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Flow injection spectrophotometric determination of acetylsalicylic acid in tablets after on-line microwave-assisted alkaline hydrolysis
Airton Vicente Pereira, Clezio Aniceto and Orlando Fatibello-Filho* Grupo de Qu mica Anal tica, Departamento de Qu mica, Centro de Ci encias Exatas e de Tecnologia, Universidade Federal de S ao Carlos, Caixa Postal 676, CEP 13.560-970, S ao Carlos, SP, Brazil. E-mail: doff@power.ufscar.br The proposed method is based on the on-line microwave-assisted alkaline hydrolysis of acetylsalicylic acid (ASA) to salicylic acid (SA) that reacts with FeIII to form a complex that absorbs at 525 nm. Samples merged with NaOH were passed continuously through a domestic microwave oven in order to accelerate the hydrolysis of ASA. Under the best analytical conditions, the linearity of the calibration equation for ASA ranged from 25 to 250 mg ml21. The precision for ten successive measurements of 200 mg ml21 ASA presented a relative standard deviation of 0.40%. The detection limit was 4.0 mg ml21 and recoveries of 99.1101.0% were obtained for ASA. No interference was observed from the common excipients of tablets and other active substances such as ascorbic acid and caffeine. The proposed FI method is adequate for a large number of samples because it is not time consuming and permits the determination of ASA in 90 samples per hour. Keywords: Acetylsalicylic acid; flow injection; microwave-assisted hydrolysis; spectrophotometry Microwave oven pre-treatment of samples has gained widespread application in different areas and samples.1720 However, few applications in pharmaceutical analysis have been reported.21 FIA combined with microwave-assisted alkaline hydrolysis has been applied to the spectrophotometric determination of paracetamol in pharmaceuticals.22 On-line microwave-assisted alkaline hydrolysis of vitamin A to retinol and its detection at 325 nm was also reported.23 This paper reports the application of a FI spectrophotometric method for determination of ASA using a microwave-assisted hydrolysis incorporated in the system in order to minimize the time of analysis and increase the sensitivity of the method. In the method presented, it is unnecessary to perform two determinations, one before and one after hydrolysis, to obtain a value for the ASA concentration in the samples. The proposed method is based on the on-line microwave-assisted alkaline hydrolysis of acetylsalicylic acid to salicylic acid that reacts with FeIII in acid medium to form a complex that absorbs at 525 nm. Experimental Apparatus Acetylsalicylic acid (ASA) is widely used in the relief of headaches, fever, muscular pains and inflammation due to arthritis or injury. In solution, the rate of decomposition of ASA to salicylic acid (SA) is dependent on the pH. At pH 1112, ASA is immediately hydrolysed; at neutral and acid solution (pH 48), the hydrolysis rate is slow, and the maximum stability is attained at pH 23.1 The conventional back-titration method2 for ASA determination is simple and economical, but requires heating over reflux for 10 min. It has been replaced by HPLC in the more recent editions of USP.3 UVVIS spectrophotometry,46 fluorimetry,7,8 amperometry9 and HPLC1013 methods were reported for the determination of ASA in pharmaceuticals. HPLC is preferred over other methods because of the possibility of simultaneous determination of ASA and SA. However, this technique is time-consuming and requires sophisticated equipment. Juhl and Kirchhoefer14 developed a semiautomated-UV detection method for determination of ASA in tablets but a complex system was used because extraction with chloroform was necessary. The FeIIIsalicylate reaction has been used for quantitative determination of SA in ASA samples and appropriate conditions for the reaction were established. The maximum colour intensity was obtained at pH 2.53.5.15 Lopes-Fernandez et al.16 proposed an asymmetrical FIA with dual injection for the simultaneous determination of SA and ASA, which is online hydrolysed to SA in a longer channel and two peaks were obtained by complexation between FeIII and SA and the resulting coloured product was measured at 520 nm. The linear range for ASA varied from 300 to 1800 mg ml21 and 30 samples were analysed per hour. The flow injection manifold is shown in Fig. 1. A Panasonic 600 W (Manaus, Brazil) model NN 5556 B domestic microwave oven equipped with a magnetron of 2.450 MHz was used. A twelve-channel Ismatec (Zurich, Switzerland) model 7618-50 peristaltic pump supplied with Tygon pump tubing was used to pump all solutions. The manifold was constructed with polyethylene and PTFE tubing (0.8 mm id). Sample and reagent solutions were injected manually into the carrier stream using a laboratory-constructed three-piece injectorcommutator made of Perspex, with two fixed side bars and a sliding central bar, that is moved for sampling and injection. A Femto (S ao Paulo, Brazil) spectrophotometer model 435 equipped with a glass flow-cell (optical path 1.0 cm) was used for the absorbance measurements. Peaks were recorded using a Cole Parmer (Chicago, IL, USA) two-channel strip-chart recorder model 1202-0000. Reagents and solutions All reagents used were of analytical-reagent grade and all solutions were prepared with water obtained from a Millipore (Bedford, MA, USA) Milli-Q system (model UV Plus UltraLow Organics Water). A stock solution of ASA (2.0 mg ml21) was prepared by dissolving 500 mg of ASA (Synth) in 250 ml of water. Solutions of desired concentrations were obtained by diluting the stock solution with water. All solutions to be analysed were prepared just before injection into the FIA system in order to avoid ASA hydrolysis to SA. The solution containing 0.50% (w/v) ferric nitrate in 0.4 mol l21 nitric acid was prepared by adding Fe(NO3)39H2O

