Determination of Aspirin by Pre-Column Transacetylation Reaction of 3-Aminophenol and Reversed-Phase High-Performance Liquid Chromatography: Simultaneous Determination of Aspirin, Acetaminophen, and Caffeine

Determination of Aspirin by Pre-Column Transacetylation Reaction of 3-Aminophenol and Reversed-Phase HighPerformance Liquid Chromatography: Simultaneous Determination of Aspirin, Acetaminophen, and Caffelne KRISHNAK. VERMA', SUNIL K. SANGHI, ARCHANA JAIN, AND DAYASHANKER GUPTA Received December 5, 1986, from the Department of Chemisfry, Rani Durgavati University, Jabalpur 482001, India. Accepted for publication April 21, 1987.

0 The accuracy of the measurement of aspirin by highperformance liquid chromatography (HPLC) is reduced by its hydrolysis into salicylic and acetic acids during sample preparation. The ready and quantitative transacetylation of 3-aminophenol by aspirin, giving 3hydroxyacetanilide (which is stable), has been utilized as a pre-column reaction for the determination of aspirin either alone or in the presence of acetaminophen and caffeine by reversed-phase HPLC. Abstract

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Reversed-phase high-performance liquid chromatography (HPLC) provides a convenient method for the simultaneous assay of aspirin (acetylsalicylic acid) and other active ingredients,1-5 but hydrolysis of aspirin into salicylic and acetic acids can limit the accuracy of the determination.C8 This degradation reaction occurs under the anchimetric assistance of the ortho carboxyl group and is catalyzed by the presence of moisture or basic substances and other materials used in aspirin products.a10 Though salicylic acid remains a valuable indicator of aspirin degradation, recent studiesll-'" have confirmed that under certain conditions a large number of other products can be formed. Reported means of correcting errors from this source include separation of aspirin from product ingredients t h a t promote degradation,lb injection of sample solution without appreciable delay after its preparation,16 use of standards with matching amounts of aspirin,'? extrapolation of results back to time zero,l0 and selection of solvents to minimize salicylic acid formati0n.~.9,15 The aim of the present work is to make use of t h e chemical reactivity of the acetyl group of aspirin as a pre-column reaction in a direct approach to its determination. I t has been found that aspirin quickly transacetylates 3-aminophenol producing 3-hydroxyacetanilide, which is a stable compound and can be determined by reversed-phase HPLC without any interference from salicylic acid a n d other ingredients of drug formulation. Other aromatic primary amines can also be employed for the transacetylation reaction, but 3-aminophenol is preferred because of its faster elution and its chromatographic behavior, which is close to but distinct from that of acetaminophen (4-hydroxyacetanilide). The proposed method can be used for the simultaneous determination of aspirin, acetaminophen, and caffeine.

Experimental Section Apparatus-A Shimadzu LC-5A solvent delivery pump was used with a Shimadzu SPD-PA ultraviolet detector and a manual loopvalve injector (10-pL loop). The detector signal waa integrated and recorded with a Shimadzu C-R2AX integrator. The 250 x 4.6-mm Zorbax ODS column contained 5-pm octadecylsilane as the stationary phase. The peak area was used for quantitation. Reagents and Chemicals-Aspirin (acetylsalicylic acid), acetaminophen, and caffeine were of USP grade, 3-aminophenol and 0022-3549/87/0700-055 1$01.00/0 0 1987,American Pharmaceutical Association

