by small amounts of monoethyl compounds as impurities. Because of the relative size of the peaks a t m/e 30, deuterodiborane is ruled out as a possible impurity. Again, the ions CzHbBzH4+ and (C, H&B+ are present in the mass spectra of 1,l-diethyldiboranes. One major unpredicted peak occurs in each of the spectra in which hydrogendeuterium rearrangements are possible. I n the spectrum of 1,l-diethyldeuterodiborane the anticipated peak at m/e 42 is accompanied by a larger peak one mass unit lower. On the other hand, the spectrum of 1,1-diethy1-d6-diborane s h o w the anticipated peak a t m/e 46 followed b y a large peak one mass unit higher. The proton attached to the boron atom in the ions C2H5BH+ as illustrated, is a bridge hydrogen. The interpretation of these peaks given here is that this proton can also attach itself to the boron atom by rearrangement from the fragmented ethyl groups. This type of rearrangement has been observed in the mass spectra of trimethyl- and triethylboranes-viz., the formation of CH3BH+ and CzH5BH+ ions, respectively (4). Thus the CzHbBH’ ion in the third spectrum could account for the high peak a t m/e 41. Likewise, the ion C2D5BD+ could give rise to the prominent peak at m,’e 47 in the spectrum of 1,l-diethyl-dlo-diborane. Obviously, these rearrangements could not be observed in the other three spectra. 1,2-DIETHYLDIBORAKES (Figure 4). These spectra proved to be the most complex. TVhile the anticipated peaks appear, there are also several other large unexpected peaks. A. significant peak
a t m/e 69 occurs in the spectrum of the isotopically normal compound, which might be rationalized a s the loss of a methyl group from the parent compound -i.e., 84-15369. If this argument were valid, the same fragment mould appear a t mle 67 in the BlO-enriched spectrum. The shift is only to m ’e 68. The diethylboron ion b y rearrangement is therefore assigned to these peaks. The other spectra show that rearrangement of ethyl groups takes place. KO anomalies occur in the regions of m/e 49 to 64; however, the regions of the spectra from m/e 38 to 47 are comparatively ambiguous. Each of the large predicted peaks is accompanied by one or more prominent peaks in the next lower mass numbers. I n the spectra where no hydrogen-deuterium rearrangements are possible, the ions CzHSB1Of, CSHSB-, and C2D5Baccount for the unexpected high peaks a t m,’e 39, 40, and 45, respectively. Apparently the 1,2 - diethyldiboranes produce an abundance of CBHIB+ions, while the 1,l-isomers favor C2H5BH+ ions by rearrangement of a proton from the fragmented ethyl group. The other two spectra are coniplicated by the possibility of deuterium-hydrogen rearrangement. The high peak a t m,/e 41 in the spectruni of 1,2-diethyldeuterodiborane might be explained as a n abundance of CzH4DB* ions. Additionally, the oversized peaks a t m l e 43 and 44 might arise from the ions CzH4DBD- and C2H6BDz-or C2H3D2B D + , respectively. I n the spectrum of 1,2-diethyl-dlo-diborane the ions C2D3H2B+, C2D4HB+, CzD5B+, and CBDsBH- might account for the high peaks a t m,’e 43 to 46, respectively.
The peak a t m/e 47 could conceivably be due t o the C2D5BHzf ion. Again, these rearrangement peaks could not be detected in the other three spectra. ACKNOWLEDGMENT
The authors thank June F. Gray for the preparation of illustrations and TV. J . Lehmann for assistance in interpreting the infrared spectra. LITERATURE CITED
(1) Florin, R. E., Kall, L. &I., Mohler, F. L., Quinn, Edith, J . .Im. Chem. Soc. 76, 3344 (1954).
(2) Lehmann, W.J., Wilson. C. O., Jr., Shapiro, I., J. Chent. Phys. 28, 777 (1958). (3) Ibid., p. 781. (4) Lehmann, W.J., Wilson, C. O., Jr., Shapiro, I., J . Inory. &: S u c l e a r Chem. in press. ( 5 ) McLaffertp, F. ST., d p p l . Spectroscopy 11, 148 (1957): A x ~ L CHEM. . 28, 306 (1956); 29, 1782 (195i); 31, 82
(1959).
