The same type arguments could be applied to a cyclic case such as succinic where the anhydride is 0.5 ppm downfield relative to the acid. The greater rigidity of the cyclic anhydride compared to the linear anhydride increases the degree of downfield shift. This could be expected for magnetic anisotropic effects. The inductive effects should be the same for the cyclic us. linear situation. The alpha hydrogens for cyclic maleic anhydride are 0.8 ppm downfield from maleic acid. The ethylenic and anhydride functions make a completely rigid structure for maleic anhydride. Furthermore, the hydrogens are in the same plane as the carbonyl bond. This is the region of maximum deshielding for the carbonyl group. However, in addition, there may be a second effect operating in this case. Because maleic anhydride is planar and there are six pi electrons in a ring, the possibility exists that it has some aromatic character. Circulating pi electrons cause deshielding of the hydrogens in benzene and would act similarly in this case. Fumaric acid (trans-butenedioic) has its alpha hydrogens 0.4 ppm downfield from maleic acid (cis-butenedioic). The inductive effects should be the same in both cases. As the carboxyl groups rotate in fumaric acid, first one of the two
hydrogen atoms and then the other comes under the deshielding influence of any given carbonyl group. This is not true for maleic acid. Consequently, the hydrogens 'are deshielded for a lesser amount of time in maleic acid than in fumaric acid. While this study was limited to carboxylic acids and anhydrides, other carboxylic type compounds may show similar distinctions in their proton spectra. It would be interesting to compare the above results with those of the corresponding sulfur analogues. Other interesting comparisons might be made between carboxylic acids, amides, and imides. The quantitative implications of being able to distinguish between acids and anhydrides by proton magnetic resonance is also of some importance. Quantitation is especially simple in those cases where only singlet absorption bands are involved. This includes the following acids and anhydrides in Table I : acetic, chloroacetic, dichloroacetic, maleic, fumaric, succinic, and chlorendic. The analysis of mixtures of maleic acid, fumaric acid, and maleic anhydride is not easily accomplished by other means. RECEIVED for review February 26, 1969. Accepted April 16, 1969.
Differentiating Spectrophotometric Titration of Phenobarbital-Diphenylhydantoin Combinations in Nonaqueous Medium Suraj P. Agarwal Department of Biochemistry, The Chicago Medical School, University of Health Sciences, Chicago, Ill. 60612 Martin I. Blake' Department of Pharmacy, Unicersity of Illinois at the Medical Center, Chicago, Ill. 60612 DIPHENYLHYDANTOIN is often administered with phenobarbital in anticonvulsant therapy. Because of the structural similarity of these two compounds, any analytical procedure for their simultaneous determination is complicated by the problem of one interfering with the other. Nevertheless, a variety of procedures (titrimetric, spectrophotometric, and chromatographic) have been reported in the literature. These have been recently reviewed (Z-3). In the present report a differentiating spectrophotometric titration procedure is described for the determination of the individual components in phenobarbital-diphenylhydantoin mixtures. The method is simple and quantitative for the two drugs over a wide range in concentration ratios. Conditions for the spectrophotometric titration of the individual components are also presented. The procedure is applicable to the analysis of commercially available dosage forms after treatment with a cation exchange resin to convert the sodium diphenylhydantoin to the free acid. 1
Author to whom inquiries should be sent.
(1) S . P. Agarwal and M. I. Blake, J. Assoc. Ofic.Anal. Chem., 51, 1013 (1968). (2) S. P. Agarwal, Ph.D. Dissertation, University of Illinois at the Medical Center, Chicago, Ill., 1969, pp 7-11. (3) D. H. Sandberg, G. L. Resnick, and C. 2. Bacallao, ANAL. CHEM.,40, 736 (1968).
