A New Method for the Determination of Ethanol in the Blood andUrine by Pulse Heating
Taizo Nagano, Tatsunori Takayasu and Hitoshi Maeda
Department of Legal Medicine, Kanazawa University School of Medicine, Takara-machi
13 1, Kanazawa 920, Japan(Received May 22, 1989 ; accepted July 18, 1989)
Abstract
Abstract.
We have established a new method for the determination of ethanol and othergaseous substances in even minute amounts of biological material by utilizing a Curie-point
pyrolyzer (Model JHP 3, Japan Analytical Industry) connected to GC or GC-MS. No pretreatment of
the biological material is needed and the procedure is based on the principle that volatile or gaseous
substances can be evaporated from biological materials by pulse heating and introduced for analysis
directly to a GC or GC-MS.
The blood from rats that had been administered ethanol-d6 orally, as well as blood and urine
collected from healthy volunteers that had ingested alcoholic drinks and blood and tissue specimens
taken from autopsied cadavers were examined for ethanol concentrations by both this new method
and by the usual head space-GC method.
To test this new method, one microliter of the blood or urine was injected into a tubularly folded
ferromagnetic alloy (Pyrofoilｮ) and heated pulsatively five times at 160°C (pulse heating). The vapor
that evaporated from the sample then was directly introduced to the GC or GC-MS, and the timerequired to complete the analysis took approximately 5 min. In parallel, for comparison purposes,
portions of the same samples (0.2-0.5g) were examined by means of the head space method as well.
The measured values of ethanol-d6 and ethanol in the rat blood and in the human whole blood,
blood plasma, and urine that were obtained by this pulse heating procedure correlated almost
perfectly with those obtained by head space method. These results show the accuracy andreproducibility of this new method. Further, “pulse heating” makes it possible to determine thepresence of ethanol very quickly from a very small amount of the specimen.
We have termed this new investigative method for determining volatile and gaseous substances
the “pulse heating method”. Informatively, nitrous oxide (N 2 O, laughing gas) was identified by thispulse heating GC-MS procedure from the blood and organ specimens of a victim that had succumbed
to gas asphyxia.
Key words : Toxicology, Ethyl alcohol, Mass fragmentography

Introduction
The analysis of ethanol in biological materials isa routine procedure in the medico-legal practiceand it has a great significance when investigating
traffic incidents. For determining the presence ofethanol, several methods are available, though
they are relatively complicated and time-consuming. Procedures that use gas chromatography forthe detection of ethanol and other volatile poisonsin biological materials have been reported 1)-7) .

Among them, a head space method 1)-4) is now
widely utilized because of its many advantages.
This method, however, also has some disadvantages that will be mentioned in the Discussion
portion of this paper. Further, Ueda and Fukui8'
and Ishizawa et al.9),10) have reported on a possible
method for determining the presence of ethanol bya process of evaporation that requires only a verysmall amount of the specimen to be tested anduses an electric furnace-type pyrolyzer. While
their methods are theoretically appropriate, they

have not been practically utilized because of thereasons that also will be mentioned in the
Discussion.
Recently, a Curie-point pyrolyzer has beendeveloped that uses ferromagnetic alloys whichvery quickly generate heat by electromagnetic
induction and can be warmed to an accurately
defined Curie temperature 11)12) . Thus, we havemade an attempt to establish a new method for
determining the presence of ethanol and othervolatile and gaseous substances by pulse heating,utilizing this Curie-point pyrolyzer and connecting it to a gas chromatograph (GC) apparatus or agas chromatograph-mass spectrometer (GC-MS).
In this study, we have carried out a fundamental animal experiment using ethanol-d6 andwere able to determine the presence of alcohol inhuman blood and urine by this method, which wehave termed the “pulse heating method”, andhave compared our results with the results
achieved by the head space method.
Materials and Methods

