CLIN. CHEM.
26/1,
66-68
(1980)
Extraction and Determination of Chloroform in Rat Blood and Tissues by
Gas Chromatography-Electron-Capture
Detection: Distribution of
Chloroform in the Animal Body
Corazon R. Vogt, John C. LIao, and Albert V. Sun1
We have developed a simple, sensitive, and accurate
method for the determination
of chloroform in rat blood,
brain,kidney,liver,
and fat. The detectionlimitis 2.5 ng of
chloroform
per gram of tissue. Studies of in vivo distributionof chloroform
in rat blood and target tissues after intragastric
intubation
of chloroform/water
show that the
amount of chloroform accumulated
in the different tissues
increases
with increasing
doses. Fat tissue contains the
greatest
amount of chloroform.
The accumulation
of
chloroform
in rat blood and target tissues seems to be
maximum
1.5 h after administration,
and the apparent
chloroform
concentration
is almost at baseline value 8 h
later.
in viva deposition of chloroform
chloroform in drinking water
AddItional Keyphrases:
Several
analytical
methods
have been developed
for the
quantitation
of halogenated
anaesthetics
in animal tissues.
Duncan (1) and Burns et al. (2) used reducing agents to convert the halothanes
to halides, which in turn were determined
photometrically.
Chloroform
and other anaesthetics
have also
been determined
by the infrared method (3, 4). In this
method, the anaesthetics
are extracted
by soaking the tissue
in carbon disulfide for up to 24 h and distilling the extract to
remove the dissolved
lipids; the resulting
distillate
is then
analyzed by infrared spectroscopy. Both the photometric and
the infrared methods are time consuming
and are sensitive
only to milligrams per liter. More recently, gas chromatography has been used to determine chloroform in blood (5, 6).
Davies (5) used n-heptane
as the extracting solvent and an
electron-capture
detector as the detection
system of the gaschromatographic
effluent;
Poobalasingam
(6) used carbon
disulfide and a flame-ionization
detector. The former author
reported
a detection
of 10 ng of halogenated
anaesthetic
per
gram of sample; the latter, detections
in the micrograms
per
gram range.
Gas chromatography
interfaced
with mass
spectrometry
has also been used to analyze for chloroform and
other halogenated
hydrocarbons
in blood plasma after en-
richment
in an adsorbent
by a purge method (7).
In 1974, Rook (8) showed that haloforms were found in the
chlorinated
natural waters. In 1976, a study on the reaction
of chlorine with water-soluble
organic compounds
found that
chloroform
was the predominant
haloform produced and that
removal
of organic compounds,
possibly
by air stripping,
would minimize
the haloforms
in drinking
roform and chiorophenol
were reported
decarboxylation
of naturally
occurring
ation (10).
Chloroform
has been shown
is reported
to be nephrotoxic
to be carcinogenic
(11) and recently
to rats (12). In view of this,
Environmental
Trace Substances Research
Comparative
Medicine Research Farm and
chemistry; University of Missouri, Columbia,
Received July 5, 1979; accepted September
66
water (9). Chlo-
to be produced by the
acids during chlorin-
Center; and
Department
MO 65201.
19, 1979.
CLINICALCHEMISTRY,Vol. 26, No. 1, 1980
Sinclair
of Bio-
the deposition
of chloroform
in the physiological
system is of
concern. We have investigated
the distribution and elimination of chloroform in blood and body target tissues of rats after
the administration
of water containing
chloroform
and have
developed a simple, sensitive, and accurate analytical method
for the determination
of chloroform
in blood, brain, kidney,
liver, and fat.
Materials and Methods
Apparatus
We used a Model
Austin, TX 78721)
electron-capture
8176 electrometer.
MT-220 gas chromatograph
(Tracor, Inc.,
equipped
with a Ni63 high-temperature
detector,
a dc power supply,
A glass column,
and a Model
1.83 m X 4 mm i.d., was
packed with 80/100 mesh HP Chromosorb
W (Applied
Science Lab., Inc., State College, PA 16801) coated with 100
mg of polyethylene
glycol (average M 20 000; Union Carbide
Corp., New York, NY 10017) per gram of Chromosorb.
The
temperatures
of injection
port, column oven, and detector
were 150, 75, and 300 #{176}C,
respectively.
Nitrogen
flow was 60
mL/min through the column and 30 mL/min for purging the
detector. A four-port
valve was installed between the column
and the detector
for introducing
the chloroform
peak to the
detector
and venting the rest of the column effluent
(this
keeps the detector clean). We used a 1-mV recorder (Linear
Instrument Corp., Irvine, CA 92714) for signal recording, an
Omni-Mixer (Ivan Sorvall, Inc., Norwalk, CT 06856) to homogenize
the samples,
and a clinical
centrifuge
(Model
CL;
International
Equipment Co., Needham, MA 02192) to separate the organic layer from the aqueous phase of the homogenate.
