T H E AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Vol. 44, No. 4
Copyright © 1965 by The Williams & Wilkins Co.
Printed in
A SPECIFIC PROCEDURE FOR SERUM GLUTETHIMIDE
DETERMINATION
U.S.A.
(DORIDEN)
R. M. DATJPHINAIS, M.D., AND 11. McCOMB, P H . D .
Clinical Chemistry Laboratory, Department of Pathology, Hartford Hospital, Hartford, Connecticut
Since the development of glutethimide
(Doriden*) 10 years ago, this drug has enjoyed widespread use as a "nonbarbiturate"
sedative and hypnotic. 6 Reports soon
followed its initial clinical trial, however, implicating glutethimide in an increasing number of fatal as well as nonfatal poisonings,1, 2'8"10 and the need for a procedure to
determine the amount present in biologic
fluids was recognized.
The earliest methods for glutethimide
analysis were nonspecific and lengthy. In
1956, Sheppard and associates11 established
a method for the determination of glutethimide and its metabolite, a-phenyl glutarimide, in urine. The method required approximately 2 days for determination of the
total metabolite, and when applied to cases
of overdose of glutethimide, the resulting
metabolite would occasionally require multiple dilutions before attempting a final color
reaction.9
In 1960, Goldbaum and colleagues4 introduced a highly sensitive spectrophotometric
method for glutethimide determination
which required only 4 hours for completion.
Although the Goldbaum procedure offers
marked advantages over earlier methods, the
time required for complete analysis of serum
glutethimide levels is still unduly long. The
accuracy of analyses may also be impaired by
interfering substances in serum, which presumably accounted for the fact that 2 patients seen in this hospital center with high
serum glutethimide levels were actually
found to be fully conscious and alert.
This report describes a rapid, specific procedure for determination of glutethimide in
serum. Interfering substances are removed
by hexane, and the absorbances of the analysis mixtures in neutral and basic media
are determined. Once the extract is preReceived, April 27, 1965.
* Ciba Pharmaceutical Company, Summit, New
Jersey.
440
pared, the actual concentration of glutethimide can be measured and reported within
a few minutes rather than waiting for 3 hr.
as in the original Goldbaum method.4
EXPERIMENTAL
PROCEDURE
Development of the Method
Extractions of "normal" sera prepared by
the Goldbaum procedure will often show a
change in absorbance at 235 m«. when
treated with KOH, which varies with the
particular serum specimen used. Examples
of this phenomenon which occurred in serums from 3 separate individuals are shown as
curves A, B, and C in Figure 1, measured
with a Beckman DU spectrophotometer. If
the same serum extract is treated with 5 ml.
of hexane before adding KOH, no significant
change in absorbance is observed between
zero time and 3 hr. later; cf. Curves A and
A'. Curves D and E represent change in
absorbance of 2 additional serum specimens
after hexane treatment.
When samples consisting of a known glutethimide standard, normal serum, and standard-serum mixture were analyzed by the
Goldbaum procedure and the results compared with the modified procedure using
hexane, the results listed in Table 1 were
obtained. The total change in absorbance of
standard-serum mixture (AA3) by the original Goldbaum procedure was the same order
of magnitude as the sum of separate decays
of standard (AAi) and of serum (AA2). When
hexane was used, only glutethimide contributed to the total change in absorbance,
there being no change in absorbance due to
serum. The removal by hexane of substances
which add to absorbance as well as contribute to the over-all change in absorbance, at
235 mju, has increased the specificity and
precision significantly.
The hexane layer used to remove interfering substances from an extract of serum was
itself subjected to KOH treatment. Changes
Oct. 1965
0.2
0.1
0
V
,- .
rr*—*-
441
SERUM DORIDEN DETERMINATION
B
t£
L
A'
r
D
E
v
u
FIG. 1. Time course of absorbance decay of
unknown substances extracted from randomly
selected serums with and without hexane treatment. Curves A, B, and C, hexane step omitted.