( 6 ) Schlesinger, H. I., Walker, A. O., J . A m . Chem. Soc. 57, 621 (1935). ( 7 ) Shapiro, I., Keiss, H. G., Schmich, M.,Skolnik, S.,Smith, G. B. L., Ibid., 74,
901 (1952).
(8) Shapiro, I , Kilson, C. O., Jr., Lehmann, W.J., J . Chem. Phys 29, 237
(1958).
( 9 ) Shapiro, I., Wilson, C. O., Jr., Ditter,
J. F., Lehmann, W.J , Ahstracts, 133rd Meeting, American Chemical Society, p. 39L, San Francisco, Calif., April 1958. (10) Solomon, Irvine J., Klein, Morton J., Hattori, Kiyo, J . A m . Chein. Soc. 80, 4520 (1958).
RECEIVED for reviexv May 18, 1959. Accepted October 19, 1959. Sixth Annual Meeting, ASTM Committee E-14 on l\Eass Spertrometry, S e w Orleans, La., June 1958.
Determination of Glutethimide in Biological Fluids LEO R. GOLDBAUM and MELVIN A. WILLIAMS Armed Forces Institute of Pathology, Washington 25, D. C. THEODORE KOPPANYI Department of Pharmacology, Georgefown University School o f Medicine and Dentistry, Washington, D.
b An ultraviolet spectrophotometric procedure i s described for determining 2-ethyl-2-phenylglutarimide (glutethimide) in blood, plasma, and urine. The method consists of extracting glutethimide from the sample with chloroform, removing interfering substances, and determining its characteristic ultraviolet spectrum and rate of hydrolysis a t 235 mp. The method i s specific, rapid, and accurate; i t detects less than 2 y per ml. of glutethimide with an error of approximately 5%.
The application of the procedure for determining glutethimide levels in animals and humans i s described.
T
Surp, and Hoffmann ( 9 ) reported on the preparation of various glutaric acid imides. Among the compounds described, 2-ethy1-2phenylglutarimide. glutethimide (Doriden), prored on the basis of detailed pharmacological and clinical studies (2) to be a mild hypnotic. Because of BGMANN,
C.
its spreading use as sedative and hypnotic, a procedure for its determination in biological samples is needed. Sheppard, D’Asaro, and Plummer ( 7 ) have described a method for the detection of glutethimide and a metabolite thereof in dog urine. Their method is nonspecific, requiring much higher concentrations of glutethimide than those found in many biological specimens. The present paper presents a simple, rapid, highly sensitive, and specific procedure for the determination of VOL. 32, NO. 1, JANUARY 1960
81
0.8
Table I. Effect of Alkali Concentration on Rate of Hydrolysis of Glutethimide Time,
0.6
I
iA
I
0.6
B
SerondR _._.
Normality of KOHa 0.05 0.1
0.2
0.3
0.2
2000 ~~~.
0.1
0.61 1000 0.61 740 0.61 260 One milliliter of different concentrations of aqueous KOH was added to 3 ml. of alcohol containing 10 y per ml. of glutethimide. Table 11.
Effect of Heat on Stability of Glutethimide Absorbance at Mixing Per Time at Cent Procedure 235 M p Loss Standard (control) I ,20 .. 0 Low volume" 1.20 2 Air-dried 1.18 4 70" C. to dryness 1.15 7O9 C. for 5 mimb 1.10 8 13 78" C. for 16 min.5 1.05 23 100' C. for 10 min. 0.92 32 100" C. for 15 min. 0.82 38 100' C. for 20 min. 0.74 Evaporated on steam bath to 2 to 3 ml. and air-dried. bAir-dried and then heated at that temperature.
