H Tavallali* & M Sheikhaei. Indian Journal of Chemistry Vol. 48A, June 2009, pp

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1 Indian Journal of Chemistry Vol. 48A, June 2009, pp Simultaneous kinetic determination of paracetamol and caffeine using Cu(II)- neocuproine in presence of dodecyl sulfate by H-point standard addition method H Tavallali* & M Sheikhaei Payame Noor University (PNU), Shiraz, , Iran tavallali@pnu.ac.ir, tavallali@yahoo.com Received 6 Aug 2008; re-revised and accepted 18 May 2009 A simple, feasible and selective kinetic spectrophotometric method for simultaneous determination of paracetamol and caffeine using H-point standard addition method is described. The method is based on difference in the rate of oxidation of the compounds with Cu(II)-neocuproine system and formation of Cu(I) neocuproine complex, which is monitored at 453 nm and at ph 5.0 in the presence of sodium dodecyl sulfate. Experimental conditions such as ph, reagents concentrations, ionic strength and temperature have been optimized. Paracetamol and caffeine can be determined in the range and µg ml -1 respectively. The proposed method has been applied for the determination of paracetamol and caffeine in pharmaceutical samples with satisfactory results. Keywords: Analytical chemistry, Spectrophotometry, H-point standard addition, Paracetamol, Caffeine, Neocuproine IPC Code: Int Cl. 8 G01N21/25 Paracetamol (4-acetamidophenol) is one of the most common drugs used in combination with aspirin an analgesic. Caffeine (1,3,7 trimethylxanthine) is mainly ingested by drinking coffee, cola-beverages, and tea and acts both as diuretic and stimulant to the central nervous and cardiovascular system. The use of the mixture of paracetamol and caffeine as an analgesic and antipyretic is well established in pharmaceutical formulations. In order to achieve better curative effect and lower toxicity, it is very important to control the amounts of paracetamol and caffeine in pharmaceutical preparations. Various methods like spectrofluorimetric determination in solid phase using partial least squares multivariate calibration 1, flow injection solid phase spectrometry using C 18 silica gel as a sensing support 2, flow-injection spectrophotometry 3 and high performance liquid chromatography (HPLC) 4,5, have been reported for the determination of paracetamol and caffeine in various biological and pharmaceutical preparations. Capillary electrophoresis (CE) 6-8 and 1 H-NMR spectroscopy 9 have been developed for simultaneous determination of a few drugs in their formulations. Reports on simultaneous spectrophotometric determination of paracetamol and caffeine are rare, and sensitivity 10,11. Thus, there is a need for a simple, accurate and sensitive method for simultaneous determination of paracetamol and caffeine. H-point standard addition method (HPSAM) is a modification of the standard addition method and permits direct correction of both proportional and constant errors produced by sample matrix. The HPSAM has been applied with analytical spectroscopy to resolve mixtures of two components with extensively or fully overlapped spectra 12. HPSAM can remove errors resulting from the presence of an interferent and blank reagent Application of H-point standard addition method to kinetic data has been proposed for the simultaneous determination of binary mixtures. In the present study, a variant of the HPSAM is used which is based on the assumption that only analyte X evolves with time and the other species Y or interference does not affect the analytical signal with time. In this case the variables to be fixed are two time values, t 1 and t 2, at which the species Y, which does not evolve with time or over the range between these times, has the same absorbance 16. The method is very simple, selective, precise, accurate, and is a low cost procedure for simultaneous spectrophotometric determination of paracetamol and caffeine. The method is based on the difference in the rate of the reactions of paracetamol and caffeine with Cu(II) in the presence of neocuproine. The ability to measure trace amounts of Cu(I) in the presence of an excess of Cu(II) is used for indirectly quantifying substances that reduce Cu(II)-Nc reagent at a suitable ph and in the presence of surfactant SDS. The colored Cu(I)-Nc chelate has been measured at 453 nm as a function of time. The method is also applied to commercial formulations.

