Simultaneous Spectrophotometric Determination in Solid Phase of Aspirin and Its Impurity Salicylic Acid in Pharmaceutical Formulations A. VILLARI*,

N. MICALI*,M. FRESTA~, AND G. P U G L I S I ~ ~

Received April 4, 1991, from the 'Dipartimento Farmaco-Chimico,UniversitS di Messina, V. le Annunziata 98010, Messina, the *lstituto di Tecniche Spettroscupiche CNR Messina, Salita Sperone 31 villagio S. A ata 98166 Messina, and the 51stitutodi Chimica Farmaceutica e Tossimlogica, UniversitS di Catanla, V. le A. Doria 6, 95725 Catania, Itat. Accepted for publication October 9, 1991.

Abstract 0 We report the simultaneous determination of aspirin and its hydrolysis product, salicylic acid, in solid phase by fluorescence spectrophotometry. Aspirin is often the most labile component in a combination-type analgesic compound. Therefore, its stability is often the initial concern in any formulation-screeningprogram. Preliminary screening of a large number of potential formulations can be arduous, because most current methods of analysis generally consist of several steps: extractions or column separations followed by UV, colorimetric, or gas-liquid chromatographic assays. The method proposed here is quite suited to large numbers of assays because it is not time consuming, it is straightforward,and it is not subject to interference from the substances present in the pharmaceutical formulations. In addition, the method is nondestructive, not dependent on the sampling procedure, and, above all, quite sensitive.

Aspirin (ASA) is an analgesic agent widely used both alone and in combination. On exposure to moisture, ASA is hydrolyzed to salicylic acid (SAX The content of SA as an impurity in pure samples of ASA in pharmaceutical formulations is limited to 0.1-3% in various pharmacopoeiae.1" Several methods have been developed for the determination of SA and ASA in pharmaceuticals and biological specimens. Among the techniques used in the past are colorimetric assays involving complexation with ferric ions,b7 diazotization with p-nitroaniline and nitrous acid,8 use of the Folin-Ciocalteu phenol reagent,g complexation with cupric ion in nitrous acid,lOJl and conversion to the nitro derivative.12 A second class of analytical methods is the group of UV-visible assays. However, by all these techniques, either SA alone or SA impurities in ASA are present after a physical separation step.lS16 Both ASA and SA have been simultaneously determined on the basis of the pH-dependent shift in their individual absorption spectra. Such a hypsochromic shiR technique results in a partial overlap of the absorptions of SA and ASA, and corrections may be required for these spectral interferences.16 Chromatographic methods have also been used for the simultaneous determination of SA and ASA: thin-layer chromatography,17.18gas-liquid chromatography (GLC),1%23 liquid chromatography (LC),24-25 and highperformance liquid chromatography (HPLC).26 With these chromatographic methods, separation of the pharmaceuticals from tablet additives (excipients and antacids) is essential. In fact, GLC methods require chemical derivatizations of ASA and SA, and separation is necessary to avoid interferences of the additives with the chemical derivatizations. Estimation of ASA and SA as the free acids by GLC20-22 is difficult because of the low vapor pressures of ASA and SA and the presence of polar functional groups, which cause absorption and tailing. Masking the functional groups by derivatization makes these molecules much less polar, more volatile, and, consequently, more amenable to GLC analysis. Among the derivatives that OO22-3 5 4 9 / 9 ~ ~ - 0 89~2.50/0 0 1992, American Pharmaceutical Association