glutethimide based on its ultraviolet absorption spectrum and rate of hydrolysis in alcoholic potassium hydroxide. EXPERIMENTAL
Ultraviolet Absorption Spectrum of Glutethimide. Attempts t o dissolve glutethimide in dilute alkali t o measure its ultraviolet absorption spect r u m gave erratic results which could not be duplicated. Absorbance readings decreased quickly and soon disappeared, This was probably due t o a rapid hydrolysis with opening of the piperidine ring. T h e reaction in t ~ a t e rwas too fast to make dependable measurements, but when glutethimide was dissolved in alcohol and then treated with dilute alkali, the reaction was slowed to the point where the absorbance at various wave lengths could be measured. Figure 1 shons the spectrum of glutethimide in alcoholic potassium hydroxide 90 seconds after mixing time. The solution started absorbing at 260 nlp, reached a maximum peak at 233 to 235 niw, and reached a minimum peak a t 223 to 225 mp; glutethimide can be measured with high sensitivity at the maximum 235 ( E : 2, = 880). There was a decrease in the absorbance at 235 nip with time, the rate of which appeared to be a property that is characANALYTICAL CHEMISTRY
9
4000
0.3 0.5
82
;0.5 $ 0.4
Absorbance for 60% at Mixing Hydrolysis of Time Glut,ethimide 0.30 0.50
c.3
0.7
0 225 230 235 240 245 W A M LENGTH IN Mli
220
2%
Figure 1 . Ultraviolet a b sorption spectrum of 3 ml. of absolute ethyl alcohol containing 10 y of glutethimide with 1 ml. of 0.2N potassium hydroxide
Relationship of Concentration to Absorbance. Solutions of glutethimide in alcohol containing 2.5, 5 , 10, 15, 20, 25, and 50 y per ml. were
280
so3
Figure 2. Ultraviolet absorption spectra-of glutethimide-free samples 1. 2. 3.
Determined b y Beckman D-K 2 recording spectrophotometer 90 seconds after mixing
teristic of glutethimide. Thus, the ultraviolet spectrum and the rate of hydrolysis of a n alcoholic alkaline solution offered a n excellent method for characterizing and quantitating glutethimide. Effect of Alkali on Hydrolysis of Glutethimide. Potassium hydroxide vias used for hydrolyzing glutethimide. Solutions of sodium hydroxide as weak as O.1N developed a turbidity when mixed with absolute ethyl alcohol, whereas potassium hydroxide solutions a s strong a s 0 . 5 s remained clear on mixing and standing. Table I s h o w t h e effect of varying concentrations of potassium hydroxide on the rate of hydrolysis of glutethimide. The most effective solution over a wide range of glutethimide concentrations n as 1 part of 0.2N potassium hydroxide in water mixed with 3 parts of absolute ethyl alcohol solution. This combination gave maximum absorbance readings without developing turbidity even after 24 hours, and a t the same time gave a steadymeasurablerate of hydrolysis. Half the concentration of glutethimide hydrolyzes approvimately every 1000 seconds. There is a decrease of 200 seconds in the half life for every increase of 6" C. Effect of H e a t on Decomposition of Glutethimide. KOloss occurred when a solvent containing glutethimide was evaporated on a steam bath t o a lorn volume and then air-dried. Residues subjected to further heating shorved decomposition proportional to the temperature a n d t o the length of time exposed. If care is not taken to prevent heating of t h e residue after evaporation of solvent, glutethimide will decompose (Table 11).
WAW 240 LENGTH 260 IN M r
Water Urine Plasma
c
0.2
-
0.1
-
I
-.
. 1
D-
215 220
2% 235 240 2% 260 WAVE LENGTH IN MY
270
J
280
Figure 3. Ultraviolet absorption spectra of solution of 3 ml. (10 y per ml.) mixed with 1 ml. of 0.2N potassium hydroxide 1, 3.
2-ethyl-2-phenylglutarimide 2-Phenylglutarimide
(glutethimide)
prepared. Three milliliters of each solution viere mixed with 1 ml. of 0.2.V potassium hydroxide and the absorbances were recorded immediately and every 1000 seconds. The absorbances were plotted on semilogarithni paper to obtain the initial concentration a t mixing time. A straight-line relationship of concentration to absorbance a t mixing time n-as obtained for glutethimide up to 25 y per ml. Determination in Blood and Urine. MATERIALS. Glutethimide (Ciba Laboratories, Summit, PI'. J.) standards: strong, 500 y per ml. in absolute ethyl alcohol; T e a k , 500 y per ml. diluted to 10 and 20 y Der ml. in absolute ethyl alcohol. Chloroform, analytical grade, distilled through a 2-foot Vigreux column and the myddle portion saced. Chloroform was chosen as the solvent for extracting glutethimide from biological materials because of the following properties: It quantitatively extracts glutethimide from water solution, i t is heavier than water and has a low mutual solubility, it has a low volatility and is nonflammable, and it is easily purified by fractional distillation through a Vigreux column over an open flame.