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(Riedel, Hannover, Germany) and an appropriate volume of concentrated nitric acid to a 500 ml calibrated flask and diluting to volume with water. Methods Analysis of pharmaceutical samples To prepare commercial samples, a known number of tablets were ground to a fine power and an accurate mass corresponding to about 500 mg of ASA was transferred to a 250 ml Erlenmeyer flask and stirred with about 200 ml of water. These solutions were filtered through a filter paper into a 250 ml calibrated flask and made up to volume with water. This solution was used for determination after dilution with water to the desired concentration. A volume of 250 ml of sample was injected immediately into the carrier stream to prevent errors due to the conversion of ASA into SA. Reference method In order to compare the results obtained by the FI procedure, the back-titration method described for aspirin tablets in the British Pharmacopoeia2 was carried out with minor modifications. An accurate amount of powder from the tablets was treated with 0.5 mol l21 NaOH and heated in a microwave oven for 3 min at lower power level to ensure complete hydrolysis and to avoid boiling. After cooling, the solution was titrated with standardized 0.501 mol l21 hydrochloric acid solution, using phenolphthalein as indicator. Flow injection procedure The flow injection system for on-line ASA hydrolysis is shown in Fig. 1. When the central bar of the injector is moved to the injection position, sample (L1, 250 ml) and R1 reagent (0.2 mol l21 NaOH; L2, 250 ml) are injected as individual zones into the water carrier streams (C1 and C2; both flowing at 3.4 ml min21) which merged downstream synchronously. At confluence point x, the water carrier streams merged so that alkaline hydrolysis of ASA occurs in the PTFE tube reaction coil B1 (200 cm, 0.8 mm id) placed inside the microwave oven turned on at the maximum power level (600 W). For safety reasons, these PTFE tubes were passed through the walls of the microwave oven using the external oven air vents. The hydrolysed sample passed through the PTFE coil B2 (100 cm, 0.8 mm id), immersed in a water bath. To avoid the presence of bubbles in the detector, produced during the passage of the sample in the coiled reactor inserted in the microwave oven, a T-shaped glass debubbler (aspiration rate, 0.2 ml min21) was incorporated between the water bath and the confluence point y. Then, the sample zone is mixed with the R2 reagent [0.50% (w/v) Fe(NO3)3 in 0.4 mol l21 nitric acid, flowing at 1.3 ml min21] in the reactor coil B3 (100 cm, 0.8 mm id). The