Materials
Sprague-Dawley
rats (Charles River Lab., Wilmington,
MA
01887), weighing about 300 g, were fasted overnight before the
administration
of 5 mL of distilled
water or chloroform
water.
Methylcyclohexane
was purified by being passed through
a column packed with activity 1 basic alumina (Fisher Scientific Co., Fair Lawn, NJ 07410) that had been previously
activated
at 600 #{176}C
for 15 h. Glass culture tubes (15 mL) with
Teflon-lined
screw caps were used for extraction.
Procedure
We homogenized
1 g of tissue in 5 mL of distilled water with
5 mL of methylcyclohexane,
then transferred
the homogenate
into a culture tube with a Teflon-lined
screw cap and shook
the mixture
layer,
vigorously
for 10 mm. The methylcyclohexane
containing
the chloroform,
was separated
from the
aqueous
layer by centrifugation
for 10 mm at 4000 rpm. If any
suspended
lipid remained in the organic phase, we cooled the
mixture in a slurry of solid CO2 and acetone to freeze the lipid.
The tube was centrifuged
again to sharply separate
the two
layers. We injected 1-4 zL of methylcyclohexane
extract onto
the gas chromatograph.
The chloroform
peak had a retention
Table 1. Reproducibility of Chloroform Determination and analytical Recovery in Rat Blood and
Tissues a
Chloroform. ua/L ± SD
Sample
Blood
Determined
89.3 ± 3.2
40.3 ± 3.6
Brain
Recovered
81.3
40.0
%
104.2 ± 4.6
102.3 ± 10.3
99.0 ± 5.0
16.7 ± 1.0
20.0
19.8 ± 1.0
Liver
31.5
± 3.3
40.0
31.9 ± 2.0
380.0
± 6.8
387.5
recovery
84.7 ± 3.8
40.9 ± 4.2
Kidney
Fat
a
Added
394.0
97.8 ± 5.1
± 3.2
101.7
± 0.8
Mean ± SD; five determinations for each.
time of 2.5 mm, after which it went through
the electroncapture detector. The solvent peak and the effluent after the
chloroform
were vented, to keep the detector
clean.
We constructed
a calibration
curve from known amounts
(2-400 pg) of chloroform
in methylcyclohexane
by plotting
response peak height vs amount injected. The plot was linear
over that range. A 4-iL injection of the extract gave a detection limit of 2 pg of chloroform,
with a signal-to-noise
ratio of
2. Thus the procedure
of extracting
1 g of tissue with 5 mL of
methylcyclohexane
has a detection
limit of 2.5 ng of chloroform per gram of tissue.
Reproducibility
and recovery:
Six rats that had been used
in a different study and were suspected
to contain chloroform
in their systems were killed, and the blood and other tissues
pooled to obtain composite
samples. Each of the composite
samples was divided into two portions. Five replicates of each
sample in one portion were assayed, from which we calculated
standard
deviations
and reproducibility.
To other 1-g portions of blood, brain, kidney, liver, and fat
we added 50.8, 25, 12.5, 25, and 242 L of chloroform to yield
samples with additional
chloroform
of 81.3, 40.0, 20.0, 40.0,
and 387.5 g/L,
respectively.
These additional
amounts
doubled the concentration
of the chloroform
in the composite
samples. The samples were shaken and let stand for about an
hour. We determined
chloroform
on each sample in five replicates. Standard
deviations
and percent analytical
recovery
were calculated
from the difference
of the supplemented
and
nonsupplemented
samples.
In vivo deposition
of chloroform: Three different concentrations of chloroform
in 5 mL of water were administered
to
two rats each by intragastric
intubation
with a ball-pointed
curved
needle. The control rats were given distilled water. All
of the rats were killed 1.5 h after administration.
Blood, brain,
kidney, liver, and fat were collected and analyzed for chloroform.
Time exposure: We gave eight rats 5 mL of water containing
0.5 mg of chloroform,
and killed two rats at 0.5, 1.5, 4, and 8
h after administration.
Blood and tissues were analyzed
for
chloroform.
Results and Discussion
Because it is sensitive and selective to chlorinated
hydrocarbons, we chose electron-capture
detection for detecting the
chloroform
at the gas-chromatographic
effluent.
Two picograms of chloroform
per injection could be detected
at a signal-to-noise
ratio of 2. The injection of methylcyclohexane
tissue extract severely contaminated
the detector,
probably
because
of the presence
of the lipid; thus, the response
of
chloroform
was not reproducible.
To abate this problem,
we
allowed
only the chloroform
peak to enter the detector,
venting the solvent and the rest of the column effluent by use
of a four-port
valve. A period of 20 mm between sample injections was necessary
to obtain reproducible
response. Presumably, within 20 mm most of the volatile solutes that could
contaminate
the detector were eluted out of the column.
