smaller samples, the sample could be diluted with carbon disulfide which is not seen by a flame detector and large sample volumes could then be used. Using a 10% solution of the aromatics in CS2, a 1 - ~injection 1 of the CS2 solution can be made. Using small sample volumes, column efficien-
cies of 800-900 platedft were obtained as measured from the n-propylbenzene peak.
RECEIVEDfor review May 2, 1974. Accepted August 12, 1974.
Quantitative Analysis of a Multicomponent Analgesic Product Containing Butalbital, Using High Speed Reverse-Phase Liquid Chromatography Daniel Rosenbaum Sandoz Pharmaceuticals,fast Hanover, N.J. 07936
High-speed liquid chromatography previously has been applied to the quantitative analysis of analgesics using ion exchange columns (1, 2). A fast, dependable, and accurate method, using the new high-efficiency reverse-phase packing now commercially available, is reported in this article. The present work describes the successful quantitative analysis of an analgesic tablet and the advantages of this method over partition column chromatographic procedures ( 3 -5) which are currently employed in most laboratories. Differences with previously reported ion-exchange liquid chromatography methods are evaluated.
EXPERIMENTAL Apparatus. A Model ALC202 Liquid Chromatograph with Model 6000 pump (Waters Associates, Milford, Mass.) was used with the 254-nm detector. Samples were introduced onto the column by syringe injection. A digital integrator (Autolab Model 6300) was used to determine peak areas and retention times. Column. A 30-cm X 4-mm id pBondapak C ~ (Waters R Associates) column was used. WBondapak C ~ has R a monomolecular layer of octadecyltrichlorosilane chemically bonded to Porasil beads having an average particle size of 10 microns. Reagents. Aspirin USP,phenacetin USP, caffeine USP,and butalbital N F were used as standards. The internal standard, p chloroacetanilide, was of analytical grade from Eastman Kodak. Acetonitrile (distilled in glass) was obtained from Burdick and Jackson Laboratories. Fiorinal Tablets and Fiorinal Capsules, containing aspirin, phenacetin, caffeine, and butalbital, are products of Sandoz Pharmaceuticals, East Hanover, N.J. Mobile Phase. Acetonitrile/O.Ol% ammonium carbonate in water (4050) was used. Internal Standard Solution. The solution was 0.05 mg/ml p chloroacetanilide in acetonitrile. Sample Solution. A sample of the powder equivalent to one tablet or one capsule net content weight (taken from a composite weight of tablets or capsules) was accurately weighed and transferred to a 200-ml volumetric flask. About 100 ml of Internal Standard Solution was added and solution was achieved in 15 minutes using a sonic bath. The samples were then diluted to volume with Internal Standard Solution. Standard Solution. Similarly, a standard solution containing aspirin, caffeine, phenacetin, and butalbital was prepared equivalent to the theoretical concentration of each component in the sample solution. Dissolution was also effected by means of the sonic bath.
(1) J. S. Mayell, C. F. Hiskey, and L. Lachman, Anal. Chem., 46, 449 (1974). (2) A. F. Michaelis, D. W. Cornish, and R. Vivilecchia, J. Pharm. Sci., 62, 1399 (1973). (3) National Formulary Xlll Edition. (4) R. F. Heuermann and J. Levine. J. Amer. Pharm. Ass., 47, 276 (1958). (5)T. D. Doyle and J. Levine, Anal. Chem.,39, 1282 (1967).
2226
&j 20
10 R E i E N T l O N T I M E IM8nut.i)
Figure 1. Fiorinal on 122 cm X 2.3 mm Zipax SAX, 0.01Msodium borate and 0.01MNH4N03, 1.0 ml/min, 0.16 AUFS, ambient. Acetaminophen was used as the internal standard. Benzoic acid w a s added for information only Peak identity: 1, Caffeine: 2, Acetaminophen (APAP); 3, Phenacetin: 4, Aspirin: 5. Benzoic acid: 6, Butalbital
The analysis of the components present in the product was performed under the optimum conditions given under Figure 2 . The concentrations of the active constituents present in the analgesic product were determined by peak area using a digital integrator. Area ratios of each component were obtained in relation t o the Internal Standard. The accuracy of the method was checked with laboratory prepared samples of known amounts. The results are summarized in Table I.