anhydrous ethanol were of reagent grade, and methanol was HPLC grade. 3-Hydroxyacetanilide was obtained from Sigma Chemical Co. Other chemicals were of analytical reagent grade. The mobile phase was prepared by dissolving 17.7 g of potassium dihydrogen phosphate dihydrate in 700 mL of deionized distilled water, mixing with 300 mL of methanol, and adjusting the pH to 3.60 * 0.05 with concentrated HCI. The solution was filtered through a 0.45-pm membrane filter. Solution StandardpAspirin-An accurately weighed 10-mg portion of aspirin was mixed with a solution of -20 mg of 3-aminophenol in 5 mL of anhydrous ethanol in a 10-mL round-bottomed flask fitted with a reflux water condenser. The contents were shaken to dissolve the solid, and refluxed gently over a boiling water bath for 15 min. The cooled solution was diluted to 25 mL with methanol in a calibrated flask. Acetaminophen and Caffeine-A mixed standard was made by dissolving accurately weighed amounts of acetaminophen (5 mg) and caffeine (2 mg) in 25 mL of methanol in a calibrated flask. A mixed solution standard was made by mixing a 200-pL aliquot each of aspirin, acetaminophen, and caffeine solutions in a 10-mL calibrated flask and diluting to volume with mobile phase. Conditions for Chromatographic Separation and Quantitation-The mobile phase was pumped through the ODS column at a flow rate of 1 mL.min-' (pressure = 50 kgcm-?) until a stable baseline was obtained. For the standard solutions and the analysis of aspirin, acetaminophen, and caffeine in pharmaceuticals, replicate 10-pL idections of each solution were made using a loop injector. The eluate was passed through a UV detector set at 240 nm. Tablet Formulation Assay-A known number of tablets were weighed and triturated to a fine powder. An accurately weighed aliquot equivalent to 10 mg of aspirin was mixed with a solution of 20 mg of 3-aminophenol in 5 mL of anhydrous ethanol. The contents were shaken for 5 min, refluxed for 15 min, and the cooled mixture was centrifuged at 3000 rpm. The suspension was filtered through a 0.45-gm membrane filter, and the residue was washed with three 3mL portions of methanol. The filtrate and washings were collected in a 25-mL calibrated flask and made up to volume with methanol. A 200-pL aliquot of this solution was diluted to 10 mL with the mobile phase, and 10 pL (or more for caffeine analysis) was injected by the loop iqjector into the liquid chromatograph.

Results and Discussion Aspirin has been assayed by nonaqueous alkalimetric titration,17J8 reaction with nitrous acid followed by alkali treatment to give a color which is measured,'g and bromimetry.?O These general reactions cannot be used to assay aspirin in the presence of salicylic acid or in its compound tablets. Spectrophotometry,21.22fluorimetry,lO GC,2%25 and room-temperature phosphorimetry26have been used in many methods, and 13C NMR27 a n d 'H NMR28 determination of aspirin, phenacetin, and caffeine mixture have been described. However, HPLC2Qremains a convenient method for monitoring aspirin simultaneously with other active ingredients of the formulation. To overcome the stability problems, especially in aqueous Journal of Pharmaceutical Sciences / 551 Vol. 76,No. 7,July 1987

Preliminary studies showed that the best separation was achieved on an ODS column by a 30:70 (v/v) mixture of methano1:water. Adjustment to pH 3.6 decreased the retention time of unused 3-aminophenol and avoided overlapping with the acetaminophen peak. Acetanilide and its derivatives absorb strongly near 238 nm.30The absorption spectra

OH

COOH

B NHAC

C OOH

OH

or partially aqueous solvents, aspirin has been subjected to a pre-column reaction with 3-aminophenol during which 3hydroxyacetanilide is formed by transacetylation. The possibility of acetylation of the hydroxyl group is only remote since the amino group is a more efficient nucleophile, and in the proposed method, the mass of 3-aminophenol is double the mass of aspirin. 3-Hydroxyacetanilide was confirmed as the product of transacetylation in the following manner. An authentic sample of 3-hydroxyacetanilide and the reaction product of aspirin and 3-aminophenol had the same HPLC retention times. These compounds of corresponding retention times had identical ultraviolet and infrared spectra. Also, the agreement (within ? 3%) between the peak areas of equivalent molar masses of authentic 3-hydroxyacetanilide and of aspirin (after transacetylation) confirmed the quantitative nature of the reaction. The transacetylated solution was found to be stable for several days.

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Figure 1 High-performance ljquid chromatogram of (A) 3-aminoacefaminophen; (C) 3-hydroxyacetanilide (from aspirin);and phenol; (6) (D) caffeine. Row rate, 1 mL.min- UV detection at 240 nm with detector sensitivity of 0.02 AUFS.