c
I"'"
KIDNEY
2.0 KiDhiY 0
0.6 0.b
0.4
c1
stopcocks. The solution is shaken vigorously for approximately 1 minute, allowed to settle, and the sample is aspirated off a n d discarded. The solvent extract is run through a fast filter (Whatman No. 41), washed tnice b y shaking with 5 ml. of 0.5-1- sodium hydro\ide, and once b y shaking with 5 nil. of 0.5X hydrochloric acid. Each time, the wash is aspirated off and discarded. A clear aliquot (20 ml.), usually obtained after filtering through a K h a t m a n No. 41 filter, is evaporated on a steam bath to low volume ( 2 to 3 ml.) and air-dried. The d r y residue is dissolved in 4 nil. of absolute ethyl alcohol. A 3-ml. aliquot is pipetted into a stoppered test tube containing 1 ml. of 0.2N potassium hydroxide. The mixing time is recorded; the absorbances are deterniined in a Beckman quartz spectrophotometer at wave lengths 280, 240, 235, and 225 mp for the characteristic glutethimide curve, using as the reference solution 3 ml. of absolute ethyl alcohol mixed with 1 ml. of 0.2N potassium hydroxide. .4 glutethimide standard, 10 y per ml. in absolute ethyl alcohol, is determined under identical conditions for comparison. Glutethimide in alcoholic potassium hydroxide 1%ill have a characteristic absorption curve with maximum a t 235 m t (E::, = 880) and minimum at 220 mp (Figure 1). The absorbance a t 235 nip is recorded repeatedly a t intervals of approximately 500 seconds for the rate of change. The absorbance a t 235 nip after 10 half-life periods, approximately 10.000 seconds, represents absorbing materials other than glutethimide and is subtracted from each reading. The corrected absorbances are plotted on semilogarithm paper. The absorbances a t mixing time and the initial concentration are obtained b y extrapolation. If the unknown solution contains glutethimide, the slope should be similar to that of the glutethimide standard. Recovery from Biological Samples. A stock solution of glutethimide in alcohol containing 500 y per ml. was used to niake dilutions in water, urine, and plasma, varying in amounts from 25 to 100 y per 2-ml. sample. The 2nil. samples rTere carried through the extraction procedure as described (Table 111). Almost 1 0 0 ~ o of the added glutethimide is recovered in the extraction procedure: the recovery error is less than 5%.
Table 111.
KO. of Ex.2-111. periments Sample Water 10 4 Plasma 5 Urine 10 Water 12 Plasma 5 Urine 4
L\& 0
0
30
20
-3
43
/O
t0
hOJRS
Figure 4. Plasma glutethimide levels in a dog after administration of 200 mg. per kg.
0.I
, 0
I0
I
I
20
I
,
,
30
40
,
,
53
,
, , 60
~
70
HCJRS
Figure 5. Plasma levels from patient who had ingested 10 grams of glutethimide 12 hours before entering hospital
I n the extraction of glutethimide from biological samples, no adjustment of p H is necessary, as glutethimide can be quantitatively removed from aqueous solution b y chloroform a t any pH. However, above p H 10 there is a tendency for glutethimide to decompose. Yeutral fats and acidic and basic conipounds will also be present in the chloroform extract. K h e n acidic substances were removed b y washing the solvent with dilute alkali, glutethimide n a s too weak a n acid to forni a salt and remained in the solvent layer. This property of glutethimide is useful in decreasing absorbing materials by removing acidic and basic compounds from the chloroform extract. T n o alkaline washings n ill remove almost all acidic compounds, and one acid wash \vi11 remove almost all basic compounds. The first alkaline extract may be saved for determination of acid drugs, such as barbiturates.
PROCEDURE. From 1 to 5 ml. of blood, plasma, or urine are extracted with 25 ml. of chloroform in glassstoppered mixing cylinders or in separatory funnels fitted n ith silicon-greased
DISCUSSION
Interfering Substances. Glutethimide-free plasma a n d urine, when carried through t h e extraction procedure, sho\ved n o significant change in absorbance with time a n d t h e readings remained constant for several hours. T h e absorption spectra showed no absorption peaks b u t increased in absorbance with decreasing wave lengths, as shown in Figure 2. TKOclosely related substituted glutaric acid imides were investigated t o ascertain whether they interfered with
Recovery of Known Amounts of Glutethimide
5
Plasma
Water
Table IV.