absorbance resulting from the FeIIIsalicylate complex, measured at 525 nm, is proportional to the ASA concentration of the sample. To avoid damage in the magnetron a beaker containing 500 ml of water was placed inside the microwave cavity. The volume of water that evaporated during the hydrolysis step was continuously replaced by pumping water in at a flow rate of 4.8 ml min21 (C3 channel). Results and discussion ASA can be hydrolysed rapidly in an alkaline medium to its constituent salicylic and acetic acids.1 In the conventional analytical procedure,2 complete hydrolysis is attained after 10 min heating at reflux, followed by back-titration with standardized acid solution. After hydrolysis the SA content is proportional to the ASA concentration present in the sample. SA can be quantitatively determined by complexation with FeIII ions and the resulting colour read at 525 nm. In this work, a domestic microwave oven was exploited as the energy source to accelerate the hydrolysis of ASA (about 20 s) in order to minimize the time of analysis and increase the sensitivity of the detection. Study of chemical parameters Initially, the FI manifold was used to investigate the conditions of ASA hydrolysis. In all preliminary experiments, the coil lengths of B1, B2 and B3 were 300, 200 and 100 cm, respectively. The L1 and L2 loop volumes were 250 ml and fixed flow rates of 3.0 ml min21 were used for C1 and C2 and 3.9 ml min21 for R2 reagent. The maximum power (600 W) of the microwave oven was used. The acidity of the R2 reagent is important because the nitric acid concentration should be sufficient to neutralize the sample zone and to give an acid pH in which the reaction between FeIII ions and SA occurs. Increment of NaOH (R1 reagent) concentration requires an increase in the R2 reagent acid concentration. The effect of the alkalinity on the baseline stability was studied replacing the sample (L1) by water. Within the concentration range 0.050.25 mol l21 NaOH studied no baseline drift was observed maintaining a concentration ratio of 2 : 1 (HNO3, R2 : NaOH, R1). A ratio lower than 2 : 1 could not be used because it caused baseline noise, due to incomplete neutralization of the injected alkaline zone. The effect of NaOH concentration (R1 reagent) on the ASA hydrolysis was evaluated over the concentration range from 0.05 to 0.25 mol l21. In this experiment, the nitric acid concentration of R2 reagent [1% (w/v) Fe(NO3)3] was twice that of NaOH (R1). ASA standards in the concentration range from 50 to 400 mg ml21 were used. Fig. 2 shows that the absorbance intensity increased with an increase in NaOH concentration up to 0.20 mol l21, above which it increased only slightly. In this

Fig. 1 Schematic diagram of the FI system for spectrophotometric determination of ASA. The three rectangular pieces represent a scheme of the sliding bar injector. S, sample or standard solutions; L1, sample loop (50 cm, 250 ml); L2, R1 reagent (0.2 mol l21 NaOH) loop (50 cm, 250 ml); C1, C2 water carriers streams, flowing at 3.4 ml min21; C3, water auxiliary channel pumped at 4.8 ml min21; B1, B2 and B3, reactor coils (200, 100 and 100 cm, respectively); R2 reagent solution, 0.5% (w/v) Fe(NO3)3 in 0.4 mol l21 nitric acid, flowing at 1.3 ml min21; Db, glass T-debubbler, aspiration at the top 0.2 ml min21; D, spectrophotometer at 525 nm; x and y, confluence points; W, waste. The numbers in parentheses are the flow rates in ml min21.