have been quantitatively prepared are the methyl ether or methyl ester derivatives27-29 and the trimethylsilyl derivatives.30-32 However, ASA samples, when analyzed for SA as an impurity by GLC, show a higher SA content than that obtained by spectrophotometry. This result is due to the generation of SA by the slight hydrolysis of ASA during the methylation or other derivatization processes. Therefore, a column chromatographic separation of trace amounts of SA from ASA is necessary prior to methylation and GLC determination.33 The most specificquantitative methods used to date are LC estimations. With LC and HPLC methods, insoluble additives should be removed completely to prevent the column from being blocked. The extraction procedures must be carried out with great care so as not to induce hydrolysis of ASA to SA, especially when the tablets contain buffers or antacids.23 Furthermore, with LC and HPLC methods, mobile phases with methanol and water, even in small quantities, may cause hydrolysis of ASA.26 Substantial improvements in chromatographic performance may be achieved also by optimization of the sample solvent composition. In fact, in reversed-phase LC, the sample is normally prepared as a solution in water or in the aqueous mobile phase. However, it could often be more convenient to use some other miscible solvent because of stability or solubility considerations. For example, to assay ASA formulations, it is usually preferable to prepare solutions in methanol or a similar nonaqueous solvent to avoid hydrolysis. It is also important in a quantitative work to consider the effects of injection technique34 and sample solvent on peak profiles and performance. Salicylates have also been estimated fluorometrically. ASA may be tested directly or, more commonly, as SA. Determination as SA has been accomplished by direct hydrolysis or after a separation step followed by hydrolysis. Fluorometric assays in the liquid phase generally require a hydrolytic procedure and a dual determination to estimate ASA. In developing a fluorometric method for SA in ASA tablets, it was found that ASA does fluoresce, although much less intensely than does SA. This phenomenon allows direct measurement of ASA content under conditions determined to avoid any interference in the ASA fluorescenceemission band caused by the presence of an SA impurity.35 To obtain more specific fluorometric data, the fluorescencereaction of SA and its derivatives with boric acid was successfully applied to their microanalysis.36 All these methods are time consuming, complex, and/or nonspecific for the evaluation of intact ASA stability. Only a few studies have been reported about the simultaneous determination of ASA in the presence of SA by fluorometric methods in the liquid phase, and none has been published on the determination by fluorometry in the solid phase. ThereJournal of PharmaceuticalSciences I 895 Vol. 8 1, No. 9, September 1992

fore, we extended this analytical procedure to the simultaneous determination of ASA and its degradation product, SA, in solid pharmaceutical formulations. The fluorometric assays of ASA and SA in the solid phase have several advantages; the techniques are nondestructive, and the results are not dependent on the sampling methods. In combination with high sensitivity and specificity, these features result in fluorometric methods that are powerful, selective, sensitive, accurate, and worthy of further development. The data obtained by fluorescence spectrophotometry are as comparable and as reproducible as those obtained by other methods; the results are not influenced by the assay, because the substances in the formulation analyzed do not interact and the sample is not manipulated extensively.

Experimental Section ChernicalsASA (USP grade) was obtained from J. T. Baker Chemical Company, (Phillipsburg, NJ). SA crystals (USP) were purchased from Sigma Chemical Company (St.Louis, MO). To check the purity of these substances, melting points were determined and compared with those in the literature. When melting points did not agree with literature values, ASA was recrystallized twice from benzene and washed with hexane, and SA was recrystallized twice from cyclohexane. The effectiveness of the double crystallization was monitored by thin-layer chromatography with acetic acid:chloroform: methylene chloride (8:42:50,v/v/v) as eluant. To reach a finer purity control, samples were submitted to thermal analysis by differential scanning calorimetry (DSC). All the other substances used were of analytical grade. Sample Preparation-To determine the amount of fluorescent drug, alone or in a pharmaceutical formulation, we conducted experiments based on a n addition method that involved diluting the nominally pure substance with a controlled amount of impurity. We diluted the nominally pure ASA or the solid pharmaceutical formulations with exactly weighed amounts of SA. This method allows the determination of any small amount of impurity. The mixtures of ASA and SA at different concentrations were compressed by 8 tons of pressure to yield dies with diameters of 13 mm and weights of -200 mg. Analysis of the solid pharmaceutical formulations was carried out after pulverization, homogenization, and further compression of the samples. In this way, overestimation of the SA content in the ASA tablets, which would be caused by the more facile hydrolysis a t the surface of the tablet, is avoided. ApparatueDSC-ASA and SA were submitted to DSC analysis for purity control (see Chemicals). Each sample, containing -5 mg of substance, was sealed in an aluminum pan and submitted to a DSC scan in the temperature range 50-250 "C and a t a scanning rate of 5 "C/min. Calorimetric data were obtained with a Mettler TA 3000 differential scanning calorimeter equipped with a DSC 30 cell and a TC 10 processor. The plotting range, as full scale deflection, was set a t 1.71 mW. Indium was used to calibrate the temperature scale and the enthalpy change values. An empty pan was used as a reference. The presence of ASA and SA was indicated by fusion peaks centered, respectively, a t 141.1and 159.2"C. The possible presence of SA in ASA as its hydrolysis compound, even in very small quantity, decreases the fusion temperature and the enthalpy change values; furthermore, when the impurity in SA was >1%, another peak is evident, centered at a lower temperature (Figure 1). Fluorescence Method-The apparatus used for fluorescence measurement, which was assembled in our laboratories, consisted of a 200-W X e H g light source correlated with an Oriel model 7340 diffraction holographic grating monochromator with a passing band of 5 nm and a fiber optic interface. The detection system consisted of a B&M Spektronic monochromator equipped with a diode array and a Hamamatsu image amplifier. The detection instrumentation was thermostated at -30 "C. The sample holding system permits controlled and repeated setting of different samples at 45"angles (with five degrees of freedom) with a micrometer control (purchased from Microcontrol). In this equip ment, depending on the holding system, samples with different morphology (e.g., powders, films, and tablets) can be supported. This analytical method of measuring the intensity ratio of two fluorescence bands is absolutely unrelated to sample geometry, position, and physical state. We used this method to determine SA as 896 I Journal of Pharmaceutical Sciences Vol. 8 1, No. 9, September 1992