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ANALYTICAL CHEMISTRY
EXPERIMENTAL
Apparatus. All spectrophotometric titrations were performed with a Carl Zeiss, Model PMQ I1 spectrophotometer equipped with titration cell (silica, 18 x 35 mm), cell carriage, and magnetic stirrer. A special cell cover was provided which permitted the introduction of the buret tip through a small aperture in the cover. A 5-ml buret (Kimax) graduated in 0.01 ml was employed for delivery of titrant. A 25-ml buret with Teflon (DuPont) stopcock was used as an ion exchange column. Reagents. Reference standard phenobarbital and diphenylhydantoin were obtained from Parke, Davis and Co. Acetonitrile (Matheson, Coleman and Bell) was employed as solvent. Other chemicals and solvents were reagent grade. All dosage forms used in this study were obtained from commercial sources. Triethyl-n-butylammonium hydroxide was prepared as a 0.1N solution in benzene-methanol according to Fritz and Yamamura (4). The solution was standardized by photometric titration against a solution of reference standard benzoic acid in acetonitrile at 360 mp. One drop of thymolphthalein (0.1 methanolic solution) was employed as the indicator. Five synthetic mixtures each containing 100 mg of diphenylhydantoin and 100, 50, 32, 25, and 16 mg, respectively, of phenobarbital were prepared. Procedure. DETERMINATION OF PURECOMPOUNDS.Ten milliliters of a sample solution, containing approximately 0.1 (4) J. S . Fritz and S . S . Yamamura, ANAL.CHEM.,29, 1079 (1957).
meq of pure drug dissolved in acetonitrile, was transferred to the titration cell containing a small plastic covered bar magnet. The instrument was set to zero absorbance a t the desired wavelength (phenobarbital, 293 mp ; diphenylhydantoin, 275 mp). The solution magnetically stirred was titrated with 0.1N triethyl-n-butylammonium hydroxide solution. The titrant was added in 0.1-ml portions and the absorbance of the sample was recorded after each addition of the titrant. The end point of the titration was determined from a plot of absorbance cs. volume of the titrant. The intersection of the two straight line segments was taken as the end point. DETERMINATION OF SYNTHETIC MIXTURES. A stock solution of the diphenylhydantoin-phenobarbital mixture was made by transferring an accurately weighed amount (0.35 to 3.5 meq) of the mixture to a 100-ml volumetric flask. The powder was dissolved in acetonitrile and sufficient solvent was added t o bring the volume to the mark. A 10-ml aliquot of the stock solution was transferred t o the titration cell and was titrated with 0.1N triethyl-n-butylammonium hydroxide a t 293 mp, The titration procedure and the treatment of the data were the same as described above. I n those instances where large volumes of titrant (greater than 1.5 ml) were used, the absorbance was corrected for dilution as follows: Corrected absorbance = absorbance X
v+v vo ’
where V , = volume prior t o titrant addition, and V = volume of titrant added. APPLICATION TO DOSAGE FORMS.The commercially available dosage forms contain phenobarbital as the free acid and diphenylhydantoin as the sodium salt. I n order to determine the two components, it is necessary first to convert the sodium diphenylhydantoin to the free acid. The following ion exchange procedure was used and is similar to that described by Vincent and Blake (5). Ten grams of the air-dried resin (Amberlite IRC-50, C.P.) were soaked in distilled water for 24 hours. The slurry was poured into the column and the resin was regenerated by passing 50 ml of 1N HCl through the column. The column was washed free of hydrochloric acid with distilled water, the water was drained, and methanol was added t o the column. The resin was allowed to stand in methanol overnight prior to use. The column was washed with a small portion of methanol prior to a determination, The contents of twenty capsules of a dosage form were removed as completely as possible and weighed. An accurately weighed aliquot containing approximately 3 meq of total acidity was suspended in methanol (20 ml) and transferred quantitatively to the ion exchange column with the aid of additional methanol. Sufficient methanol was passed through the column until 150 ml of the eluate was collected in a 250-ml beaker. The methanol was evaporated completely by keeping the beaker under a stream of air. The air-dried residue which consisted of diphenylhydantoin and phenobarbital was dissolved in acetonitrile and transferred to a 250-ml volumetric flask. Additional solvent was added to bring the volume to the mark. Ten milliliters of this solution were transferred t o the titration cell and the titration was performed in the manner described earlier. RESULTS AND DISCUSSION
The usual methods of analysis for the combination of diphenylhydantoin and phenobarbital require a separation of the two components prior to their determination. Column partition chromatography was employed by Lach, Bhansali, and Blaug (6) and by Marino (7) to separate diphenylhydantoin and phenobarbital. These procedures are lengthy and (5) M. C . Vincent and M. T. Blake, Drug Std., 26, 206 (1958). (6) J. L. Lach, K . Bhansali, and S. M. Blaug, J. Amer. Pliarm. Assoc., Sci. Ed., 47, 48 (1958). (7) V. Marino, J . Assoc. O’ffic. A m / . Clrem., 48, 582 (1965).