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work, a 0.2 mol l21 NaOH solution and Fe(NO3)3 in 0.4 mol l21 nitric acid solution were chosen for the R1 and R2 reagents, respectively. Study of microwave oven conditions The efficiency of hydrolysis of the ASA is influenced by the microwave oven power level (Fig. 3). The microwave oven has five power levels corresponding to 60600 W output. Increasing the power level (60600 W) gave continuous increase in the sensitivity followed by irreproducible results (probably due to the pulsed way that the energy is supplied by the magnetron) was observed up to 420 W. The best hydrolysis efficiency and more reproducible results were obtained using maximum power (600 W). However, when maximum power was used bubble formation occurred and caused problems in the measurements. Efficient removal of the bubbles generated during the microwave-assisted hydrolysis was obtained when a T-debubbler was incorporated in the FI system maintaining a higher sensitivity. Therefore, a maximum power level was selected for this work. To obtain reproducible results, it is recommended that sample injections are started 5 min after turning on the microwave oven.

Study of FI parameters The effect of the reactor coil length B1 placed inside the microwave was studied between 100300 cm using PTFE tubing (0.8 mm id) and maintaining constant all other parameters as described above. Increasing the length of B1 coil, a continuous increase of the sensitivity was observed (Fig. 4).

There was an increase of only 15% in sensitivity when B1 coil length was increased from 200 to 300 cm. Therefore, a length of 200 cm was chosen as a compromise between sensitivity and sample throughput. The effect of the sample and R1 reagent volumes (L1 and L2) was studied by changing the lengths of L1 and L2 loops in the manual injector. The volumes were varied equally between 62.5 and 312.5 ml. The absorbance was found to increase with the sample volume up to 250 ml, without meaningful increase to 312.5 ml. Thus, 250 ml volumes were chosen for the sample and R1 reagent. The influence of the reactor coil lengths B2 and B3 on the sensitivity of the FI method was studied in the range 100300 and 50200 cm, respectively. The results showed that the absorbance decreased with increasing length due to the dispersion of the sample zone. A length of 100 cm was chosen for B2 in order to minimize the dispersion and maintain an efficient cooling time. A length of 100 cm was chosen for B3 in order to obtain ideal mixing of the hydrolysed sample with Fe(NO3)3 solution in 0.4 mol l21 nitric acid and attain an appropriate acid medium for the solution flowing through the detector. The C1 and C2 water carrier flow rates were optimized by varying simultaneously these streams while keeping constant the flow rate of R2 reagent stream at 3.9 ml min21. When flow rates were increased from 1.5 ml min21 to 4.0 ml min21 it was found that the sensitivity increased up to 3.4 ml min21, above which it showed a slight decrease. Therefore, flow rates of 3.4 ml min21 were selected for both C1 and C2 water carrier streams. The effect of flow rate of ferric nitrate reagent solution on sensitivity was studied using 1.0% (w/v) Fe(NO3)3 in 0.4 mol l21 nitric acid and varying the flow rate from 1.0 to 4.0 ml min21. A decrease was observed in the sensitivity with an increase of the flow rate probably due to a decrease in the final pH. The intensity of absorption of the FeIIIsalicylate complex depends on the pH value and it has been reported that maximum intensity occurs at pH 2.53.0 and is five times higher than that at pH 1.5.15 In all further experiments, a flow rate of 1.3 ml min21 was selected for the R2 reagent. No baseline drift was observed for blank runs (injecting water instead of ASA solution). Effect of the Fe(NO3)3 concentration The influence of the Fe(NO3)3 concentration was studied in the range 0.125 to 1.0% (w/v). The results showed that the absorbance signals corresponding to 250 mg ml21 ASA increased with increasing concentration of the R2 reagent up to

Fig. 2 Effect of the NaOH concentration (R1 reagent; L2, 250 ml) on the determination of ASA (200 mg ml21; L1, 250 ml) using 1% (w/v) Fe(NO3) in 0.4 mol l21 nitric acid (R2 reagent flowing at 3.9 ml min21).

Fig. 3 Effect of the microwave power (%) on the efficiency of hydrolysis of ASA (400 mg ml21) using NaOH (0.2 mol l21). Reactor coils B1, B2 and B3 were 300, 200 and 100 cm, respectively.