Blood and tissues were homogenized
with water and
methylcyclohexane.
Methylcyclohexane
is relatively
less
volatile than heptane
(5) and carbon disulfide (6), solvents
used previously
in chloroform
analysis,
and its evaporation
during the entire procedure is negligible. Further, methylcyclohexane
fim
has been shown
to efficiently
extract chloroform
blood and tissues with
the extraction
efficiency.
The
water (13).The homogenizatiorrof
methylcyclohexane
improved
methylcyclohexane,
of chloroform.
being the upper layer, prevents
the loss
Reproducibilities
and analytical
recoveries
for the determination
of chloroform
are shown in Table 1. The standard
deviations
were all within 10%. The procedure
was repro-
ducible for blood, brain, kidney, liver, and fat. Recoveries were
about 100%, which indicates that the extraction of chloroform
from blood and tissue homogenates
was complete.
With this procedure,
we studied
in vivo distribution
of
chloroform
in rat blood and target tissues. Table 2 shows the
amount
of chloroform
in blood and tissues 1.5 h after administration
of various amounts
of chloroform
in water. The
standard
deviations
shown are satisfactory
for biological
systems,
where
than analytical
individual
variations
could
be much
greater
precision. The chloroform content of blood and
tissues increased
with increasing
dosage of chloroform
in
water. Fat showed the highest deposit of chloroform.
The
amount of chloroform
found in blood, brain, kidney, and liver
did not differ much from each other, which is analogous to the
observation
that, 24 h after the cessation
of anaesthetic
inhalation,
equal amounts
of chloroform
were found in brain,
blood, liver, and kidney of experimental
animals (14).
Table 3 shows the amount of chloroform
in blood and
tissues at various times after administration
of 5 mL of water
with 0.5 mg of chloroform.
When compared
with values from
the control rats in Table 2, the chloroform
in the different
tissues were back almost to baseline values in 8 h. Except in
Table 2. Chloroform in Blood and Tissues in Two
Rats Administered Various Amounts of
Chloroform a
Chloroform,
mgI5 mL
of water
0
0.1
0.5
1.0
a
Chloroform detected, ig/L
Brain
Kidney
Liver
Blood
Fat
4.8
4.0
2.5
2.9
3:2
2.5
2.9
6.6
6.4
7.4
3.9
3.9
10.8
42.8
6.0
4.5
3.9
4.6
54.4
25.8
20.0
934
2.0
20.8
15.9
14.8
8.0
19.0
8.0
49.5
31.3
34.1
34.7
44.0
34.5
38.6
42.0
1699
1.5 h after administration.
(‘I
IkIIf’At
IIITV
tI,I
‘
Il
I
IGOfl
C?
Table 3. Chloroform in Rat Blood and Tissues
after Administration of Chloroform/Water
Tim. after
admin., h
0.5
1.5
4
8
‘0.5
a
Blood
Chloroform d.tect.d,
uqIL
Brain
Kldn.y
Liver
14.2
8.1
12.3
5.2
12.5
4.9
65.0
25.8
14.8
20.6
8.0
20.8
19.0
15.9
8.0
934.0
6.7
3.7
6.8
14.8
13.8
16.8
15.8
32.2
312.7
342.9
N.D.
11.0
10.6
4.9
4.6
4.0
15.4
22.6
30.3
32.7
Fat
728.2
b
in 5 ml of water. b Erroneous experiment value
none detected; detection lImit 182pg.
mg of chloroform
omitted. N.D.
=
the liver, deposition of chloroform in the tissues seems to be
greatest 1.5 h after administration.
Elimination may occur by
excretion; however, metabolic conversion of chloroform into
carbon dioxide and urea, as observed in rats (15, 16), or into
other metabolic products
(17) is another
pathway
of elimination. We did not study metabolic excretion.
The analytical procedure we present has been used successfully in the study of the formation
of chloroform,
in vivo
and in vitro, in animals exposed to acute doses of free chlorine
in the form of sodium hypochlorite
solution
(18). In vivo
chlorination,
intended
to reflect the chemical activity
of high
residual amounts of chlorine in drinking water and, eventually,
the effect on membrane functions, shows the presence of
chloroform in blood, brain, liver, kidney, and fat tissues 1.5
h after the intragastric administration
of the sodium hypochlorite (18).Thus chlorine, as well as chloroform-or
perhaps
instead of chloroform-may
need closer scrutiny in studies
of the carcinogenicity
We greatly appreciate
of chlorinated
water.
the valuable assistance
of Dr. Shubhender
Kapila. Supported by grant B-119-MO, awarded by the Office of
Water
Resources
awarded
by the
and
Technology,
USD1;
grant
National Science Foundation; and
awarded by the National
Cancer Institute,
MPS74-18084,
CA26586,
grant
DHEW.
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88
CLiNICAL CHEMISTRY. Vol. 26. No. 1. 1980
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