12.0, and 12.0 cm., respectively. I\leasurements above 90#{176} were imowing to release of water vapor from the hydrated copper sulfate, and subsequent condensation of the water in the plastic tubing which connects the outlet needle to the port of the gas chromatograph. Inasmuch as optimum sensitivity alld precision were obtained at 80#{176}, this tenuperature was selected for routine use. Time optimum times for Ileatillg and mixing tile dehyclratioll tube and precise,
purging tile vaporized ethanol with carrier varying tile duration of one step while keeping
gas were ascertained by the duration of the other steps constant. Tile optimum interval for heating the dehydration tube was determined to be 120 sec. at 80#{176}, with the Vortex mixing performed during the final 30 sec. Greatest sensitivity was obtained with 90 sec. of purging with carrier gas. The dimensions of the dehydration tube \vere fouiid to be relatively critical, since tile 115C of lai’ger tubes resulted for
iii broadening
of tile
chromatogi’aphic
tubes was occasionally attended crystals of copper sulfate.
peaks,
by clogging
and
the
use
of the outlet
of
smaller
needle
with
V01. 14,
o.
2. 1968
139
SERUM ETHANOL
Recovery Tests
r1easurenuel1ts of ethanol recovery were i)erforlned b additiouis of ethanol to pooled serum in quantities equivalent to 75 and 130 mg./ 100 ml. As indicated in Table 1, the mean recovery of ethanol was 98.7%. Sensitivity Studies
When the gas chromatograph was operated at its maximum sensitivity settings, an aqueous standard solution containing 5 pg. of ethanol per 100 ml. yielded a peak height of 12 cm. For practical purposes, tile limit, of sensitivity for detection of ethanol in serum was found to l)e approximately 0.1 mg./100 ml. in comparison, the limit of detection of etilanol by the original Natelson-Stehlate l)rocedure was 3-3 mg./i00 ml. (14).
Interference Studies
Compounds which are commonly 01’ occasionally present ill serum were tested for interference in the analysis of ethanol (Table 2). The compounds were dissolved or suspended iii water in concentrations of 200 nig./100 ml. Fifty-microliter samples of these solutions or suspensions were subjected to gas cilromatography as previously described. None of these compounds interfered in the measurement of ethanol. It was observed that acetaldehyde, acetone, benzene, chloroform, diethyl ether, halotilaule, isopropanol, metilallol, and n-propanol yielded symmetrical peaks with specific retention times. Therefore, tile gas chromatographic method may be adaptable to the qualitative detection and quantitative determination of these compounds. A chromatogram obtained by the described procedure with an aqueous solutioll containing a mixture of acetaldehyde, methanol, acetone, ethanol, isopropanol, and n-propanol is illustrated in Fig. 5. More complete resolution of these compounds was obtained 1)y decreasing the flow rate of helium carrier gas from 75 to 43 ml./min. (Fig. 5). With this decrease iii flow rate of helium, tile retention time of ethanol was increased from (1.7 to 13.4 mm., and the retention times of the other constituents were proportionately prolonged. For routine measurements of serum ethanol, the high flow rate and shorter retention time are preferred, Since the Table
I.
MEASUREMENTS
OF THE RECOVERY Ethanol
Analt,’-e
(En.)
15 15 *
Mean
± S.D.
Added
75 150
(mg/lOO
OF ETHANOL
ADDED
TO SERUM
ml.) Pou,Id5
Rerorery*
74.0 ±20 14S.0±5.7
PS.7±2.7 9S.7±3.S
(O#{149})
140
fT AL.
SAVORY
Table
2. COMPOUNDS
Clinical
TESTED!FOR’INTERFERENCE MEASUREMENTS
Compound
IN GAS
OF SERUM
CHROMATOCRAPHIU
ETHANOl. Ttm
Retention
PeaL*
Acetaldehyde Acetic acid Acetone Benzene Beazoic acid Chloroform Cholesterol Creatinine 1)iethyl ether Ethanol Formaldehyde Formic acid
+
Galactose
-
-
-
-
+
8.7
-
-
Glucose Halothane -Hydroxybutyric
-
+ +
5.4 12.0
-
-
+
13.5
-
-
-
-
:.o
+ +
Isopropanol
6.7
-
-
-
-
+
7.9
Lactic acid Methanol
-
-
+
4 .9
Phenobarhital
-
n-Propanol
-1-
Pyruvie
Salicyclic Urea Uric acid
acid acid
Water *
-
12 .5
-
-
-
-
-
-
-
-
-
+
=
Chromatographic
chromatographic
analyses are determinations
peak
observed
with
(mln.)
3.1
-
acid
Chemistry
the hydrogeti-flome
iotiizatioi,
detect
or.