Curves A', D, and E, hexane step included. A and
A' were extracts from the same serum sample.
in its absorbance were measured photometrically over a 5-min. period and compared with
changes seen in hexane similarly prepared
from a glutethimide standard and distilled
water instead of serum (Table 2). In this
experiment, 3 ml. of hexane layer were
evaporated to dryness, reconstituted with 3
ml. of ethyl alcohol, and mixed with 1 ml. of
0.2 N KOH and measured in a Beckman DU
spectrophotometer, using matched quartz
cuvets. As indicated in Column 1 of
Table 2, the hexane extract of glutethimide
standard shows no evidence of changing absorbance, indicating that little glutethimide
if any was carried over into this hexane layer.
The reason for the relatively high, stable absorbance in Column 1 was probably due to
impurities extracted from chloroform itself.
An extract of chloroform and water prepared
under similar conditions, without serum or
glutethimide standard, was found to have an
absorbance of 0.095 at 235 m^. In marked
contrast the hexane extract of serum (Column 2) showed a significant change in absorbance.
Glutethimide in a neutral ethanol-water
solution exhibits no significant absorbance
at 235 m/i (Curve A, Fig. 2), whereas in an
alkaline solution there is a strong absorbance
maximum at this wave length (Curve B,
Fig. 2). Spectra in this experiment and that
shown in Figure 5 were obtained using a
Beckman DK-1 recording spectrophotometer. The feasibility of using this absorbance
difference in neutral and alkaline mediums
as the basis for glutethimide assay was investigated as an alternate to estimating total
decay of glutethimide in alkaline solution as
described by Goldbaum.4
Glutethimide absorbance at 235 m^ in
KOH cannot be determined directly because
of rapid hydrolysis of the drug under these
conditions. However, as demonstrated by
Goldbaum,4 an accurate estimate of the absorbance under alkaline conditions may be
made by following the initial time course of
the absorbance decay and extrapolating
these values back to zero time. An example
of this technic is shown in Figure 3, Curve A
illustrating the absorbance decay in alkaline
medium. Also shown is the time course of
absorbance measurements under neutral conditions, Curve B, where no decay is evident.
TABLE 1
COMPARISON OP ABSORBANCIES AT 235 M/J OP
ANALYSIS MIXTURES WITH AND WITHOUT
HEXANE TREATMENT
Goldbaum
method
Hexane
method §
AAi
(Standard)*
AAs
(Serum)t
AAi &
AAj
AA3 (Standard
Serum Mixture)}
0.727
0.636
1.363
1.495
0.429
0.000
0.429
0.423
* Standard = CHC13 extract of 1 ml. of 10 mg.
per 100 ml. glutethimide standard in ethyl alcohol
and 3 ml. of distilled H 2 0.
f Serum = CHC13 extract of 1 ml. of ethyl
alcohol and 3 ml. of serum.
i Standard-serum mixture = CHClj extract of
1 ml. of 10 mg. per 100 ml. glutethimide standard
in ethyl alcohol and 3 ml. of serum.
§ The actual absorbance readings differ from
those of the Goldbaum procedure above because
of sampling and dilution differences in the 2
methods. AA by the Goldbaum method refers to
the difference in absorbance of sample at zero
time and 3 hr. later.
442
Vol.U
DAUPHINAIS AND MCCOMB
TABLE 2
ABSOIIBANCE AT 235 MM OF SUBSTANCES EXTRACTED
INTO H E X A N E
FROM F I N A L ANALYSIS
MIXTURE
Absorbance at 235 mji
Time after Adding
KOH
Glutethimide standard
(5mg./100 ml.)
Serum
min.
1
2
3
4
5
0.117
0.118
0.117
0.115
0.115
0.780
0.720
0.665
0.623
0.595
The difference between these values (AA)at
zero time is indicated by the arrow.