1. Standard solution.
Deuterium-labelled ethanol-d6 (MSD Isotopes,
Canada; 0.2-2.0 mg/ml) and natural ethanol(0.2-5.0 mg/ml) were added to distilled water.
2. Ethanol-d6 containing samples.
Ethanol-d6 (25 v/v% aqueous solution) was
administered orally to rats (Wister strain, ca. 200
g in weight) at a dose of 1 g per kg body weight.
The blood was extracted from 15 to 90 min after
this administration, and stored in small glass
vials at 4°C.
3. Human blood and urine samples.
Healthy adult volunteers were given alcoholic
drinks: beer in the amount of 750 and 1,300 ml;
and, Japanese sake in the amount of 700 and 950
ml. Blood and urine samples then were collected atabout 30 min after ingestion. The whole blood,plasma, and urine were stored at 4°C until use.
Blood samples also were collected from 12
autopsy cases and examined as well.
4. Equipments and analytical conditions.
A Curie-point pyrolyzer (Japan Analytical Industry, Model JHP-3) was connected to a GC
(JEOL, Model GCG-06, FID) or a GC-MS system(JEOL, Model JMS-DX303 & DA5100 Data
System). Fig. 1 is a schematic diagram that showscarrier gas flow (He)45 ml/min
MS El (positive) mode
accel volt
3 kV
ionization volt
70 eV
ionization current
0.3 mAionization chamber temperature
150°C
5. Analytical procedure.
For calibration curves, 0.5, 1.0, 2.0, 3.0, and 5.0//I of standard solutions of ethanol-d6 and ethanol
were used. Ethanol standard solutions of 0.2, 0.5,
1.0, 2.0, 3.0, 4.0, and 5.0 mg/ml were used todetermine within-run and day-to-day precision.
Each sample was divided into two portions andanalyzed in parallel by both the head spacemethod and the “pulse heating” method.
Pulse heating method : One microliter of eachsample was extracted with a microsyringe andinjected into a Pyrofoil ® which was folded in atubular form. The foil was immediately inserted
into a “sample tube” which was fixed on a holder
and set on the “GC-prove” of the pyrolyzer (Fig.
1). The sample then was heated at 160°C for 4 sec
five times pulsatively at intervals of 20 sec inorder to completely evaporate the volatile substances. The vapor was analyzed by the Py-GC or-GC-MS.
Head space method : According to the method of
Nanikawa and Kotoku4), samples (0.5 ml) pipetted
into vials were incubated with an internal standard (0.2 mg/ml tert-butanol) at 55°C for 20-25
min. The head space gas (about 1 ml) then was
analyzed by GC or GC-MS.
6. A case of determination of gaseous anesthetics
in human blood and tissue specimens.
During our investigations, we also had theopportunity to analyze the amount of gaseousanesthetics present in the heart blood and thebrain, and also in the heart muscle, lung, liver,and kidney by means of both methods. The above
materials were obtained from a cadaver of a 9year-old, who had died in an anesthetic accident. A
small amount of each material was sealed into avial, and frozen at –30°C immediately after being
taken from the cadaver. Two days later thematerials then were examined by the two methods
described above.
Results
1. Mass chromatographic analysis of ethanl-d6

from animal blood by the pulse heating method.
Rat blood containing ethanol-d5, into which
ethanol-d6 immediately changed in water, wereanalyzed by the pulse heating method. Mass
chromatogram of ethanol-d5 (ion current profiles
at m/z 49 and 51) that was extracted by acomputer-data process from a series of consecutively recorded mass spectra is shown in Fig. 2.
When the blood samples were examined by 5 pulse
heatings, only one peak was noted at a retention
time (1.6 min) of ethanol-d6 (-d5) in the mass
chromatogram (Fig. 2). The El mass spectrum of
the peak showed predominant ions of ethanol-d5
at m/z 33, 49 and 51. Additionally, very small ion
peaks at m/z 50, 46 and 48 were observed, whichwere also detected in standard solutions. No otherions, including natural ethanol (m/z 31 and 45),
were noted.
2. Calibration Curves.
Using 0.2-2.0 mg/ml of an ethanol-d6 solution
as the standard solution, calibration curves weredrawn from an estimation of the peak area andpeak height of their mass-chromatograms. Eachline was linear through the zero-point as shown in
Fig. 3. By means of the peak area, the following
equation was obtained : Y=4.40 X 106X, where Y is
the peak area per 1ìl, and X is the concentrationof ethanol-d6. Similar results were obtained whennatural ethanol (0.2-5.0 mg/dl) was used insteadofethanol-d6. When 0.5, 2.0, 3.0, and 5.0ìlof eachstandard solution was applied, the calibration
curves were almost identical to that seen in theuse of 1 ìl of the standards.
For within-run precision, the coefficients ofvariation (CVs) ranged from 2.7 to 5.1% at 0.2-5.0
mg/ml level. For day-to-day precision, CVs ranged
from 5.5 to 8.6%.
3. Measured values of ethanol d6 concentrationsin the animal blood.
Ethanol-d6 (changed into -d5) concentrations in
the rat heart blood measured by the pulse heating
and the head space methods are given in Table 1.
The measured values by both methods very
closely approximated each other. The correlation
between the values by both methods is shown in
Fig. 4 and the regression curve was estimated per
the following formula: Y=0.03 + 1.03 X (Y, themeasured value by the pulse heating method; X,
the measured value by the head space method)with a correlation coefficient (r) 0.965 (sample sizen=15).
4. Measured values of ethanol concentration inhuman whole blood, plasma, and urine.
Ethanol concentrations of the whole blood,plasma, and urine collected from healthy volunteers were measured by both the pulse heating
and the head space methods. The data obtained
are shown in Table 2. The correlation between themeasured values by both methods is shown in Fig.
5 and the regression curve was estimated per thefollowing formula: Y=0.017 + 1.03X (r=0.998,n=12).
5. Measured values of ethanol concentration inwhole blood in autopsy cases.
In autopsy cases as well, the measured values ofthe blood ethanol concentrations by means of thepulse heating were almost identical with the head
space methods (Table 3). For the correlation between both methods, the regression curve wasestimated as follows : Y=0.020 + 0.952X (r=0.997,n=12).
All processes to complete the analysis required