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Table CHlgh-Performance Liquid Chromatographic AnalySl8 of Aspirin, Acetaminophen, and Caffeine Mixtures ~

Mixture Composition, mg"

Mixture No. _____

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1 2 3 4 5 6

Found by HPLC, mgb

Aspirin

Acetaminophen

Caffeine

Aspirin

Acetaminophen

Caffeine

10.6 22.4 39.8 113.1 56.5 67.9

50.2 41.7 86.3 60.7 150.2 101.5

13.5 29.6 45.1 25.8 33.5 14.2

10.7 22.1 39.5 112.5 56.9 67.1

50.0 41.5 86.0 61.2 150.8 102.1

13.4 29.8 45.4 25.1 33.9 14.4

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"The weights were accurate to 20.2 mg. From the pooled data, the relative standard deviation (7') was found to be 1.8 for aspirin, 1.3 for acetaminophen, and 2.1 for caffeine. Table iCDetermination of Asplrln, Acetaminophen, and Caffeine in Various Drug Formulations Amount Found, mgltabiet Tablet

Present Method' Aspirin

Micropyrin" Disprin Anacin Coldarind Painkill' Panjon" Panbine

345 (1.9) 362 (1.9) 412 (15) 581 (1.9) 308 (1.a) 350 (1.7) 326 (2.0)

Acetaminophen

Comparison Method9 Caffeine

-

19 (2.1)

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31

143 (1.3) 141 (1.1) 131 (1.7)

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(14

28 (1.6) 28 (1.8) 27 (2.1) 31 (1.9)

Aspirin

Acetaminophen

348 (22) 357 (4) 408 (4) 579 (22) 302 (3) 355 (4) 321 (4)

146 (32) 140 (4) 135 (32)

Caffeine 21 (3)

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29 (3) 28 (4) 30

(3)

26 (4) 29 (4)

'Aspirin (300 mg)and caffeine (20 mg). bAspirh (350 mg), calcium carbonate (105 mg), and anhydrous citric acid (35 mg). 'Aspirin (400 mg) and caffeine (30 mg). dAspirin (600 mg), caffeine (30 mg). phenylephrinehydrochloride(10 mg), terpin hydrate (30 mg), and calcium carbonate (200 mg). 'Aspirin (300 mg), acetaminophen (150 mg), and caffeine (30 mg). 'The relative standard deviation (%) is given in parentheses (n = 6). 9The reference number of comparison method is given in parentheses. 552 / Journal of Pharmaceutical Sciences Vol. 76,No. 7,July 1987

of purines are very sensitive to changes in the pH of the medium;31 in the mobile phase used in this work, caffeine exhibits a band around 220 nm and another at 262 nm. Acetaminophen, 3-hydroxyacetanilide, and caffeine all absorb significantly at 240 nm. Under these conditions, a mixture of aspirin (pre-column reacted with 3-aminophenol), acetaminophen, and caffeine produced the chromatogram shown in Figure 1. Calibration standard solutions of aspirin (reacted with 3aminophenol), acetaminophen, and caffeine were prepared and the following quantities of each compound were injected: 0.2,0.4,0.6,0.8,1,2,3,4, and 5 pg. A plot of peak area versus ~ the drugs, with the amount injected was linear up to 5 l z for correlation coefficients of 0.999 or better. Solution standards were doped with pharmaceutical excipients and subjected to HPLC analysis. In all cases, satisfactory recoveries and reproducibility of peak areas were obtained. No interference due to excipients was detected in the chromatogram produced. Results for the analysis of mixtures of aspirin, acetaminophen, and caffeine are shown in Table I; six solutions, prepared in the same way as the standards and with the given concentration per 100 mL, were tested. Results of the determination of drugs in their pharmaceutical formulations are presented in Table 11.

