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the postulated mechanism of action a t the molecular level in t h e treatment of cystinuria (20).
ACKNOWLEDGMENT The authors are indebted to F. F. Cantwell of the University of Alberta for his many helpful discussions, and gratefully acknowledge the cooperation of the Rheumatic Disease Unit of t h e Department of Medicine, University of Alberta, and their technician, A. Saetre, for providing numerous urine and blood samples from their patients on penicillamine therapy.
LITERATURE CITED J. M. Waishe, Am. J . Med., 21, 467 (1956). J. M. Walshe, Br. J . Hosp. Med., 4, 91 (1970). S.Sehnder, K. Crarner. and L. Hallberg. Br. J. Ind. Med., 23,282 (1966). J. C. Crawhall, E. F. Scowen and R.W. E. Watts, B r . Med. J., 1, 1270 (1973). Multicenter Trial Group, Lancet, 257 (1973). F. Bakir, S.F. Damluji, L. Arnin-Zaki, M. Murtadha, A. Khalidi. N. AI-Rawi. S. Tikriti, H. I. Chahir, T. W. Clarkson, J. C. Smith, and R. A . Doherty, Science, 181,230 (1973). G. Tisman, V. Herbert, L. Teng Go, and L. Brenner, Proc. SOC. Exp. B o / . Med., 139, 355 (1972). K. Gibbs and J. M. Walshe, 0.J . Med., 40, 275 (1971)
(9) D. Perrett, W.Sneddon and A. D. Stephens, Biochem. Pharmacal., 25, 259 (1976). (lo) D. L. Rabenstein and R. Saetre. Anal. Chem., 49, 1036 (1977). (11) J. C.Crawhail, E. F. Scowen, and R. W. E. Watts, B r . Med. J., I,141 1 (1964). (12) P. C. Jocelyn, "Biochemistry of the SH Group", Academic Press, New York, N.Y., 1972, pp 94-110. (13) Ref. 12,p 121. (14) I. M. Koithoff, W.Stricks, and R. C. Kapoor, J . Am. Chem. SOC.,77, 4733 (1955). (15) K. Bir. J. C. Crawhail, and D. Mauidin, Clin. Chim. Acta, 30, 183 (1970). (16) K. Borner. Z . Physiol. Chem., 341, 264 (1965). (17) P. R. Pal, J . Bid. Chem., 234, 618 (1959). (18) E. Jellum, V. A. Bacon, W. Patton, W. Pereira, and B. Halpern, Anal. Biochem., 31, 339 (1969). (19) E. S. K. Assem and M. R. Vickers, Postgrad. Med. J . , Suppl., 50, 10 ( 1974). (20) J. C.Crawhail, E. F. Scowen, and R.W. E. Watts, Br. Med. J . , 1, 588 (1963).
RECEIVED for review September 12,1977. Accepted November 4, 1977. This research has been supported by the University of Alberta and the National Research Council of Canada. Financial support to R.S. by a University of Alberta Scholarship is gratefully acknowledged.
Liquid Chromatographic Analysis of Analgesics on Amberlite XAD-7 Robert G. Baum and Frederick F. Cantwell" Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2G2
Amberlite XAD-7, a non-ionic polyacrylate resin, is used as a statlonary phase for the separation of mixtures of analgesics. Binary and ternary solvent mixtures prepared from chloroform, ether, ethanol, and hexane are used as mobile phase. From plots of log adjusted retention volume of each compound vs. solvent composition, the optimum solvent mixture is chosen to achleve a given separation. Commercial products containing varlous combinations of ASA, salicylic acid, caffeine, phenacetin, salicylamide, and acetaminophen are analyzed. A mlxture of all six compounds is base-line resolved on a 30 cm X 0.28 cm column. By choosing mobile phases in which separation factors are large, analgesic mixtures are also separated on short (10 cm) columns at low pressures. These systems represent a low-cost alternative to existing analytical methods.
Continued widespread use of multicomponent pain relief preparations has led to a long standing interest in improving assay methods for analgesics. Although methods based on direct spectrophotometry have been popular in t h e past ( 1 , 2 ) a n d occasionally appear in the current literature (3), chromatographic techniques constitute the bulk of recent methods. Gas chromatography has been used for the determination of analgesics without t h e formation of derivatives, but compounds such as acetylsalicylic acid (ASA), salicylic acid, and acetaminophen which possess acidic hydrogens show considerable tailing on most columns (4, 5 ) . Consequently these compounds are usually derivatized prior to gas chromatographic analysis (6). Perhaps for this reason. gas chromatography is not widely used in the analysis of multicomponent analgesics. Column partition chromatography 0003-2700/78/0350-0280$01.00/0
was used by Levine (7)for the separation of ASA, phenacetin, and caffeine with subsequent quantitation by ultraviolet spectrophotometry. This approach has been extended to more complex mixtures (&IO), and partition chromatography is the basis of some current official methods (11). High efficiency liquid chromatography has been employed more frequently in recent years. Several investigators have used pellicular anion exchangers with aqueous buffers as eluents (12-14), and one group employed a pellicular cation exchanger (15). Walton et al. (16) investigated the use of macroporous and conventional gel-type ion-exchange resins for the separation of several analgesics using electrolyte-free alcohol-water mobile phases. Caffeine, phenacetin, ASA, and acetaminophen mixtures have been separated on an octadecylsilyl bonded phase using acetonitrile-water mobile phase containing ammonium carbonate ( I 7 ) , and these four compounds plus salicylamide were analyzed on a column of pellicular silica gel using organic solvent mobile phases (18). T h e resin Amberlite XAD-7, a nonionic macroporous polymer of poly(methy1 methacrylate) (19),has previously been studied as an adsorbent. Adsorption isotherms of fatty acids (20) and the adsorption of organic contaminants from drinking water (21)have been reported. I t has been used as a stationary phase for chromatography of phenols and other acids with aqueous-organic mobile phases (22, 23) and of a variety of compounds with both aqueous and nonaqueous mobile phases (24). A similar poly(methy1 methacrylate) polymer, Hitachi Gel 3030, was used to separate aromatic hydrocarbons, dyes, and steroids (25). In the present study Amberlite XAD-7 is used as t h e stationary phase for chromatographic separation and determination of ASA, salicylic acid, phenacetin, caffeine, salicylamide, and acetaminophen in analgesic tablets. T h e chromatographic systems described herein are more rapid and 0 1978 American Chemical
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FROM CONSTANT PRESSURE SOLVENT RESERVOIRS t (
OLVENlS r i i ~
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Figure 1. Diagrams of the liquid chromatographs (descriptionin text)
efficient than traditional partition chromatography, while a t the same time they are low-cost alternatives t o higher pressure liquid chromatographic methods.
EXPERIMENTAL Apparatus. A diagram of the liquid chromatograph that was used in most of the studies is shown in Figure 14. Pump P1 (Model CMP-2VK),all valves, chromatographic columns, fittings and Teflon tubing were obtained from Laboratory Data Control, Riviera Beach, Fla. (Model numbers shown in parentheses). The water jacketed glass column C1 was either 50 cm X 0.28 cm i.d. or 30 cm X 0.28 cm i.d. (Type MB) and was dry packed with 0.82 or 0.47g of