-
=
No
peak observed.
performed more rapidly, is slightly improved.
and
the
precision
of replicate
Comparisonwith a Reference Method In Fig. 6, determinations of serum ethanol by gas chromatography are contrasted with measurements by tile alcohol dehydrogenase procedure of Bonnichsen (20). In analyses of 15 samples, the slope of the regression line was 0.98 and the standard error of estimate was ± 6.3 mg. of ethanol per 100 ml.
Discussion Tile method for ethanol analysis which was described by Natelson amId Stellate in 1965 (14) overcame many of the difficulties which had been encountered with previous gas chromatographic technics for determination of ethanol in blood or serum. Dehydration and simultaneous deproteinization of the sample with anhydrous copper sulfate
Vol. 14. No. 2, 1968
141
SERUM ETHANOL
was more rapid and more precise than the procedures which involved diffusion, distillation, solvent extraction, or protein precipitation. Moreover, the Natelson-Stellate technic enabled 100-l. samples of blood to be analyzed instead of the 1- or 2-l. samples which were
ho
I Helium Flow -75 ml per mm
8 F
ABC D E F-
4 -.%.
E
Acetaldehyde Methanol Acetone Ethanol Isopropanol n - Propanol
2
‘3 ‘S
0
5
0
5
20
25
of volatile
compounds.
30
Minutes Fig.
5. Gas
chromatograms
of
a mixture
usually employed in gas chromatographic technics involving direct “on-column” injections. Quantitative addition of a volatile internal standard was avoided by the Natelson-Stellate procedure, and the presence of large solvent peaks was excluded. The modifications of the Natelson-Stellate procedure which are described in the present paper have resulted in significant improvements in sensitivity, precision, and convenience. The major differences between the present modification and the original Natelson-Stellate method (14) are summarized in Table 3. Analysis of serum samples has proved to be preferable to analysis of whole blood, since blood tends to form air-locks in the microsyringe, and blood reacts with anhydrous copper sulfate to form a dense cake which is difficult to disperse with the Vortex mixer. Use of anhydrous copper sulfate powder has been
142
SAVORY
fT AL.
Clinical
Chemistry
found to be more convenient than the glass beads impregnated copper sulfate which were employed by Natelson and Stellate Contamination of the chromatographic column with copper sulfate has been avoided by use of narrow test tubes rather than the
with (14).
dust wide
200
150 -
30
E
C
a) 0
00
II:
50
50 Ethanol Fig. alcohol
6. Comparison
of measurements
dehydrogenase
procedure
penicillin limiting
00
(mg/lOOmll of
serum
Gas ethanol
150
2 CO
Chromatocjraph’ by
gas
chromatography
and
by
an
(.O).
bottles employed by Natelsoll and Stellate, the amount of anhycirous copper sulfate to
and also by 150 mg. This quantity of copper sulfate provides a sufficient excess above tile 90 nig. which is required to combine Witil 50 l. of water. rFlle cliromatographic column employed in tile present modification gives sharper peaks and better resolution of volatile constituents than tile combined Carbowax and Hailcomid columns described in tile original method. By use of a hydrogen-flame ionization detection system instead of thermal couuductivity detection, the water vapor peak has been eliminated and the sensitivity of measurements of ethanol has been increased. During the past year, the gas chromatographic I)roCedllre described in this paper has been used routinely iii our laboratory to furnish medicolegal evidence of alcoholic intoxication. The high sensitivity of the gas chromatographic method for analysis of serum ethanol has enabled traces of ethanol to be detected in serum one or more days tufter the ingestion of alcoholic beverages. For this reason, measure-
Vol. 14, No. 2, 1968
Table
3. l)IFFERENCEN
SERUM
BETWEEN NATELSON
Parameter
hating
THE PRESENT -STELLATE
?%ate! ron-Stetlate
Type of sample \olume of sample I)ehydrating agenl assembly
\aporization temperature I teating time Purging time 1-lelium flow rate Column packing
143
ETHANOL
MODIFICATION
PROCEDURE
AND THE
method
Whole blood 100 Cu504 (250 mg.) on perforated glass beads Air-jacketed aluminum healing block 50#{176} 3 miii. 0.3 mm. 20 ml./mmn. Carhowax & Hailcomid Itit 35-mesh Teflon 6
ORIGINAL
(14) Present
modification
Serum 50 l. CuSO4
powder
Water-jacketed heater
(150 mg.) circulating
2 mm. 1.5 miii. 75 nil/mitt. I )iisodecylphthalat e, polyethyleneglyeol, & Flexol SNS on C-22 fIrebrick
Column I)etector
I empet-at
ure
500
‘l’hermal
conductivity
hydrogen-flame
100#{176} ionization
-
meiits of serum ethanol may be used to monitor abstinence from alcohol in patients who are being treated for alcoholic cirrhosis or chronic alcoholism. Confronted with graphic proof of the presence of ethanol in their sei’uni samples, such patients usually admit to the ingestion of alcohol. Patients are niore conscientious in observing abstinence Irom alcoholic beverages when they recognize that the concentration of serum ethanol in their blood is being monitored. The gas chromatographic technic has also been useful in detecting ethanol produced endogenously in cerebrospinal fluid by Gryptococcus neoformans. Ethanol concentrations ranging from 0.4 to 1.7 mg./100 ml. have been reported in the cerebrospimmal fluid of patients with cryptococcal meningitis (21). Tn two patients who have been studied in our hospital, the diagnosis of cryptococcal meningitis has been corroborated by the demonstration of traces of ethanol ill tile cerebrospinal fluid.