References and Notes 1. Ascione, P. P.; Chrekrian, J . P. J. Phurm. Sci. 1975,64,1029. 2. Gupta, V. D.J. Pharm. Sci. 1980,69,110. 3. Jalal, I. M.; Sa'sa', S. I. Talanta 1984.31,1015. 4. Bevitt, R. N.; Mather, J. R., Sharman, D. C. Analyst 1984,109, 1327. 5. Yang, S.L.; Wilken, L. 0.;Clark, C. R. Drug Dev. Znd. Pharm. 1985,11, 799. 6. Flore , K. In Analytical Profiles o Drug Substances; Florey, K., Ed.; icademic: New York, 1979; 01. 8, 1. 7. Connors, K. A.; Amidon, G. L.; Kennon, Chemical Stability of Pharmaceuticals; Wiley: New York, 1979;p 151.

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f.

8. Kelley, C. A. J. Phurm. Sci. 1970,59, 1053. 9. Bundgaard, H.Arch. Pharm., Chem. Sci. Ed. 1976,4,103. 10. Baum, R. G.;Cantwell, F. F. J.Pharm. Sci. 1978,67,1066. 11. Kirchhoefer, R. D.;Reepmeyer, J. C.; Juhl, W. E. J.Phurm. Sci. 1980,69,550. 12. Taguchi, V.Y.; Cotton, M. L.; Yaks, C. H.; Millar, J. F. J. Pharm. Sci. 1981,70,64. 13. Mroso, P.V.;Li Wan Po, A.; Irwin, W. J. J . Phurm. Sci. 1982,71, 1096. 14. Reepmeyer, J. C.J.Phurm. Sci. 1983,72,322. 15. Galante, R. N.; Visalli, A. J.; Grim, W. M. J. Phurm. Sci. 1984, 7 .1-1, -195 --. 16. Kirchoefer, R.D.; Juhl, W. E. J.Phurm. Sci. 1980,69,548. 17. Lin, S.-L.; Blake, M. I. Anal. Chem. 1966,38,549. 18. Walash, M. I.; Agrawal, S. P.; Martin, M. I. Can.J. Pharm. Sci. 1972,7,123. 19. Belal, S.F.; Elsayed, M. A. H.; Elwalily, A.; Abdine, H. Analyst 1979,104,919. 20. Boudeville, P.;Burgot, J. L.; Chauvel, Y.Analusis 1983,1I,406. 21. Harris, P.A.; Riegelman, S. J. Phurm. Sci. 1967,56,713. 22. Verma, K. K.;Jain, A. Anal. Chem. 1986,58,821. 23. Hoffman, A.J.; Mitchell, H. I. J. Phurm. Sci. 1963,52,305. 24. Nicker1 , J. G. Anal. Chem. 1964,36,2248, 25. Tam, J K . ; Au, D.S.L.; Abbott, F.S. J. Chromutogr. 1979, 1974,239. R. P.;Winefordner, J. D. J.Pharm. Biomed. Anal. 1983, 26. Bateh, * 4,"

1 . llJ.

27. Chiang, H. C.; Imanari, M. T'ai-wan Yao Hsueh Tsa Chih 1981, 32,83;Chem. Abstr. 1982,96,24,862r. 28. Eberhart, S.T.; Hatzis, A.; Rothchild, R. J. Pharm. Biomed. Anal. 1986,4, 147. 29. Hamilton, R.J.;Sewell, P. A. Introduction to Hi h Pe orrnance Li uid Chromatography, 2nd ed.;Chapman and hail: &w York, 1912:D 201. 30. Rao,'c. N. R. Ultraviolet and Visible S ctroscopy: Chemical Applications, 3rd Ed.; Butterworths: Longn, 1975;p 87. 31. Dyer, J. R. A plications o Absorption S ctroscopy of Organic Compounds;f;entice-HalL En l e w d Cfp,, NJ, 1974;p 18. 32. Sa'sa', S.; Rashid, A.; Jalal, I. falanta 1984,31,397.

Acknowledgments Thanks are due to the Council of Scientific and Industrial Research, New Delhi, for the award of a Senior Research Fellowship to A. J.

Journal of Pharmaceutical Sciences / 553 Vol. 76, No. 7, July 1987