Fig. 4 Effect of the reactor coil B1 length (PTFE tubing, 0.8 mm id) placed inside the microwave oven on the hydrolysis of ASA (400 mg ml21) with reactor coils B2 and B3 of 200 and 100 cm, respectively. The other variables were as described in Fig. 2.

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0.50% (w/v), above which it remained constant. Consequently, a 0.50% (w/v) Fe(NO3)3 in 0.4 mol l21 nitric acid was chosen. Interferences and recovery studies The selectivity of the proposed method was evaluated by studying the effect of common excipients used in pharmaceutical preparations and other active substances on the determination of 200 mg ml21 ASA. No interference in the flow procedure was observed up to a tenfold excess of caffeine, lactose, starch, sodium carbonate, sucrose, saccharin and glucose. However, when 2.0 mg ml21 citric acid was added, the absorbance decreased about 20% because of the complexation of FeIII ions by this compound. Since FeIII is reduced to FeII by ascorbic acid, a negative interference could be expected, but ascorbic acid is easily decomposed at high pH even at room temperature and produced a slight error in the determination only when present in concentrations four times higher than ASA. The limit of free SA that could be present in ASA samples due to hydrolysis was tested by adding standard salicylic acid solution to ASA samples and results compared to unspiked standard solution. ASA samples added with salicylic acid up to 0.3% (w/v) did not cause any interference in the proposed FI procedure. Otherwise the content of SA as an impurity in plain tablets is limited to 0.10.3% w/v. Thus, SA content correction is not needed. Additionally, the effect of the interference caused by SA was tested by processing ASA samples similarly but without the hydrolysis step [replacing NaOH (L2) by water]. No AS signals were obtained even at the lower acidity of R2 reagent 0.50% (w/v) [Fe(NO3)3 in 0.2 mol l21 nitric acid]. Therefore, the interference from SA in the ASA determination was negligible for all samples analysed. The accuracy of the proposed flow injection method was evaluated by analysing tablets spiked with known amounts of aspirin. In this study, 50 100 and 150 mg ml21 of ASA were added to each tablet sample solutions (Table 1). Recoveries of 99.1101.0% of ASA from four commercial tablets (n = 5) were obtained using the FI procedure. This is good evidence for the accuracy of the proposed FI procedure. Calibration graph and applications In order to demonstrate the applicability of the proposed FI method to commercial samples it was applied to the determination of ASA in tablets. Fig. 5 shows typical transient signals corresponding to a linear calibration graph for ASA and

injections of seven samples of tablets. The results of the analysis of the tablets given in Table 2 compared favourably with results obtained using the standard titration method (FI = 3.6 3 1021 + 1.00 RM; r1 = 0.9997, where FI and RM are flow injection and reference methods, respectively) and also agreed with those declared on the labels (FI = 1.0 3 1021 + 0.99 DV; r2 = 0.9996, where DV is the declared value) confirming the accuracy of the FI spectrophotometric method with on-line microwave-assisted alkaline hydrolysis. The calibration graph for ASA was linear in the concentration range from 25 to 250 mg ml21 (A = 4.3 3 1022 + 3.1 3 1023 C; r = 0,9998, where A is the absorbance and C the concentration of ASA in mg ml21) with a detection limit (three times the signal blank/slope) of 4.0 mg ml21 ASA and a throughput of 90 samples h21. The repeatability for ten successive measurements of 200 mg ml21 ASA presented a relative standard deviation of 0.40%. Conclusion The proposed FI method with on-line microwave hydrolysis of ASA is simple, precise, accurate and sensitive enough to permit the determination of ASA in tablets. The results obtained by the FI method compared well with those obtained by a reference method. However, while the back-titration method for the determination of ASA requires heating over reflux for 10 min, the proposed method is less time consuming increasing the

Fig. 5 Transient absorbance signals obtained for ASA standards and sample solutions of tablets. From left to right: triplicate signals for six reference solutions (25250 mg ml21) followed by six consecutive signals for A, AAS; B, Melhoral; C, Doril; D, Aspirina; E, Cibalena; F, Bufferin; G, Fontol and standard solutions again.