That this difference in absorbance under
alkaline and neutral conditions was proportional to glutethimide concentration is shown
in Figure 4. Several dilutions of a 5.0 mg.
per 100 ml. glutethimide standard in chloroform were prepared by adding known different amounts of standard to 2.0 ml. of distilled water and adding enough chloroform to
make a total of 25 ml. of chloroform. After
extraction by the modified hexane procedure,
1.0 ml. of the final dilution mixture was
added to 2.0 ml. of ethyl alcohol and either
1.0 ml. of distilled water or 1.0 ml. of 0.2 N
KOH. The change in absorbance of each
mixture was measured at 235 nut by taking
readings at 1-min. intervals for 5 min. in the
neutral and alkaline media and plotting the
difference in absorbance at zero time (AA)
against glutethimide concentration. The results indicate a linear relation between AA
and glutethimide concentration over the
whole range of glutethimide concentrations
likely to be encountered in serums.
For an analytical procedure based on the
above relationships to hold, there must be
no interference because of carry-over of
other 235 m^ absorbing substances during
extractions from serum. The absorption
spectra of the final analysis mixtures from
serum and glutethimide-serum mixtures are
shown in Figure 5. In neutral media, only a
low absorbance (0.05) at 235 myu was found
in either specimen (Curves A and B, respectively). When KOH was added to each
of these specimens and the blank, an absorbance maximum was observed at 235 myu only
in the specimen with glutethimide (Curve
B'). The shape and magnitude of the spectrum of this analysis mixture in KOH medium closely resembles that of extracts of
glutethimide standard alone treated in the
same manner (Curve B, Figure 2).
Since the changes in absorbance of different amounts of a glutethimide standard in
chloroform are proportional to their respective concentrations, an attempt to show this
proportionality in the presence of serum was
made. The only other factor which would
interfere with this procedure would be the
absorbance of nonglutethimide substances
extracted from the serum. Data in Table 3
show the results of determining, by the hexane procedure, known quantities of a 5 mg.
per 100 ml. glutethimide standard in chloroform added to 2-ml. samples of pooled serum
with per cent recovery. Each specimen was
extracted in the same manner, with the re-
200
250
300
350
MILLIMICRONS
F I G . 2. Absorption spectra of glutethimide in
neutral aqueous-ethanolic solution (Curve A),
and alkaline aqueous-ethanolic media (Curve B).
Absorbance a t 235 ITIM portion of t h e spectral scan
was recorded 3 min. after addition of K O H .
Oct. 1965
443
SERUM DORIDEN DETERMINATION
0.6
i
0.4
i
i
i
LO
o
i
i
i
1 0.4
CD
• 0.2
0.05
3
MINUTES
F I G . 3. Time-course of glutethimide decay in
alkaline (A) and neutral (B) media.
200
250
350
300
MILLIMICRONS
F I G . 5. Absorption spectra of final analysis
mixtures of extracts of serum and serum-glutethimide. A, neutral serum extract, B, neutral serumglutethimide, A', alkaline serum, B', alkaline
serum-glutethimide.
TABLE 3
RECOVERY STUDY O F G L U T E T H I M I D E IN S E R U M ,
U S I N G G L U T E T H I M I D E STANDARD IN CHLOROFORM
AND P O O L E D S E R U M (2
2
3
GLUTETHIMIDE
F I G . 4. Relation of glutethimide concentration
to difference between absorbance a t 235 m/i in
neutral and alkaline media (AA).
suits reported as glutethimide concentration
in blood.
Interference studies with commonly used
drugs and various physical serum conditions
were performed, both in the absence and
ml.)
Glutethimide
Added
Glutethimide
Found*
Recovery
mg./lOO ml.
mg./lOO ml.
Per cent
5.0
5.0
5.0
5.0
5.0
5.0
2.5
2.5
2.5
1.25
1.25
1.25
5.0
5.1
5.0
4.9
5.2
4.9
3.0
2.6
2.7
1.50
1.29
1.30
100
102
100
98
104
98
120
104
10S
120
103
104
* With reference to a s t a n d a r d in water carried
through the entire procedure.