f
I /
Y
Y
Y
0 0 2 04 0 6 0 0 10 12 14 16 18 2 0 2 2 24 26 28 30 32 Milliliters of 0 I N Triethyl n-butylommonium hydroxide
Figure 1. Differentiating spectrophotometric titration curves for various ratios of phenobarbital and diphenylhydantoin A . 1:2 B . 1:3
C. 1:4 D . 1:6
d o not give satisfactory recoveries when widely disproportionate quantities of the two ingredients are present. Spectrophotometric titrations have been utilized successfully for differentiating mixtures containing components of similar acidic or basic strength. Goddu and Hume (8) titrated mixtures of acetic acid, p-nitrophenol, and m-nitrophenol and also a mixture’ of p-nitrophenol and m-nitrophenol. Hummelstedt and Hume ( 9 ) determined the individual amines in a mixture of di-n-butylamine, N,N-diethylaniline, aniline, and o-chloroaniline. The latter authors compared the differentiating ability of potentiometric titration and spectrophotometric titration methods. A mixture of 2-methyl-5nitroaniline and 4-methyl-2-nitroaniline gave poor separation of the components when titrated potentiometrically but excellent recoveries of both components were obtained by the spectrophotometric procedure. I n preliminary studies attempts were made to titrate the phenobarbital-diphenylhydantoin mixture using a differentiating potentiometric procedure. Various solvents--e.g., acetone, methyl isobutyl ketone, methyl ethyl ketone, ethyl acetate, acetonitrile, isopropyl alcohol, isobutyl alcohol, t-butyl alcohol, chloroform, ethylenediamine, pyridine, and dimethylformamidewere used to dissolve the mixture and either sodium methoxide or triethyl-n-butylammonium hydroxide was used as the titrant. Glass and sleeve-type calomel electrodes were employed throughout. In all cases only one break in the titration curve was observed which corresponded to the total amount of the two components. A possible explanation for failure of differentiation may be attributed to close proximity of the pK, values of phenobarbital [pK, = 7.42 ( l o ) ]and diphenylhydantoin [pK, = 8.31 (ZZ)]. (8) R. F. Goddu and D. N. Hume, ANAL.CHEW,26, 1679 (1954) (9) L. E. I. Hummelstedt and D. N. Hum-, ibid., 32, 576 (1960). (10) T. D. Edmonson and J. E. Goyan, J. Amer. Pharm, Assoc., Sci. Ed., 47, SlO(1958). VOL. 41, NO. 8,JULY 1969
1105
Table I. Differentiating Spectrophotometric Titration of Phenobarbital and Diphenylhydantoin Mixtures Mixture Added, mg Recovery, Phenobarbital 3.861 100.73 f 0.50" Sodium diphenylhydantoin 3.690 99.88 f 1.60 Phenobarbital 10.012 101.82 f 0.53 Sodium diphenylhydantoin 20.046 99.01 f 2.12 12.521 Phenobarbital 99.97 f 0.60 37.563 Sodium diphenylhydantoin 101.66 f 0.06 Phenobarbital 12.032 101.06 f 0.49 Sodium diphenylhydantoin 48.128 98.41 f 1.35 Phenobarbital 10.275 99.67 f 2.58 60.165 Sodium diphenylhydantoin 97.98 3t 1.38
1.4 -
x
a
Standard deviation based on at least 4 determinations.