Table 1 Recovery of ASA from tablets spiked with three different concentrations by the proposed FI method ASA/mg ml21* Tablets Melhoral Cibalena Fontol Bufferin Added 50 100 150 50 100 150 50 100 150 50 100 150 Found 49.8 99.8 149.7 49.5 99.3 150.3 49.7 98.9 149.6 50.6 100.7 148.9 Recovery (%) 99.6 99.8 99.8 99.1 99.3 100.2 99.5 98.9 99.7 101.0 100.7 99.3

Table 2 Comparison of results obtained by reference and proposed FI methods for ASA ASA/mg tablet21 s Relative Flow error (%) Label value/ Reference injection Tablets mg tablet21 method* method Re1 Re2 AAS 500 498.8 3.9 503.5 3.6 +0.9 +0.7 Melhoral 500 499.6 2.1 494.4 4.5 21.0 21.1 Doril 500 500.2 2.4 498.2 4.0 20.4 20.4 Aspirina 500 494.6 4.4 495.9 4.9 +0.3 20.8 Cibalena 200 198.4 1.0 200.8 1.3 +1.2 +0.4 Bufferin 500 503.6 3.3 +0.7 Fontol 650 646.7 5.5 651.6 5.7 +0.8 +0.2 * Average of three measurements back-titration method according to BP 1980. Average of five measurements. Buffered tablets containing CaCO3 and MgCO3. Re1 FIA-spectrophotometric vs. back-titration method value; Re2 FIA-spectrophotometric vs. declared value.

n = 5.

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speed of the sample pre-treatment, and therefore is useful for routine analysis of the ASA in tablets. Additionally, it decreases the possibility of interference caused by SA, the major decomposition product of ASA, which is critical in ASA determination. A sample throughput of 90 samples h21 was obtained. Financial support of FAPESP (Funda ao de Amparo a ` Pesquisa do Estado de S ao Paulo, Processes 91/2637-5 and 92/2637-5), PADCT/CNPq (Process 62.0060/91-3) and also the scholarship granted by CNPq to A.V.P. are gratefully acknowledged. References
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Bevitt, R. N., Mather, J. R., and Sharman, D. C., Analyst, 1984, 109, 1327. Verstraeten, A., Roets, E., and Hoogmartens, J., J. Chromatogr., 1987, 388, 210. Kirchhoefer, R. D., J. Pharm. Sci., 1980, 69, 1188. Fogel, J., Epstein, P., and Chen, P., J. Chromatogr., 1984, 317, 507. Juhl, W., and Kirchhoefer, R. D., J. Pharm. Sci., 1980, 69, 544. Kelly, C. A., J. Pharm. Sci., 1970, 59, 1053. Lopez-Fernandez, J. M., Luque de Castro, M. D., and Valcarcel, M., J. Autom. Chem., 1990, 12, 263. Haswell, S. J., and Barclay, D., Analyst, 1992, 117, 117. Arruda, M. A. Z., Fostier, A. H., and Krug, F., J. Braz. Chem. Soc., 1997, 8, 39. Morales-Rubio, A., Mena, M. L., and McLeod, C. W., Anal. Chim. Acta, 1995, 308, 364. Burguera, M., and Burguera, J. L., Anal. Chim. Acta, 1986, 179, 351. Mart nez-Calatayud, J., Flow Injection Analysis of Pharmaceuticals, Taylor & Francis, Hants., 1996, p. 130. Bouhsain, Z., Garrigues, S., Morales-Rubio, A., and de la Guardia, M., Anal. Chim. Acta, 1996, 330, 59. Luque P erez, E., and Haswell, S. J., Anal. Proc., 1995, 32, 85.

Paper 8/00036K Received January 2, 1998 Accepted February 17, 1998