1

120

I

I

I

140

l60

180

Temperature, "C Figure 1-Typical DSC heating curves of (A) SA; ( 8 )ASA; and ASA containing (C) 1% SA, (D) 2% SA. and (E) 3% SA.

nm

Figure 2-Fluorescence spectrum of nominally pure ASA in the solid phase.

an impurity in ASA. ASA and SA present maximum excitations at 298 and 319 nm, respectively. The excitation wavelength was experimentally fixed at

330

roc

355

450

500

nm

flgum %Fluorescence spectrum of pure SA in the solid phase.

ASA Sampleb 1 2 3c 4 5 6 7 8

ooc

of SA In ASA Samples by Three Analytical

Tabb I-Rocovery M6ttlOd8

350

.00

,50

200

CJCP

Amount of SA (mg) Determined by Indicated Method' Fluorescence 0.090 (1.O) 0.100 (0.8) 0.110 (1.1) 0.200(0.9) 0.280 (0.9) 0.290(1 .O) 0.310 (0.9) 0.380(0.8)

USP

FUI

0.093 (3.2)

0.088 (3.4) 0.101 (3.5) 0.115 (3.4) 0.209 (3.3) 0.294(3.3) 0.298 (3.0) 0.315 (3.1) 0.372 (3.2)

0.096(3.1) 0.119 (3.1) 0.211 (3.2) 0.291 (3.0) 0.301 (3.2) 0.316 (2.9) 0.377 (3.0)

Values are the averages of nine measurements. Values in parentheses are CVs ("YO). bSarnple~were submitted to different storage procedures. Sample used for Figures 4 and 5.

Figure Malibration line for ASA sample 3 (Table I). The ratio varies linearly with C,:&.

/Jp

Results and Discussion The unique feature of our method is that the samples are in the solid phase. For this reason, it is not possible to have h e control of the sample area involved in the fluorescence process. Consequently, it is not possible to normalize the fluorescence amplitude from one sample to another. This problem can be resolved by the linear relation between the ratio (ZiY of the fluorescenceintensity (aftereach addition of impurity7 of the band associated with the impurity (ZJ to that of the band associated with the nominally pure compound (Z ) and the ratio (C,:Cp)of the concentration of the impurity to that of the nomnally pure substance (C 1. The fraction of impurity (a)contained in tLe pure substance can be determined with the ratio of the intercept a t the origin to the angular coefficient of the straight line obtained by linear regression of the experimental data, as previously indicated. Furthermore, when a C 1,the amount of drug can be determined from the following equation:

(6)

295 nm.To eliminate the excitation wavelength, we used a MellesGriot WG 305 filter with a cutoff of 305 nm.The fluorescence bands associated with ASA and SA were identified at 320 and 440 nm, respectively (Figures 2 and 3). No interference was observed from active substances (ascorbic acid, acetaminophen, and caffeine) and other ingredients (glycine, citric acid, sodium carbonate, sodium benzoate, cellulose, starch, and saccharin) in the formulations analyzed. Preliminary testa showed no significant variation in the precision and sensitivity of the method during a 1-week period.