444
Vol. 44
DATJPHTNAIS AND MCCOMB
TABLE 4
E F F E C T OF D R U G S AND O T H E R SUBSTANCES ON T H E ANALYSIS OF G L U T E T H I M I D E
Concentration
of Drug
Without Glutethimide
V"
AA6 min.
With Glu tethimide
Ac216
AA5 m i n .
per cent
mg./lOO ml.
I. Drug
Phenobarbital
Salicylate
Sulfadiazine
Chlorothiazide
II. Serum condition
"Normal" (pool)
Jaundiced
Lipemic
Hemolytic
10
50
20
25
Recovery of
Glutethimide*
0.630
0.060
0.066
0.056
0.000
0.000
0.000
0.000
0.530
0.470
0.500
0.520
0.080
0.086
0.110
0.102
100
94
98
106
0.088
0.080
0.090
0.061
0.000
0.000
0.000
0.000
0.505
0.530
0.505
0.500
0.096
0.090
0.090
0.108
94
102
96
100
* Based on averaged values of normal " p o o l e d " serum with 5 mg./lOO ml. glutethimide (Ao K O H
0.500 and Ao H j 0 = 0.060).
presence of glutethimide, to determine what
effect they had on glutethimide measurement
by the modified procedure, using hexane.
These drugs were: phenobarbital, salicylate,
sulfadiazine, and chlorothiazide; the serum
conditions included high bilirubin, hemolysis, lipemia, and "normal" pooled serum.
These materials are listed in Table 4, showing
zero-time absorbance (extrapolated) and
change in absorbance of several specimens
over a 5-min. time interval. The per cent
recovery of added glutethimide in each of
the serum samples is also listed.
Little absorbance is seen in the final extracts of drug-treated serum alone. When
glutethimide is added together with these
drugs, the high absorbance expected from
glutethimide is noted along with the predicted decay AA5 minutes. Abnormal conditions of the serums, such as jaundice, lipemia,
or hemolysis, did not interfere with the
analysis as indicated by Section I I of Table 4.
The low absorbance in the absence of glutethimide and the full expected absorbance
were obtained in these experiments.
Goldbaum reports a recovery error of less
than 5 per cent for samples containing added
glutethimide when carried through chloroform extraction and washes with weak acid
and base.4 Recovery of known amounts of
glutethimide in which the final hexane step
was added indicated 90 to 93 per cent of
glutethimide, added to serum samples, was
found in the final alcohol-water extract, with
no loss identifiable due to the added hexane
step.
MATERIALS A N D
METHODS
A. Reagents (All Maintained at Room
Temperature)
1. Glutethimide standard, 5 mg. per 100 ml.
(w/v). Ten milligrams of purified crystalline
glutethimide (Doriden*) are dissolved in
chloroform and diluted to volume with chloroform in a 200-ml. volumetric flask with
glass stopper. (Chloroform was found to be
as stable a vehicle as alcohol and more
suitable for the present procedure.)
2. KOH, 10 N. Fifty-six grams of anhydrous KOH are dissolved in distilled
water, and volume is adjusted to 100 ml. with
water.
3. Alcohol-water blank {2.0:0.5 v/v). Five
milliliters of distilled water are mixed with
20.0 ml. of ethyl alcohol (absolute).
4. HCl, 0.6 N. Dilute 25 ml. of concentrated hydrochloric acid to 500 ml. with
distilled water.
5. NaOH, 045 N. Dissolve 101 Gm. of
NaOH in 200 to 300 ml. of distilled water
and adjust volume to 500 ml.
6. Ethyl alcohol, absolute.
* Courtesy of Albert J . Plummer, M . D . , Director
of Microbiology, CIBA Pharmaceutical C o m p a n y ,
Summit, New Jersey.
