High-speed Ion Exchange Chromatography of Barbiturates, Diphenylhydantoin, and Their Hydroxylated Metabo1ites M. W. Anders and Jennine P. Latorre Department of Pharmacology, Unioersity of Minnesota, Minneapolis, Minn. 55455 of barbiturates and related compounds by high-speed ion exchange chromatography.
THE SEPARATION and determination of barbiturates, their metabolites, and related compounds can be carried out by various chromatographic techniques including paper, thinlayer and gas-liquid chromatography. Ion exchange chromatography has seen minimal application in the analysis of this group of compounds. Horvath and coworkers have described a high-speed ion exchange chromatograph that has proved useful in the analysis of nucleotides ( I ) and nucleosides and bases (2). In this technique, a very long narrow column packed with pellicular ion exchange resin is employed. The column is operated at high pressures (800-2800 psi) permitting rapid analyses (3). The success obtained in the analysis of nucleotides and nucleosides suggested that high-speed ion exchange chromatography might be applicable to barbiturate analysis. Furthermore, the technique is applicable to the analysis of polar materials without derivatization. In contrast, reports of gas chromatographic analysis of metabolites of amobarbital ( 4 ) and diphenylhydantoin (5) employed a derivatization step. The present investigation describes conditions for the analysis
EXPERIMENTAL
(I) C. G. Horvath, B. A. Preiss, and S. R. Lipsky, ANAL.CHEM., 39, 1422 (1967). (2) C. Horvath and S. R. Lipsky, ibid.,41,1227 (1969). (3) C. Horvath and S. R. Lipsky, J. Chromntogr. Sci., 7,109 (1969). (4) J. J. Kamm and E. J. Van Loon, Chi. Chern., 12,789 (1966). (5) G. Grimmer, J. Jacob, and H. Schafer, Arzneirn.-Forsc/z., 19, 1287 (1969).
Apparatus. A Varian LCS 1000 high-speed liquid chromatograph equipped with a pellicular anion exchange column (0.04-inch i.d. x 120-inch long, type LSF) was employed. The ultraviolet detector of the instrument operated at 254 ml.c. Mode of Operation. Experiments were conducted employing both nongradient and gradient elution. When conditions were varied, the system was flushed and the column purged with the new solution for at least 30 minutes. In the nongradient mode of operation, phosphate buffers of varying composition served as the eluents; the exact conditions are described in the text. Sodium chloride solutions were employed for gradient formation. The following conditions were utilized in most gradient elution experiments : high concentrate solution: 1 M sodium chloride, gradient chamber solution: O.lmM sodium chloride, initial volume of O.lmM sodium chloride in gradient chamber: 50 ml, high concentrate flow rate into the gradient chamber: 12 ml/hr and column flow rate: 24 ml/hr. These conditions result in the formation of a linear gradient (I). Chemicals. Reagent grade chemicals were employed for the preparation of eluents. The following drugs were studied: pentobarbital, hydroxypentobarbital [5-ethyl-5-(3-hydroxy-lmethylbuty1)barbituric acid], amobarbital, hydroxyamobarbi[5-ethyl-5-(3-hydroxy-3-methylbutyl)barbituric acid], tal
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Figure 1. Effect of eluent molarity on chromatography of diphenylhydantoin (first peak) and hydroxydiphenylhydantoin (second peak) Eluent: 0.2, 2.0, and 20.0 m M KH2P04, pH 4.5; column temperature: 80" C; flow rate: 36.3 ml/hr. Inlet pressures: 1200-1600 psig 1430
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Figure 2. Effect of column temperature on chromatography of diphenylhydantoin
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Eluent: 20 mM KHzP04,pH 4.5; column temperatures: 60, 70, and 80 "C; flow rate: 33.3 ml/hr. Inlet pressures: 900-1100 psig
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Figure 3. Chromatography of barbiturates and metabolites using linear gradient elution Eluent: 0.1 mM to 1.0 mM sodium chloride; temperature: 80 "C; flow rate: 26 ml/hr. Inlet pressures: 700-900 psig, pH 7. Peak 1, ketohexobarbital (0.3 pg); peak 2, hydroxyamobarbital (13 pg); peak 3, contaminant; peak 4, amobarbital (5.3 pg); peak 5, phenobarbital (2.5 pg); and peak 6, hydroxyphenobarbital(3.5 pg)
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hexobarbital, ketohexobarbital [1,5-dimethyl-5-(3-keto-lcyclohexeny1)barbituric acid], diphenylhydantoin, hydroxydiphenylhydantoin [5-p-hydroxyphenyl-5-phenylhydantoin], phenobarbital, and hydroxyphenobarbital [5-p-hydroxyphenyl-5-ethylbarbituric acid]. Ketohexobarbital was prepared as described by Tsukamoto et al. (6). Compounds were dissolved in distilled water for analysis. RESULTS AND DISCUSSION
Initial experiments dealt with the application of high-speed ion exchange chromatography to the separation of diphenyl(6) H. Tsukamoto, H. Yoshimura, and S. Toki, Chem. Pharm. Bull., 4, 364 (1956).