Table 11. Analysis of Capsule Dosage Forms Containing Phenobarbital and Sodium Diphenylhydantoin Label claim per unit Label Components in capsule dose, mg. claim found Capsule A Phenobarbital 32 97.40 & 1.67= Sodium diphenylhydantoin 100 96.26 f 0.56 Capsule B Phenobarbital 16 104.69 f 1.60 Sodium diphenylhydantoin 100 99.02 & 0.48 a
Standard deviation based on at least 4 determinations.
Successful differentiation was, however, achieved by the spectrophotometric titration procedure. It was found that an acetonitrile solution of the two drugs could be titrated differentially with a solution of triethyl-n-butylammonium hydroxide. The data for the titration of a series of synthetic mixtures are illustrated in Figure 1. The phenobarbital content is equivalent to the titrant consumed to the first break in the titration curve, and the diphenylhydantoin content is equal to the amount of titrant from first break t o the second break in the titration. The choice of the conditions for the titration was dictated by several factors. Because the titrations were performed in the ultraviolet region, it was desirable to have a solvent which shows very little absorption in this range. Acetonitrile was found to be the solvent of choice for several reasons : both phenobarbital and diphenylhydantoin are soluble in it, the solvent does not absorb appreciably above 210 mp, and it is commercially available in pure form. Titrants composed of a n alkali metal alkoxide generally give a precipitate, for the alkali salt of both phenobarbital and diphenylhydantoin is insoluble in acetonitrile and hence these titrants are not acceptable. Fritz and Yamamura ( 4 ) used triethyl-n-butylammonium hydroxide in the titration of many weak acids and observed the absence of a precipitate with the use of this titrant. Preliminary experiments showed that this titrant does not form insoluble salts with either phenobarbital or diphenylhydantoin. The selection of the wavelength at which the titrations were to be performed required a study of the absorption spectra of the ionized and unionized forms of the two compounds. Ultraviolet spectra of phenobarbital and diphenylhydantoin were obtained in 0.01N NaOH and 0.01N HCl. At 293 mp phenobarbital (1 X 10-4M) showed greater absorption in
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ANALYTICAL CHEMISTRY
1.21.0aJ
0.8-
2
Z 0.6 a 0.4 -
I
I
I
I
I
I
0.4 0.6 0.8 1.0 1.2 1.4 Milliliters of 0.1 N Triethyl n-butylammonium hydroxide
0.2
Figure 2. Spectrophotometric titration curves for ( A ) diphenylhydantoin at 275 rnp and ( B ) phenobarbital a t 293 mp alkaline medium than in acidic medium; while there was little difference in the absorption by diphenylhydantoin in these solutions (11). However, at 275 mp diphenylhydantoin did show a difference in absorption in alkaline and acidic media and this wavelength proved suitable for the determination of the diphenylhydantoin alone. The differentiating titrations were performed at 293 mp. During the initial stages of the titration, the absorption by the sample increased with each increment of the titrant. At this stage phenobarbital was being titrated. I n the next stage while diphenylhydantoin was being titrated, the absorbance of the sample remained practically unchanged. The rise in absorbance beyond the end point is due to the titrant. The data in Table I indicate excellent recovery of the two components in various synthetic mixtures. The results of the analysis of two dosage forms by the proposed method are reported in Table 11. The per cent recoveries are compared with the label claim. Excipients in the dosage forms apparently d o not interfere in the analysis. The spectrophotometric titration procedure was also utilized for the determination of phenobarbital and diphenylhydantoin individually. Figure 2 shows the titration plot obtained with phenobarbital. The titration was performed at 293 mp and an average per cent recovery of 101.57 was obtained. Similarly diphenylhydantoin was titrated at 275 mp and gave an average per cent recovery of 100.33 (Figure 2). ACKNOWLEDGMENT
The valuable help received from Kenneth A. Connors (School of Pharmacy, University of Wisconsin) during the initial stages of this study is gratefully acknowledged.
RECEIVED for review January 2, 1969. Accepted May 2,1969. Presented a t the 157th National Meeting, American Chemical Society, Minneapolis, Minn., April 1969. (11) S. P. Agarwal and M. I. Blake, J . Pkarm. Sci., 57, 1434(1968).