References .1. F., Gas chromatograpitic analysis of alcohol and certain other volatiles ill biological materials for forensic purposes. Proc. Soc. Exp. Biol. Med. 97, 236 (1958). 2. Cadman, W. J., and Johns, T., Application of the gas chromatograph in the laboratory of crimimalisties. J. Forensic Sci. 5, 369 (1960). 3. Chuadela, B., and Janak, J., Quantitative determinations of ethanol besides other volatile substances in blood and other body liquids by gas chromatography. J. Forensic Med. 7, 153 (1960). 4. Curry, A. S..., Walker, G. W., and Simpson, G. S., Determination of ethanol in blood by gas chromatography. Analyst 91, 742 (1966) 5. Davis, R. A., The determination of ethanol in blood or tissue by gas chromatography. J. Forensic Sri. 11, 205 (1966). 6. Drawert, F., and Kupfer, 0., Reaction gas chromatography: V. Blood alcohol analysis. Hoppe Seyler Z. Physiol. Chem. 329, 90 (1962). 1.
Fox,
144 7.
SAVORY
fT AL.
Clinical
Chemistry
J. B., and Vignau, J. A., Determination of alcohols in body fluids by gas. liquid chromatography. J. Forensic Sci. 10, 73 (1965). 8. Freund, G., Gas chromatographic determination of volatile substances in biological fluids by direct on-column injection. Anal. Chen. 39, 545 (1967). 9. Hessel, D. W., and Modglin, F. R., The quantitative determination of ethanol and other volatile substances in blood by gas-liquid partition chromatography. J. Forensic Sci. 9, 955 (1964). 10. Lyons, H., and Bord, J., Gas chromatographic determination of lower alcohols in biologic samples. Chin. Chem. 10, 429 (1964). 11. McCord, W. M., and Gadsden, R. H., The identification and determination of alcohols in blood by gas chromatography. J. Gas Chromatog. 2, 38 (1964). 12. Machata, G., Routine determination of blood alcohol concentration by gas chromatography. Milcrochim. Aeta 4, 691 (1962). 13. Maricq, L., and Molle, L., Investigations on the determination of blood alcohol levels by gas chromatography. Bull. Acad. Med. Belg. (Series 6) 24, 199 (1959). 14. Natelson, S., and Stellate, R. L., Instrumentation for the concentration of trace components of a mixture for gas chromatography: Application to the determination of acetone, ethanol, methanol, and 2-propanol in blood. Microchem. J. 9, 245 (1965). 15. Parker, K. D., Fontan, C. R., Yee, J. L., and Kirk, P. L., Gas chromatographie determination of ethyl alcohol in blood for medicolegal purposes. Anal. Chem. 34, 1234 (1962). 16. Rockerbie, R. A., The quantitative determination of ethanol in blood by gas chromatography. Can. Pharm. J. 96, 38 (1963). 17. Sturner, W. Q., and Coumbis, R. J., The quantitation of ethyl alcohol in vitreous humor and blood by gas chromatography. Am. J. Chin. Pathol. 46, 349 (1966). Freudiger,
18. Wallace, J. E., and DahI, E. V., Rapid vapor phase method for determining ethanol blood and urine by gas chromatography. Am. J. Chin. Pathol. 46, 152 (1966). 19. Savory, J., and Kaplan, A., A gas chromatographie method for the determinatioa lactic acid in blood. Chin. Che-m. 12, 559 (1966). 20.
Bonnichsen, Methods
21.
Dawson, Engl.
pp.
B., “Ethanol of Enzymatic
Determination with Alcohol Dehydrogenase Analysis, Bergmeyer, H. II, Ed. Acad. Press,
in of
and DPN.” In New York, 1963,
285-287.
D. M., and Taghavy, A., A test J. Med. 269, 1424 (1963).
for
spinal-fluid
alcohol
in torula
meningitis.
New