about 5 min.
6. Determination of gaseous anesthetics in thehuman blood and tissue specimens.
By both the pulse heating GC-MS method andthe head space-GC-MS method, nitrous oxide(N2O, laughing gas) was qualitatively identified in
the all tissue specimens tested.
Discussion
Because of its many advantages, the head space
GC method is widely used internationally. There
are many reasons for this ; the method has a highreliability and shows a fairly good reproducibility,
and the head space gas can be directly applied to a
GC-apparatus for measurement. However, particularly when the ethanol concentrations arevery low or abnormally high or when the amountof the materials to be tested is very minute, there
are also some disadvantages in utilizing thismethod. Contrary it is theoretically appropriate todetermine ethanol and other volatile or gaseous
substances which were evaporated out from thesample specimens Attempts 8)-10) have been madeto use an electric furnace type pyrolyzer to evaporate ethanol from minute samples as a method ofgas content analysis. Using this approach Ueda
and Fukui 8) have tried to analyze the ethanol
content by heating samples sealed in polyethylene
tubes that were melted by heat. Although, as they
have described, the interference from the pyrolysis products of the polyethylene containers was
negligible, some small peaks appeared that resulted from polyethylene, with a longer retention
time than that of the ethanol. Therefore, the next
sample could not be introduced for about 22 min
until all the pyrolysis product of the polyethylene
had left the column. Similarly, the gas chromatographic method reported by Ishizawa et al. 9) 10)
requires more than 30 sec to reach the necessary
temperature, by which time the ethanol will havecompletely evaporated from the specimens.
In contrast the Curie-point pyrolyzer, which we
employed, possesses a valuable heating capacitythat utilizes a self-heating capability of ferromagnetic alloys. In other words, by means ofelectromagnetic induction, hysteresis generates
heat in the metal due to friction caused by thechanging polarity. At the Curie-point, the metalthen suddenly looses its magnetism owing to adrastic change in the magnetic permeability. In
formatively, the ferromagnetic metal, Pyrofoilｮ,that was used in this study reaches the Curiepoint within 0.2 sec, whereas the temperature in a
furnace pyrolyzer rises far more slowly, taking
longer than 5 sec. Further, Pyrofoil® cools rapidly
due to the interruption of the electromagnetic
induction. Therefore, samples injected or wrappedin a tubularly folded foil can be instantaneously
and repeatedly heated to a given Curie temperature, and the heating duration can be appropriately controlled.
As shown in Fig. 2, by using a Curie-point
pyrolyzer that was connected to a GC or GC-MS, it
was possible to determine the ethanol content
very quickly from a very small sample of thematerial (ca. 0.5-5ìl), whereas the head spacemethod required ca 0.5 ml of the sample. Further,
the pulse heating method could be repeated atshort intervals until the ethanol-d5 was completely released from the specimens (Fig. 2). The
blood samples gave only one peak (retention time,
1.6 min) in spite of 5 pulse heatings, and all thenecessary processes for one analysis were completed within approximately 5 min. Informatively,
the peak width of the ethanol (or -d5) was about 20
sec. For a comparison the furnace pyrolyzer
method used by Ueda and Fukui8) and by Ishizawa
et al. 9),10) gave an ethanol peak width that was
about 45 sec and required approximately 22 and 25
min for one analysis, respectively.
This shows that a Py-GC (or GC-MS) by meansof a Curie point pyrolyzer can be performed more
rapidly than the methods that use a furnace
pyrolyzer8)~10) and that the peaks are remarkably
sharper. The Py-GC-MS analyses of ethanol-d6(-d5) in blood samples showed no other peak thanethanol-d6. This indicates that there occurred no
artifacts, such as the degradation of ethanol-d5 or
pyrolysis production of interfering substancesfrom the blood.
Further, all the calibration curves of ethanol (or-d6) obtained by repeated examinations were
almost identical, and the stability of the calibration curves is reflected in the within-run and dayto-day precision.
From these data, it can be said that the pulse
heating-GC or -GC-MS method has good accuracy
and sufficient reproducibility for practical assaysof ethanol in the blood and urine. A series of
examinations to prove the reliability of thismethod showed that the measured values fromthe blood and urine (liquid samples) very closely
approximated those obtained by the head spacemethod (Figs. 4 & 5).
In the rat whole blood and in human whole
blood, as well as in the plasma and urine fromvolunteers, the measured values obtained by thepulse heating method had a tendency to be slightly
higher than the values obtained by the head spacemethod (Tables 1& 2 and Figs. 4 & 5).
Thus, the use of a Curie point pyrolyzer issuitable for determining the ethanol content of
biological materials. Employing this new method,
the analysis can be very quickly performed andthe materials to be tested require no pretreatment.
Additionally, the quantitative accuracy and reproducibility are comparable to those obtained by
the head space method.
Further, our preliminary study has shown that
this method can be applied to the analysis of othervolatile and gaseous substances, such as petroliccomponents and gaseous anesthetics, and thedetails of our results will be published separately.
Informatively, we had an opportunity to determine gaseous anesthetics in the heart bloodand in other tissue specimens of a 9-year-old who
unexpectedly died while under anesthesia. Using
our pulse heating GC-MS method, laughing gas
was determined in all the specimens tested. This
indicate the possibility of determining the presence of volatile and gaseous drugs and poisons in
all kinds of biological materials whether liquid or
solid by the pulse heating method.
We are now seeking to establish a reliable andrapid screening system for determining volatileand gaseous substances in bioloigical materials.
Acknowledgement
This work has been supported in part by a Grant-inAid for Scientific Research (No. 60440041) from the
Ministry of Education, Science, and Culture, JapanA part of this work was presented at the 72nd
Conference of the Medico-legal Society of Japan, Akita,
May, 1988, at the International Congress of Forensic
Science, Beijing, September, 1988, and at the 67th
Annual Meeting of the Deutsche Gesellschaft fur
Rechtsmedizin Kloster Banz, September, 1988.