RESULTS AND DISCUSSION T o analyze multicomponent analgesics by existing techniques of partition column chromatography, considerable time and equipment are required. In contrast, liquid chromatography involves no prior separation and requires a minimum of sample preparation. The first studies using the procedures previously reported ( 1 , 6) were made on Dupont Zipax SAX (strong (6) R. W. Roos. J. Pharm. Sci., 61, 1979 (1972)
ANALYTICAL C H E M I S T R Y , VOL. 46, NO. 14, DECEMBER 1974
Table I. Quantitative Data-Laboratory
Thenacetin, %
Caffeine, %
Aspirin, %
Standard deviation' Confidence interval" (99% level)
Prepared Samples Butalbital, 94
Found
Actual
Found
Actual
Found
Actual
Found
Actual
82.3 92.6 99.8 97.5 113.4 123.4
81.8 92.0 99.6 96.7 112.5 122.5
80.5 91.1 99.6 98.4 116.4 132.0
79.9 90.5 99.3 98.6 116.0 130.9
82.4 91.5 98.5 98.7 109.4 118.2
81.5 91.0 98.4 97.9 110.0 119.6
75.5 97.2 100.0 109.1 107.9 121.1
75.4 96.5 100.9 108.8 108.8 12 1.2
0.792 100.21
* 0.54%
0.603
2.17
99.89 i 0.43%
99.71
* 1.75%
0.969 100.27 i 0.97%
The standard deviation and confidence interval results are based on 18 individually prepared samples.
RETENTION TIME (Seconds)
Figure 3. p-Chloroacetanilide in acetonitrile. The two small peaks are due to the solvent. Same conditions as Figure 2
500 250 0 RETENTION TIME (Seconds)
Fiorinal on 30 cm X 4 mm pBondapak Cle, Acetonitrile/ 0.01% (NH&C03 in water (40:60), 1.0 ml/min, 0.16 AUFS, ambient. Injection volume 5 PI
Figure 2.
Peak identity: 1, Salicylic acid: 2, Aspirin; 3, Caffeine: 4, Butalbital: 5, Phenacetin; 6, pChloroacetanilide
anion exchange). This packing is a quaternary amine ionexchange resin mechanically coated on the surface of spherical microbeads. Under the conditions used, aspirin and butalbital are the only components retained by ionic
exchange (Figure 1). The average chromatogram was resolved in 20 minutes, and the components retained for any length of time showed a tendency toward rounding with excessive peak broadening. In addition, the column life was only about 2 months. This problem has been reported previously by other investigators (7). The ion-exchange chromatography method ( 1 ) using a weak anion exchange packing (Dupont Zipax WAX) has a major disadvantage of using a 1.5M Na2S04 buffer which, a t that concentration, could readily clog the narrow-bore tubing used in most instruments. Furthermore, the analysis time was reported to be about 25 minutes. Nonpolar, reverse-phase packings are inherently stable but must be kept below pH 8.0 to prevent the hydrolysis of the silica gel. The anion of butalbital has 16 times greater molar absorptivity a t pH 7.8 and 48 times greater molar absorptivity a t p H 11 than the neutral molecule. Therefore, ammonium carbonate was used to maintain the mobile phase a t a pH 7.8. The samples were dissolved in acetonitrile to minimize the hydrolysis of aspirin (after 4 hours a t room temperature, there was a 1%loss in aspirin). The negative peak observed in Figure 3 is due to the difference in refractive index between acetonitrile and the mobile phase and has no effect on the separation. Free salicylic acid, the breakdown product of aspirin, elutes before aspirin which, in turn, elutes before the solvent (Figures 2, 3). Salicylic acid, a t the concentration present in most products, cannot be quantified because its molar absorptivity of 254 nm is 190 times less than aspirin. The quantitative analysis for (7) R. C. Williams, D. R. Baker. and J. A. Schmit, J. Chromatogr. Sci., 11, 618 (1973).
ANALYTICAL C H E M I S T R Y , VOL. 46, NO. 14, DECEMBER 1974
2227
salicylic acid was done by a fluorimetric procedure (8). Under optimized conditions, a chromatogram was resolved in about 10 minutes. At a higher flow rate, the time for an analysis was reduced to about 8 minutes with no loss in resolution. The number of theoretical plates for the column was 1440 (equivalent to a plate height of 0.2 mm), calculated for the most retained peak, p-chloroacetanilide, with a k' of 3.21. The average time required for one complete analysis by this method was about one hour per sample compared to four hours by partition chromatography. In addition, this system has proved extremely reliable. To date, the same column has been in operation for over three months with
(8) G. H. Schenk. F. H. Boyer, C. I. Miles, and D. R. Wirz, Anal. Chem., 44, 1593 (1972).
greater than 1000 injections, and no loss in resolution has been observed.
CONCLUSIONS High-speed, reverse-phase liquid chromatography is an effective dependable, and accurate method for the separation of analgesics. Also, by utilizing an automatic sample injection system and computer interfacing, this method should allow the complete automation of the analysis of these products.
ACKNOWLEDGMENT The author thanks D. Cornish for his help in the preparation of this paper
RECEIVEDfor review April 10, 1974. Accepted August 2, 1974.