2 NOTES 813 Experimental All chemicals and reagent (Merck) used were of analytical grade. Doubly distilled water was used throughout. Stock solution of Nc, ( M), was prepared by dissolving g of Nc in 25 ml of ethanol. Copper(II) stock solution ( M), was prepared by dissolving g of Cu(NO 3 ) 2.3H 2 O in 25 ml of water. Acetic-acetate buffer ph 5.0 was prepared by adding 1.0 M sodium hydroxide to 1.0 M acetic acid and adjusting to ph 5. Stock solution of sodium dodecyl sulfate (SDS, 0.1 M) was prepared by dissolving g of SDS in 10 ml of water. Stock solutions of paracetamol and caffeine (1000 µg ml -1 ) were prepared separately by dissolving 25 mg each in 25 ml of water. Most of the solutions were prepared daily. UV-vis absorbance spectra were recorded on a Perkin Elmer Lambda 2 (Federal Republic of Germany) scanning spectrophotometer which was equipped with a 1 cm path length glass cell and spectrophotometer attached to a Pentium 200 MHz computer. JENWAY (model 3510) ph meter with a combined glass electrode was used for ph measurements. The reagent solution was prepared in 10 ml volumetric flask by mixing stock solutions of 2 ml of Nc solution, 0.6 ml of Cu(II), 2 ml of buffer (ph 5.0, 1.0 M) and 50 µl of SDS solution, and diluting up to the mark with water. For each measurement, 3 ml of reagent solution was transferred to the spectrophotometric cell and the absorbance of this solution was set to read zero at 453 nm. Then, an appropriate amount of paracetamol and/or caffeine in the concentration ranges and µg ml 1, respectively, was injected into the cell using a 100 µl syringe and stirred manually for a few seconds and the variation of the absorbance versus time was recorded immediately. The absorbance was measured at 453 nm with 1 s time interval for each sample. Simultaneous determination of paracetamol and caffeine with HPSAM was performed by measuring the absorbances at 10 and 200 s after initiation of the reaction. Results and discussion Spectrophotometery based methods in the visible region involve redox reactions in which a colored compound is formed as result of the reaction. The present method is based on the reaction of Cu(II) in the presence of Nc and SDS in buffered medium. The Cu(I)-Nc complex formed was determined spectrophotometerically. In principle, the Cu(II)-Nc systems allows the spectrophotometric determination of a reducing agent, A red, provided the redox reaction, ncu n Nc +A red n [Cu (Nc) 2 ] + +A ox, is complete with the formation of an equivalent amount of [Cu(Nc) 2 ] + with respect to the n-electron reductant, A red. Copper (II) acts as a strong oxidizing agent only when its reduction product, Cu(I), is stabilized by a strong complex-forming ligand, e.g., Nc. Weak reductants should be determined either by masking the excess of Cu(II) or by using a dilute solution of Cu(II) 17. Difference in kinetic behavior of paracetamol and caffeine accompanied with mathematical treatment of data using HPSAM permitted simultaneous analysis of the two compounds. Figure 1 shows the applicability of HPSAM to the simultaneous analysis of paracetamol and caffeine by the proposed system. The reaction rate of paracetamol with Cu(II)-Nc system was fast, while the reaction of caffeine was very slow. The sensitivity and rate of reduction and complex formation are dependent on ph of medium. The effect of ph on the rate of Cu(I) Nc complex formation in presence of paracetamol and caffeine was studied over the range Increase in ph up to 5.0, caused an increase in the reaction rates for paracetamol and thereafter decreased with further increase in ph 5.0. However, over the studied range of ph, no significant change in the reaction rate of Fig 1 Absorbance changes of Cu (II) Nc system versus time in the reaction with (a) caffeine (1.0 µg ml -1 ), (b) paracetamol (2.5 µg ml -1 ) and (c) a mixture of caffeine (1.0 µg ml -1 ) and paracetamol (2.5 µg ml -1 ) at 453 nm.