hydantoin and hydroxydiphenylhydantoin. The effects of varying molarity and pH of the eluent as well as altering the column temperature were studied. Figure 1 shows the effect of changing the molarity of the eluent while holding pH at 4.5 and column temperature at 80 "C. It can be seen that there is relatively little difference in either peak shape or retention time when 0.2 or 2.0mMpotassium phosphate buffer is employed. However, when 20.0mM phosphate buffer is used, improved peak shape and resolution are obtained along with shortened analysis time. The pH of the eluent was varied at a buffer molarity of 20.0mM and a column temperature cf 80 "C. It was found that sharper peaks were obtained at pH 3.5 than at either 4.5 or 5.5 and retention volumes
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Table I. Comparison of Phosphate Buffer and Sodium Chloride as Eluents for Chromatography of Barbiturates and Metabolites
Retention volume, ml Compound KHQPOI" NaClb Phenobarbital 5.9 11.o Hydroxyphenobarbital 10.0 16.5 5.4 10.3 Pentobarbital Hydroxypentobarbital 1.1 4.7 Amobarbital 6.1 10.6 Hydroxyamobarbital 1.5 4.3 Hexobarbital 1.4 10.8 Ketohexobarbital ... 1.3 a Conditions: 2.0 mM KHQPO~, pH 5 . 5 , 80 "C, flow rate 32-36 ml/hr. * Conditions: Low concentrate chamber, O.lmM NaCl; high concentrate chamber, 1.OM NaC1; flow of high concentrate into gradient chamber, 12 ml/hr; column flow, 24 ml/hr; initial volume, 50 ml; temperature, 80 " C ; inlet pressures, 700-900 psig.
increased as the pH became less acidic. The effect of column temperature on the peak shape was marked and operation at higher temperatures resulted in sharper peaks (Figure 2). The finding of improved peak shape at higher temperatures is similar to the results obtained by Horvath and Lipsky (2) who observed that high column temperatures produced favorable conditions for the separation of purine and pyrimidine bases. The separation of diphenylhydantoin and hydroxydiphenylhydantoin was insensitive to changes in molarity, pH, and temperature over the ranges given above. The same conditions that proved to be optimum for the chromatography of diphenylhydantoin and its hydroxylated metabolite were also suitable for the separation of phenobarbital and hydroxyphenobarbital. Analysis of barbiturates and their metabolites, other than phenobarbital, using phosphate buffers as eluents in gradient or nongradient modes of operation was unsatisfactory inasmuch as poor peak shape was obtained. Further studies showed, however, that the use of a linear sodium chloride gradient gave satisfactory results and both barbiturates and
their metabolites eluted as sharp symmetrical peaks (Figure 3). In addition, metabolites and parent compounds were well separated. As shown in Table I, alcoholic or ketonic metabolites consistently showed retention volumes less than that of the parent molecule. On the other hand, the phenolic metabolite of phenobarbital was retained longer than the parent compound. Separation efficiency was substantially greater when a linear sodium chloride gradient was employed than during nongradient phosphate buffer operation. As expected, operation of the column with neutral sodium chloride solutions resulted in an increased sensitivity as compared to the use of acidic phosphate buffers. This is attributable to the increased absorptivity of barbiturates at more alkaline pH values. In contrast to the results obtained with barbiturates and their metabolites, diphenylhydantoin and hydroxydiphenylhydantoin were not eluted from the column by a sodium chloride gradient. In conclusion, high-speed ion exchange chromatography has been shown to be a useful method for the analysis of barbiturates, diphenylhydantoin and metabolites of these compounds. Nongradient elution with phosphate buffers proved effective for the separation of phenobarbital, diphenylhydantoin, and their phenolic metabolites. Optimum conditions for the above analysis were found to be 20.0mM phosphate buffer, pH 3.5,and a column temperature of 80 "C. Separation of barbiturates and metabolites were successfully carried out with a linear sodium chloride gradient at 80 "C. Studies are in progress to extend these techniques to the analysis of drugs in biological fluids. ACKNOWLEDGMENT
The authors thank Mehroo J. Cooper for the preparation of ketohexobarbital. Tom Flanagan, Smith Kline and French Laboratories, supplied the hydroxyamobarbital used in these experiments, and A. J. Glazko, Parke, Davis and Company, supplied hydroxydiphenylhydantoin. RECEIVED for review April 20, 1970. Accepted July 15, 1970. This research was supported by U.S.P.H.S. grants G M 17511 and G M 15477.
Identification of Barbiturates by Chemical Ionization Mass spectrometry' H. M. Fales, G . W. A. Milne, and T. Axenrod2 Laboratory of Chemistry, National Heart and Lung Institute, National Institutes of Health, Bethesda, Md., 20014 THEUSE OF BARBITURATES as sedatives is so widespread that accidental and intentional overdoses are daily occurrences. This problem is further compounded by the fact that a wide variety of substituted barbituric acids with considerably different biological activities is in common use. They are obtainable, moreover, in many different formulations and mixtures. Therefore, when an overdose has been taken, it 1 This is Part V of the series on Chemical Ionization Mass Spectrometry of Complex Molecules. NIH Special Fellow, 1969. O n leave from the City College of the City University of New York.
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is difficult to determine from an examination of the patient the identity of the drug or drugs responsible for his condition. This information is essential to a correct choice of treatment and a reliable method for the identification of specific barbiturates in stomach washings, blood, or urine would be of considerable value. A wide variety of chemical and colorimetric methods is available ( I ) for the identification of barbiturates as a class (1) See for example, D. M. Baer, Amer. J. Clin. Puthol., 44, 114 (1965) and references cited therein.
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