In eq 1,M p is the measured amount of drug (mg) and M R is the theoretical amount (mg) of drug. The following mathematical procedure was used Knowing I,, Zi,and the impurity fraction (X,)that is nominally caused by the deliberate addition of impurity, we can calculate the real impurity fraction (XJfrom eq 2:

I f Z and Ziare, respectively, proportional to the quantities (1andX, and if the respective proportionality constants (E, and E,) are the fluorescenceefficiency values, we can write the following:

fJ

_. 300

35c

1 -

403

4 50

i

500

nm

flgure &Fluorescence spectrum of an ASA-SA mixture in the solid phase.

Then, the ratio ZiYp will be as follows: Journal of Pharmaceutical Sciences I 897 Vol, 81, No. 9, September 1992

Table IHntraday Preclsion (CV) of Assay of Various Commercial Solid Pharmaceutical Formulations of ASA

Formulation A (500mg)'

Method Fluorescence USP FUI a

ASA. mg

SA, rng

CV, YO

499.0 500.1 501.1

1 .oo

0.98 1.04

0.9 2.9 3.1

Formulation 6 (330mgy ASA, mg SA, mg CV, YO 329.2 328.3 328.9

1 .o 3.1 3.0

0.80 0.87 0.83

Formulation C (200mg)" ASA, mg

SA, rng

CV, Yo

199.5 199.4 199.1

0.50 0.48 0.52

0.8 3.3 3.4

Values in parentheses are the declared ASA contents of the commercial products analyzed.

Table IiHnterday Preclslon (CV) ofA m y of Varlous Commerclal Solid Pharmaceutical Formulations of ASA

Formulation A (500rng)"

Method Fluorescence USP FUI a

Formulation B (330mg)'

Formulation C (200 rng)"

ASA, mg

SA, mg

CV, YO

ASA, mg

SA, mg

CV, YO

ASA, rng

SA, mg

CV, Yo

499.0 499.8 501.7

1.oo 1 .oo

1 .o 3.3 3.7

329.2 328.7 328.1

0.80 0.85 0.84

0.9 3.9 3.5

199.5 199.1 199.9

0.50 0.50 0.52

1.1 4.1 3.8

0.99

Values in parentheses are the declared ASA contents of the commercial products analyzed.

Ii Ei -=-.-. Zp

1

Ep (1

- U)

Xn +-.Ei u (1 -Xn) Ep (1 - U )

References and Notes (4)

The independent variable is given by the following ratio:

X,l (1 - X,) = Cj/CP In eq 5, Ci = X,, the impurity fraction caused by deliberate addition of impurity, and C, = 1 - X,, the impurity fraction in the pure substance. We assume that C, + Ci= 1. To verify the applicability of our analytical method, we carried out replicate determinations of SA as the impurity in ASA. We analyzed eight samples of ASA containing SA in different percentages. The experimental data obtained with our spectrofluorometric method were compared with those obtained with the analytical procedures reported by the United States (USPY and Italian (FUIP pharmacopeia. The data from the three methods are in good agreement (Table I). The fluorescence spectrum of the ASA-SA mixture for which the ratio Ci(SA):CNASA) is 0.1 is shown in Figure 4. The straight calibration line for sample 3 is shown in Figure 5. The sensitivity of our analytical method was experimentally estimated to be -0.01%. The SA range in ASA tablets that can be accurately assayed is 0.01-28,and the precision of the method was constant inside this range. At higher SA concentrations, because of the saturation of the spectrum signal, it is necessary to change both the mathematical approach and the experimental apparatus. We extended our spectrofluorometric method to the determination of solid pharmaceutical formulations and compared the results with those obtained by USP2 and FUP methods. Again, the analytical results are in good agreement (Tables I1 and 111).Moreover, intraday precision and accuracy of the analytical method were determined from replicate analysis (n = 6) of the three formulations reported in Table 11. Intraday precision, expressed as coefficient of variation (CV),ranged from 0.8 to 1.0%. Interday precision and accuracy were evaluated on the basis of six determinations, on each of 6 days, of the three formulations reported in Table 111. Recision, expressed as CV, ranged from 0.9 to 1.1%. The method we have developed is not only reliable but also more sensitive and less time consuming. In fact, the amount of drug in any solid pharmaceutical formulation can be determined without extensive manipulation. 898 t Journal of Pharmaceutical Sciences Vol. 81, No. 9, September 1992