3 814 INDIAN J CHEM, SEC A, JUNE 2009 caffeine with Cu(II)-Nc system was observed. Therefore, for achieving the appropriate condition for applying HPSAM, ph 5.0 was selected as the optimum ph. Use of micelles has frequently been made in catalytic and non-catalytic 21,22 reactions. The rate enhancement in such reactions is due to increased reactant concentration in the micellar pseudo phase. This concentration results in increase in sensitivity. The effect of micelles on the system was studied under optimum ph, using surfactants such as sodium dodecyl sulfate (SDS), cetyltrimethyl ammonium bromide (CTAB) and Triton X-100 (a non-ionic surfactant). SDS was found to be the best surfactant for HPSAM as it increased the rate of paracetamol reaction but had no effect on the reaction rate of caffeine. This may be due to reactant concentration in the surfactant medium 23. Therefore, M SDS was selected as the optimum concentration of surfactant in this work. A similar behavior of Cu(II) and neocuproine concentrations was observed. Both increased the rate up to M and decreased thereafter for paracetamol. The reagent concentrations had no effect on the reaction rate of caffeine. Therefore, the reagent concentrations were kept at M each. As expected, increasing temperature above 20 C increased the rate of reaction of paracetamol and caffeine, the rate of paracetamol being faster than caffeine. For ease of operation, reaction was carried out at 25 C. The ionic strength of reaction mixture was adjusted by addition of up to 0.7 M KNO 3. The ionic strength of solutions did not have any effect on the reaction rates of paracetamol and caffeine with Cu(II)-Nc system. For selection of appropriate time period for applying HPSAM, the principle followed was that the analyte signals must be linear and interferent signal must remain equal, over the range of concentration of the analytes. The analytical signals of mixture of analyte and interferent should be equal to the sum of individual signals of the two compounds. In addition, the difference of the slopes of the two straight lines obtained at t 1 and t 2 must be as large as possible to achieve good accuracy. It was possible to select several pairs of time periods in the determination of paracetamol. The pair of time periods which gave the highest accuracy, greatest slope increment, and the lowest error for analytes was selected. The following pairs of time periods were studied: , , , , , s, and the was plotted versus C added variant At 1 t 2 (Fig. 2). This yielded the concentration of paracetamol directly from the intercept on Y-axis. The time period pair that gave the maximum absorbance change at 453 nm and the highest accuracy corresponding to paracetamol concentration was s. Plot of H point standard addition method for paracetamols shown in Fig. 3. The Fig 2 Abs versus added paracetamol concentration at different time intervals at 453 nm for synthetic mixtures containing 1.5 µg ml -1 paracetamol and 1.0 µg ml -1 caffeine. [1, s; 2, s; 3, s; 4, s; 5, s; 6, s]. Fig 3 HPSAM plots for simultaneous determination of paracetamol (1.5 µg ml -1 ) and caffeine (1.0 µg ml -1 ). [1, 10s; 2, 200 s].

4 NOTES 815 absorbances corresponding to paracetamol at the two selected times of 10 and 200 s were selected to calculate paracetamol concentration in the range µg ml 1 in the presence of caffeine. The concentration of caffeine in the range µg ml 1 was calculated in each sample by obtaining the ordinate values of the H-point (A H ). The calibration graph for caffeine was plotted using ordinate values of H-points (A H ) versus corresponding caffeine concentration. According to the theory of HPSAM at H-point (-C H, A H ), C H (concentration of paracetamol at H-point) is independent of the concentration of interferent (caffeine) (Fig. 4) and so A H, the Fig 4 HPSAM plots for fixed caffeine concentration (0.8 µg ml -1 ) and varying concentrations of paracetamol. [1, 1.5 µg ml -1 ; 2, 2 µg ml -1 ; 3, 2.5 µg ml -1 ; 4, 3 µg ml -1 ]. Table 1 Application of signal increment version of HPSAM in a mixture of paracetamol (1.5 µg ml -1 ) and caffeine (1.0 µg ml -1 ) Paracetam ol (µg ml -1 ) Recovery (%) RSD (%) (n = 6) Time interval (s) absorbance value at H-point, is also independent of the analyte concentration. Table 1 gives the results obtained from employing the modified HPSAM on a mixture of paracetamol and caffeine. The results obtained by this procedure are in good agreement with the actual concentration. The reproducibility of the method was determined by five replicate analysis