GluCalculated tethiRecovery mide, and Standard Error 25 24.9 i 0 . 3 25 24 9 1 0 . 7 e ,
25 50
50
50 75 100
25.0 i 0 . 8 49 G f 0 . 3 4 9 . 2 Ik 1.0 49.6+ 1.1 74 4 f 1 . 0 (30 2 i.1 . 5
Plasma Glutethimide Levels
Time after Plasma Admission, Levels. 'Hours Mg. % Conditions At admission" 4.50 Deeply comatose, no reflex, dilated 5 ; 1 hr. on
artificial kidney
pupil
4.30 Comatose
3 . 7 5 Comatose 12; off arti- 3.15 Reflex returned ficial kidney 22.5 2.40 Semicomatose, res-
8
piration
im-
proved 26.5 2.15 Semicomatose 39 1.00 Conscious 47 0 50 Avake 59 0.35 .ln-alie Patient ingested 10 grams of glutethimide 12 hours before entering hospital.
the determination of glutethimide. They were the reported metabolite, 2phenylglutarimide (1, S), aiid the drug, 3-methyl-3-phenylglutariniide (megimide). Xlegimide is used in the treatment of barbiturate aiid glutethimide intoxication (6, 8). Figure 3 s h o w the ultraviolet spectra of these compounds and glutethimide. Aillthree have similar spectra with slight shift in maxima and minima. Honever, the rate of hydrolysis varied considerably when these compounds n ere subjected to alkaline hydrolysis. lleginiide showed no noticeable change, TT hile 2-phenylglutariniide and glutethimide FT-ere distinguished b y the difference in the rate of hydrolysis. a-Phenylglutarimide decomposed almost tnice as fast as glutethimide. I n a mixture of 2-ph~.n~-lglutarimide the concentrations of both niay be determined because of the marked difference in the rate of hydrolysis a t 235 mp with time. Initially there is a rapid decrease and the curve tapers off to a rate approximately that of glutethimide. The early decrease is due to the 2phenylglutarimide, IT hile the slower decrease may be attributed entirely to glutethimide. Extrapolation to mixing time will give the rate of change with time and the original concentrations. Both 2-phenylglutarimide and megVOL. 32, NO. 1, JANUARY 1960
83
imide are stronger acids than glutethimide. K h e n plasma samples containing these compounds were carried through the extraction procedure, there was no interference in the determination of glutethimide. Two alkaline n-ashings with 0.5S sodium hydroxide removed all the megimide and over 90% of the 2-phenylglutariniide. Urine may contain high concentrations of the metabolite, and small but significant amounts may appear in the final extracts. The characteristic rate of hydrolysis will distinguish the metabolite from the glutethimide. Determination of Glutethimide in Experimental Animals and Humans.
T h e results of a study on a dog given 200 mg. per kg. of glutethimide orally are shown in Figure 4.
(1) Bernhard. Karl. Just. M . . Vuillumier.
(5) Schreiner, George E., Berman, L. B., Kovach, R., Bloomer, H. A4.J A . M. 8 . Arch. Internal Med. 101, 899-911 (19.58). (6) Shaw, F. H., Simon, S. E., Cass, M. M., Shulman, A., Suture 174, 402-3 (1954). (7) Sheppard, Herbert, D’.\saro, Barbara S.,Plummer, Albert J., J. Ani. Pharm. ASSOC., Sci. Ed. 45, 681-4 (1956). (8) Shulman, A,, Shaw, F. H., Cass, Ii. M,, Khyte, H. >I., Brit. X e d . J . 1955, I , 1238-44. (9) Tagmann, E., Sury, E., Hoffmann, K., Helv. C h i m Acta 35, 1235-9; 1541-8 (1952).
39,596-606 (1956). (2) Gross, F., Tripod, J., hleier, R., Schweiz. med. Wochschr. 85, 305-9 (1955). (3) Kebrle, J., Hoffmann, K., Experientia 12. 21-2 (1966). (4) -iZcBa