1. British Pharmacopoeia; HMSO: London, 1988;p 901. 2. U.S.Pharmucope& 22nd rev.; US.Pharmacopdal Convention: Rockville, MD, 1990;p 113. 3. Farmacopea Ufiiale ZtulW,IX ed.; Istituta Poligra6co e Zecca Dell0 Stab:Rome, 1985;Vol. 2,p 6. 4. The Pharmaceutical Codex: Pharmaceutical Press: London. 1979;pp 63-66. 5. Trinder, P. Biochem. J. 1954,57,301403. 6. Cott , V.; Zurgola, F.; Beezley, T.; Rudgers, A. J. Phurm. Sci. 196{54,868-870 7. Cid, E.; Dresse, A.; Jaminet, Fr. Pharm. Actu Helv. 1971, 46, 377482. 8. Moss, D. G. J. Clin. Pathol. 1952,5, 208-211. 9. Weinchselbaum, T. E.; Shapiro, I. Am. J . Clin. Pathol. 1945,15 (Tech. Suppl. 91,4244. 10. Mallick, S.M. K.; Rehmaun, A. Ann. Biochem. Exp. Med. 1945, 5,97-99. 11. Reid, J. Quart.J. Med. 1948,17,139-142. 12. Volterra, M.; Jacobs, M. D. J.Lab.Clin. Med. 1947,32,1282-1284. 13. Reed,R.C.; Davis, W. W. J.Phurm. Sci. 1965,54,1533-1534. 14. Jones, M.; Thatcher, R. L. Anal. Chem. 1951,23,957-960. 15. Levine, J.; Weber, J. D. J. Phurm. Sci. 1968,57,631-633. 16. Clayton, A. W.; Thiers, R. E. J. Pharm. Sci. 1966,55,404-406. 17. Cummings, A. J.; King, M. L. Nature 1966,209,620-623. 18. Morrison, J. C.; Orr, J. M. J.Phurm. Sci. 1966,55,936-941. 19. Walter, L. J.;Biggs, D. F.; Couta, R. T. J.Phurm. Sci. 1974,63, 1754-1758. 20. Nikelly, J. G. Anal. Chem. 1964,36,2248-2250. 21. Dechene, E.B.; Booth, L. H.; Caughey, M. J. J.Pharm. Pharmucol. 1969,21,678-680. 22. Patel, S.; Perrin, J. H.; Windheueer, J. J. J.Phurm. Sci. 1972,61, 1794-1796. 23. Galante, R.N.; Egoville, J. C.; Visalli, A. J.; Patel, D. M. J. Pharm. Sci. 1981,70,167-169. 24. Stevenson, R.L.; Burtis, C. A. J.Chromutogr. 1971,61,253-261. 25. Baum, R. G.; Cantwell, F. F. J.Phurm. Sci.1978,67,1066-1069. 26. Gupta, V. D.J.Phurm. Sci. 1980,69,113-115. 27. Morris, C. H.; Christian, J. E.; Laudolt, R. R.; Hansen, W. G. J. Pharm. Sci. 1970,59,270-271. 28. Watson, J. R.;Cresculo, P.; Mataui, F. J.Pharm. Sci. 1971,60, 454-458. 29. Ali, S.L. J. Chromatom. 1976.126,651-663. 30. Horii, Z.; Makita, M.; 'hkeda, I.; Tamura, Y.;Ohuishi, Y. Chem. Phurm. Bull. 1965,13,636-638. 31. Rowland, M.;Riegelman, S.J. Pharm. Sci. 1967,56,717-720. 32. Walter, L. J.;Biggs, D. F.; Coutta, R. T. J.Pharm. Sci. 1974,63, 1754-1758. 33. Ali, S.L. Chromutogmphia 1973,6,47WO. 34. Williams, K. J.; Li Wan Po, A.; Irwin, W.J. J.Chromutogr. 1980, 194,217-223. 35. Miles, C. I.; Schenk, G. N.Anal. Chem. 1970,42,656-659. 36. Shibazaki, T.;Nishimura, T.;Hara, M.; IUima, T. Chem.Phurm. BULL. 1978,26,1737-1745.