Oct. 1965
445
SERUM DORIDEN DETERMINATION
7. Hexane (speclroqualily reagent).
8. Chloroform (spectroquality reagent).
B. Procedure
TABLE 5
Cuvet No. 1
Cuvet No. 2
Cuvet No. 3
Blank
Standard
Unknown
1. In a 125-ml. separatory funnel add 25
ml. of chloroform and 2.0 ml. of patient's
serum. Set up a standard in the same manner, replacing patient's serum with 2.0 ml. of
distilled water and replace 2.0 ml. of the
chloroform with 2.0 ml. of glutethimide
standard. (Note: Use a 25-ml. graduated
cylinder to measure out proper amount of
chloroform.)
2. Extract glutethimide in unknown and
standard by shaking contents of separatory
funnels for 3 min. (venting funnels occasionally to release built-up pressure).
3. Filter CHC13 only, through Whatman
# 541 paper, into a second separatory funnel. Add 5 ml. of 0.45 N NaOH to the filtered extract. Shake for 3 min.
4. Filter CHC13 only, as before, into a
third separatory funnel. Add 5 ml. of 0.6 N
HCl to the filtered extract. Shake for 3 min.
5. Transfer a 15-ml. aliquot of CHC13
layer to a 15 by 150 mm. test tube. Evaporate to dryness in shaker-evaporator* at
50 C , or under a stream of air in an 80 C.
water bath (remove from water bath as soon
as evaporated to avoid destroying glutethimide).4
6. Reconstitute both residues with
(a) 2.0 ml. ethyl alcohol,
(b) 5.0 ml. n-hexane,
(c) 0.5 ml. distilled water.
7. Mix in a vortex mixer for approximately 15 sec. to insure thorough mixing.
Allow layers to separate.
8. Remove hexane layer (top layer) by
suction or dropper and discard.
9. With remaining alcohol-water extract
of both standard and unknown set up quartz
cuvets as shown in Table 5.
10. With cuvet 1 (blank), zero spectrophotometer at 235 niyu. Read absorbance of
2 (standard) and 3 (unknown) at 235 m/t
vs. blank, and record absorbancies (A).
Running standard.
11. Add 0.1 ml 10 N KOH to blank
(Cuvet 1) cap cuvetf with parafilm and mix
by several inversions and again zero spectrophotometer at 235 m/x with this blank.
12. With stop-watch ready, add 0.1 ml.
10 N KOH to standard (Cuvet 2) and start
watch.
13. Cap cuvetf with parafilm and mix
solution as before.
14. Place cuvet in holder and take absorbance readings at 1, 2, 3, 4, and 5 min. against
blank.
Running unknown.
15. Repeat steps 12, 13, and 14 with unknown (Cuvet 3), using same blank.
16. Plot log absorbance vs. time for both
standard and unknown.
17. Determine zero time absorbance for
standard (A08td) and unknown (A0unk) by
extrapolation of the time course of absorbance change to zero time.
18. Subtract absorbance (A) of standard
and unknown without KOH (see "10"
above) from respective zero time absorbancies (Ao's) for standard and unknown with
KOH (see "17" above).
* Evapo-Mix, Buchler Instruments, New York 31,
New York.
t Evaporation will cause a gradual increase in
absorbance (see Curve C, Fig. 1, after 60 min.).
(a) 1.0 ml. al- (a) 1.0 ml. al- (a) 1.0 ml. alcohol-water
cohol-water
cohol-water
extract (with
extract
(2.0/0.5)
glutethimide
(patient)
standard)
(b) 2.0 ml. ethyl (b) 2.0 ml. ethyl
(b) 2.0 ml.
alcohol
alcohol
ethyl alcohol
(c) 1.0 ml. dis- (c) 1.0 ml. dis- (c) 1.0 ml. distilled water
tilled water
tilled water
C. Calculations
1. The difference in absorbance between
A0 and A equals AA, which is proportional
to glutethimide concentration.