References
1) Curry, A.S., Hurrt, G., Kent, N.R. and Powell, H.:
Rapid screening of blood sample for volatile poisonsby gas chromatography. Nature, 195, 603-604,
1962.
2) Bassette, R., Ozeris, S. and Whitnal, C.H.: Gas
chromatographic analysis of head space gas of dilute
aqueous solutious. Anal Chem, 34, 1540-1543,
1962.
3) Machata, G.: Ober die gaschromatographische
Blutalkoholbestimmung. Analyse der Dampfphase.
Mikrochim Acta, 6, 262-271, 1964.
4) Nanikawa, R. and Kotoku, S. : A routine method or
the determination of ethanol in biological materials
using gas chromatography. Jpn J Stad Alcohol, 4,27-33, 1969 (in Japanese with English abstract).
5) Chundela, B. and Jana J.: Quantitative determination of ethanol besides other volatile substances in blood and other body liquids by gas
chromatography. J Forensic Med, 7, 153-161, 1960.
6) Parker, K.D., Fontan, C.R., Yee,J.L. and Kirk, P.L.:
Gas chromatographic determination of ethyl alcoholin blood for medicolegal purposes. Separation ofother volatiles from blood or aqueous solution. Anal
Chem, 34, 1234-1236, 1962.
7) Ueda, M. : Analytical research of acute alcoholic
intoxication (Special communication). Jpn J Legal
Med, 21, 261-284, 1967 (in Japanese with English
abstract).
8) Ueda, M. and Fukui, Y.: A new method for thedetermination of ethanol in blood by pyrolysis-gas
chromatography. Jpn J Stud Alcohol, 2, 100-106,
1967.
9) Ishizawa, F., Sasagawa, K., Mashiko, K.: A study
on the gas chromatographic determination of
ethanol in blood. Rep Natl Res Inst Police Sci, 32,
33-37, 1979 (in Japanese with English abstract).
10) Ishizawa, F. : A study on the gas chromatographic
determination of ethanol in blood. Jpn J Legal Med,34, 618-625, 1980.
ll) Simon, W., Kriemler, P., Voellmin,J.A. and Steiner,
H.: Elucidation of the structure of organic compounds by thermal fragmentation. J Gas Chromatography, 5, 53-57, 1967.
12) Oguri, N. and Ohnishi, A.: Development of bioporbe Curie point pyrolyzer- GC and applications.
Proc. Pittsburgh Conference, 870, 1988.