of paracetamol (1.5 µg ml 1 ) and caffeine (1.0 µg ml 1 ) (r 2 = ). Synthetic mixtures of paracetamol and caffeine in different concentration ratios were analyzed. As evident from the results in Table 2, the accuracy and precision of the method are within acceptable limits. Limits of detection, calculated as LOD=C H +3S CH, where C H and S CH are the mean and standard deviation of five replicated measurements of a blank sample, were 0.80 and 0.05 µg ml -1 respectively for paracetamol and caffeine. To study the selectivity of the proposed method several interferants were tested for their possible interferences. Paracetamol (2.0 µg ml -1 ) and caffeine (0.5 µg ml -1 ) were analyzed in the presence of interfering species such as sucrose, glucose, starch, urea, saccharin, riboflavin and ibuprofen at different concentrations (maximum concentration tested was 300 µg ml -1 ). A species was considered as interfering, when its presence showed a variation in the change of absorbance of the sample (time period of 200 s) greater than twice the standard deviation did interfere on the simultaneous determination of paracetamol and caffeine in the system. The results show that above species do not interfere in the determination of caffeine and paracetamol. To evaluate the applicability of the proposed method, it was applied to simultaneous determination of paracetamol and caffeine in commercially available preparations in the form of tablets and capsules, viz., Table 2 Analysis of paracetamol and caffeine present in different concentration ratios A-C equation R 2 Amt taken(found) (µg ml -1 ) Paracetamol Caffeine A 200 = C A 10 = C A 200 = C A 10 = 0.024C A 200 = 0.189C A 10 = C A 200 = C A 10 = 0.026C (1.49) 1.50 (1.49) 2.00 (1.98) 3.00 (3.04) 0.80 (0.80) 3.00 (3.03) 2.50 (2.52) 2.00 (1.99)

5 816 INDIAN J CHEM, SEC A, JUNE 2009 Table 3 Simultaneous determination of paracetamol and caffeine in pharmaceutical samples Sample Cert amt. (µg ml -1 ) Found a (µg ml -1 ) (Recovery, %) Paracetamol Caffeine Paracetamol Caffeine Remidon tablet, Deva Pharm.Ind., Turkey, Batch no (100.8) 65.7(101.1) Novafen capsule, Brown & Burk Ind., UK, Batch no. NVF34E3 Pirosal tablet, Saba Pharm., Turkey, Batch no (100.6) 40.50(101.2) (99.4) 30.80(102.7) a Mean of five replicate determinations after dilution and determination by the proposed method. Remidon, containing mg of paracetamol and 65.0 mg of caffeine, capsule Novafen containing 325 mg of paracetamol, 40 mg of caffeine and 200 mg of ibuprofen per capsule and Pirosal tablet containing mg of paracetamol, 30.0 mg of caffeine and mg metamizol. The quantitative results of this analysis are shown in Table 3. Good agreement between the obtained results and certified amounts indicates the successful applicability of the HPSAM for simultaneous determination of paracetamol and caffeine. Acknowledgement The authors gratefully acknowledge the support of this work by Payame Noor University (PNU) Research Council of Iran. References 1 Moreira A B, Dias I L T, Neto G O, Zagtto E A G & Kubota L T, Anal Lett, 39 (2006) Baralles P O, Weigand R P & Dias A M, Anal Sci, 18 (2002) Knochen M, Giglio J, Reis & B F, J Pharm Biomed Anal, 33 (2003) Xue H Y, Liu J F & Liu H C, Chin J Hosp Pharm 25 (2002) Prodan M, Gere-Paszti E, Farkes O & Forgacs E, Chem Anal (Warsaw), 48 (2003) Fanali S, Pucci V, Sabbioni C & Raggi M A, Electrophoresis, 21 (2000) Zhang L, Chen G, Hu Q & Fung Y, Anal Chim Acta, 431 (2001) Ha P T T, Van-Schepdeal A, Hauta-Aho T, Roets E & Hoogmartens J, Electrophoresis, 23 (2002) Talebpour Z, Haghgoo S & Shamsipur M, Anal Chim Acta, 506 (2004) Madrakian T, Afkhani A, Borazjani M & Bahram M, Bull Korean Chem Soc, 25 (2004) Damiani P C, Moschetti A C, Rovetto A J, Benavente F & Olivieri A C, Anal Chim Acta, 543 (2005) Campins- Falco P, Bosch Reig F & Molina-Benent A, Fresen J Anal Chem, 338 (1990) Campins- Falco P, Verdu-Andres J & Bosch Reig F, Anal Chem Acta, 348 (1997) Campins- Falco P, Bosch Reig F & Blasco-Gomez F, Talanta, 47 (1998) Yang J, Zhou G, Jie N, Han R, Lin C & Hu J, Anal Chim Acta, 325 (1996) Safavi A, Abdollahi H & Hormozi Nezhad M R, Talanta, 56 (2002) Tutem E, Apak R & Baykut F, Analyst, 116 (1991) Lunar M L, Rubio S & Perez-Bendito D, Anal Chim Acta, 237 (1990) Sicilia D, Rubio S & Perez-Bendito D, Talanta, 38 (1991) Sicilia D, Rubio S & Perez-Bendito D, Anal Chem, 64 (1992) Athanasiou-Malaki E & Koupparis M A, Anal Chim Acta, 219 (1989) Sicilia D, Rubio S & Perez-Bendito D, Anal Chim Acta, 284 (1993) Perez -Bendito D & Rubio S, Trends Anal Chem, 12 (1993) 9.