2. Therefore:
, , 5 mg. per 100 ml. _
x
'
AAstd
AA unk
446
Vol. 44
DAUPHINAIS AND MCCOMB
TABLE 6
CASE 1
Goldbaum Procedure
Time
mg./lOO ml.
mg./lOO ml.
At admission
3.5
% hr. after admission
4.3
Clinical Picture
Hexane Procedure
Unresponsive, except to painful stimuli
1.4, 1.0 (same
specimen repeated)
4 hr. after admission
Moving extremities; vomiting around
Levin tube
Responds to name (opens eyes)
Answers questions verbally
Very alert (recovered)
7 hr. after admission
25 hr. after admission
28 hr. after admission
TABLE 7
CASE 2
mg./lOO ml.
hr.
3.0 (hexane procedure)
2.9 (Goldbaum
procedure)
2.1
2
5
9}i (artificial kidney for 3>£ hr.)
16
19
1.7
28 (artificial kidney for 4% hr.)
40
1.1
64
(b) X = 5 X
Clinical Picture
Serum Glutethimide Level
Time After Admission
AAunk
AAstd
mg.
per 100 ml. glutethimide in serum
REPORT OF CASES
Case 1. A 53-year-old white woman was
admitted to the emergency room in coma 1
to 2 hr. after ingesting an unlaiown quantity
of Doriden tablets. She had a 30-year history
of depression, for which she had been hospitalized 6 times.
In Table 6 are listed serum glutethimide
determinations performed on the patient by
the Goldbaum procedure as well as the modified procedure, using hexane, correlating results with the patient's clinical status.
Case 2. A 46-year-old housewife with a
Unresponsive
Vomiting small amount of fluid, color
poor, audible rales
Unresponsive
No corneal reflex
Minimally responsive; corneal reflex
present
Minimally responsive
Opens eyes on command; vital signs stable; color improved; talking jargon
Talking clearly (recovered)
history of acute drug ingestion was admitted
in coma. Her usual medications were Doriden and Librium for chronic depression, and
several drugs for asthma. The husband
stated that she awoke at 3:00 a.m. with an
asthmatic attack, medicated herself, and
was asleep when he left for work at 6:00
a.m. His repeated phone calls during the day
went unanswered and at 4:00 p.m. he found
her unresponsive, with an empty bottle of
Doriden and partially-used asthmatic medications by her bedside. The amount of
Doriden presumably ingested was estimated
at 6 to 7 Gm.
The correlation of serum glutethimide
level and clinical status is summarized in
Table 7. Both the Goldbaum procedure and
this modified procedure with hexane were
Oct. 1965
S E R U M DORIDEN
TABLE 8
CASE 3
Time after
Admission
Serum Glutethimide Level
hr
mt./100 ml.
At admission
2.8
2
4
5
14
31
Clinical Picture
Lavaged, not responding
Restless, restrained
Restless, screaming
Awake, more alert
Sleepy
Alert (recovered)
1.2
1.0
O.C
performed on the first serum specimen, yielding identical results; the remaining determinations were performed only with the
hexane procedure.
Case S. A 26-year-old white woman ingested 8 tablets (4.0 Gm.) of Doriden }/% hr.
before entering the emergency room in an
unresponsive state.
Serum glutethimide determinations, all
performed by the hexane procedure, are
listed in Table 8.
DISCUSSION
,C 2 H 5
HNy
0
V
\
CoHs
•
KOH
C
H,
Glutethimide (Doriden)
in alcohol
C2H5
>/ V
C,H 5
KO
Decomposition
C
H2
Ghitethimide (Doriden)
in alcoholic KOH
By the above equation, glutethimide
(Doriden) goes from a "ketonic" structure
to an "enolic" structure upon addition of
KOH. The change in absorbance related to
this change in structure is proportional to
DETERMINATION
447
glutethimide concentration. This being so,
a simple procedure based on determination of
absorbance of glutethimide in extracts of
serum before and after alkalinization with
KOH is feasible, provided no other interfering substances are present.