Electrochemical Determination of Arylhydroxylamines in Aqueous Solutions and Liver Microsomal Suspensions Larry A. Sternson The Bioanalytical Laboratory, Department of Medicinal Chemistry, School of Pharmacy, The University of Georgia, Athens, Ga. 30602
Certain chemical classes of drugs are converted in the body to highly toxic metabolites. Arylhydroxylamines, isolated as metabolites during enzymatic reduction of nitroaromatic compounds and bio-oxidation of primary arylamines ( I ) , are thought to initiate many toxic responses (2) including carcinogenesis (Y), metagenesis, methemoglobinemia, and hemolytic anemia. Low steady state concentrations of these intermediates and their ease of oxidation has precluded a facile procedure for their detection and quantitation. Several methods for the determination of arylhydroxylamines a t microgram levels have been described in the literature and have recently been reviewed ( 4 ) . These include spectrophotometric ( 4 , 5 ) , fluorometric (6) and gas chromatographic (7, 8) procedures. All of these methods suffer from the common disadvantage that the integrity of the hydroxylamine may be destroyed during the assay. This often prevents the quantitation of hydroxylamine in the presence of large excesses of aromatic nitro compounds and/or amines-compounds that serve as chemical and biochemical precursors to the hydroxylamines. T o detect hydroxylamines in biological fluids, the hydroxylamine must be separated from proteinaceous material by extraction and subjected to one of several rather nonspecific assays. In most cases, this has involved chemical conversion of the hydroxylamine to an amine derivative, making differentiation between hydroxylamine metabolite and amine precursors impossible. In addition, available assays are relatively time consuming. Since arylhydroxylamines are very susceptible to air oxidatlion yielding N - nitroso (1) C. C.Irving, J. Bo/. Chem., 239, 1589 (1964). (2) J. A. Miller, J. W. Cramer, and E. C. Miller, Cancer Res., 20, 950 (1960). (3) J. R. Gillette, J. R. Mitchell, and B. B. Brodie, Ann. Rev. Pharmacol., 14, 271 (1974). (4) R. E. Gammans, J. T. Stewart, and L. A. Sternson, Anal Chem., 46, 620 (1974). (5) E. Boyland and R. Nery, Analyst (London), 89, 95 (1964). (6) L. A. Sternson and J. T. Stewart, Anal. Lett., 6 , 1055 (1973). (7) H. B. Hucker, Drug Metabolism andDisposition, 1, 322 (1973). (8) A. H. Becketl and S. AI-Sarraj, J. Pharm. Pharmacol., 24, 916 (1972).
2228
compounds which are not detected by existing procedures, the accuracy of existing methods may be suspect. We wish to report the development of a rapid chronoamperometric assay for the determination of sub-microgram quantities of arylhydroxylamines directly in aqueous solutions and liver microsomal suspensions. The procedure is based on the anodic oxidation of N- substituted hydroxylamines a t a stationary carbon paste electrode. Although some studies were also conducted in human plasma, results reported herein deal exclusively with sub-fractions of liver suspensions which contain enzymes responsible for generation of transient hydroxylamine metabolites. A chronoamperometric method was chosen because it can be easily automated to accomodate multiple-sample determinations as described by Adams (9) for analysis of serum uric acid. Voltammetric behavior of phenylhydroxylamine a t dropping mercury electrodes (reduction) ( I O ) and a t the graphite electrode (reduction and oxidation) ( 1I ) have been described.
EXPERIMENTAL Instrumentation. Electrochemical measurements were made with a standard potentiostat ( 1 2 )and electrode configuration ( 1 3 ) that have been previously described. Recordings were made with an Omnigraph Model 2000 X-Y recorder. Potential scales were calibrated with a Biddle student potentiometer. All electrochemical measurements were made in all glass cells at stationary carbon paste (graphite-nujol)electrodes (geometric surface area: 0.64 cm') relative to a saturated calomel electrode with a graphite rod as auxiliary electrode. New carbon paste surfaces were prepared for each determination. Reagents. Arylhydroxylamines were synthesized by reduction (9) G. Park, R. N. Adams, and W. R. White, Anal. Lett., 5,887 (1972). (10) M. Heyrovsky and S. Vavricka, J. €lectroana/. Chem., 43, 311 (1973) and references therein. (11) L. Chuang, I. Fried. and P. J. Elving, Anal. Chem., 36, 2426 (1964). (12) B. A. Feinberg, Ph.D. Thesis, Dept. of Chemistry, University of Kansas, Lawrence, Kansas, 1971. (13) R. N. Adams, "Electrochemistry at Solid Electrodes," Marcel Dekker, New York, N.Y., 1969, p 267.
A N A L Y T I C A L C H E M I S T R Y , V O L . 46, NO. 1 4 , DECEMBER 1974