Discrepancies with the Goldbaum method
have appeared in several instances in which
the laboratory values did not correlate well
with the clinical picture.3 Although glutethimide values of greater than 3 mg. or 4 mg.
per 100 ml. of blood were reported, consistent
with deep coma,7 2 patients were found to be
alert and apparently in good health. Rechecking the procedures for each test revealed that the determinations were carried
out according to the specified conditions. It
was thought that the presence of interfering
substances in the serum, probably lipid in
nature, accounted for the discrepancy between the laboratory and clinical picture.
The present modified procedure, using
hexane, eliminates this interference characteristic of serum and also eliminates the
necessity to wait for 3 hr.
The hexane wash step is based on experimental work by Goldbaum and associates,
wherein they determined the distribution of
glutethimide in tissues of patients at autopsy
who had taken a fatal overdose.6 Fatty tissue was found to have the highest concentration of glutethimide, far exceeding other
body tissues. In order to eliminate the interference of fat, the extracted residue containing glutethimide was washed with a mixture
of ethyl alcohol and hexane (2:5 by volume),
and distilled water (0.5 volume) was then
added, causing 2 layers to form. Glutethimide present remained in the alcohol-water
layer and its concentration determined, while
the hexane layer containing fatty impurities
was discarded. In like manner, the present
work indicates that the change in absorbance of a serum extract after hexane treatment before and after addition of KOH, is
due to glutethimide only and makes possible
a rapid method for its accurate determination.
Extraction of a 5 mg. per 100 ml. glutethimide standard is performed concurrently
with the unknown. Because of the linearity
of a curve expressing glutethimide concentration vs. change in absorbance, the con-
448
DAUPHINAIS AND McCOMB
centration of glutethimide in an unknown
blood sample can be determined by comparison with the determination of a known
standard performed simultaneously.
All final solutions, including blank, standard, and unknown, are prepared so as to have
identical solvent composition. Difference in
absorbance at 235 m/j between blank and
unknown is owing almost entirely to glutethimide, as indicated by a comparison of
glutethimide absorbancies in extracts from
water and serum. The volume of KOH added
to the reaction mixture to develop the 235
m^ absorbance has been reduced to a minimum (0.1 ml.) in order to minimize concentration differences between the neutral and
alkaline mixtures. The concentration of
KOH used was more than adequate to develop maximal absorbance of glutethimide,
producing a final alkalinity slightly in excess
of that used by Goldbaum.4
The effects of temperature change and
evaporation on the concentration of glutethimide in the measured solutions is negligible, as only a 5-min. reading period is necessary.
The method proposed in this report is
highly specific for determining glutethimide
concentration in serum in the presence of
endogenous substances and several commonly used exogenous drugs. The sensitivity is
more than adequate, allowing glutethimide
levels as low as 0.5 mg. per 100 ml. of blood
to be measured with ease. Results with this
procedure have given good correlation with
the clinical state of the patient.
SUMMARY
A method for determination of serum
glutethimide is presented. Initial extraction
of glutethimide from serum into chloroform is
followed by basic and acidic washes and removal of the solvent by evaporation. Hexane
Vol. U
is then used to remove interfering endogenous "decay" substances carried along from
serum with glutethimide into the final analysis mixture. The absorbance of standard
and unknown is read against a blank before
and after addition of 10 N potassium hydroxide, and the difference in absorbance is
related to the final concentration of glutethimide. Effects of temperature change and
evaporation on the measured solutions are
negligible, as only a 5-min. reading is required to measure the absorbancies of unknown and standard. The procedure is specific, sensitive, and accurate, and requires
only 1.5 hr. for the complete analysis.
REFERENCES
1. Algeri, E. J., Katsas, G. C , and McBay, A.:
Toxicology of some new drugs: glutethimide,
meprobamate, chlorpromazine. J. Forensic
S c , 4: 111-135, 1958.
2. Blakey, H. H., Barringer, T., and Billig, O.:
Acute Doriden (glutethimide) intoxication.
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