Simple and facile methods for the determination of carvedilol in pharmaceuticals

Transcription

1 Indian Journal of Chemical Technology Vol. 16, January 2009, pp Simple and facile methods for the determination of carvedilol in pharmaceuticals T V Sreevidya & B Narayana* Department of Post Graduate Studies and Research in Chemistry, Mangalore University, Mangalagangothri , India n_badiadka@yahoo.co.uk Received 23 November 2007; revised 6 October 2008 Simple and sensitive methods for the determination of carvedilol (CAR) are described. The methods are based on the reaction of CAR with ninhydrin (NIN) in basic medium to form coloured iminium salt having absorption maximum at 402 nm and also with acetaldehyde (AA) in presence of sodium nitroprusside in basic medium to form coloured product having absorption maximum at 558 nm. The calibration graphs are found to be linear over and µg ml -1 with molar absorptivity values of and L mol -1 cm -1 for CAR-NIN and CAR-AA respectively. The optimum reaction condition and other analytical parameters were evaluated. The statistical evaluations of the methods were examined by determining intra-day and inter-day precision. The methods were successfully applied to the assay of CAR in tablet formulations. The accuracy and reliability of the methods were further ascertained by parallel determination by a reference method and by calculating the Student s t-test and F-test values at 95% confidence level. Keywords: Carvedilol (CAR), Ninhydrin, Sodium nitroprusside, Spectrophotometry Carvedilol, 1-(9H-carbazol-4-yloxy) - 3-[2-(2- methoxyphenoxy) ethylamino] propan-2-ol, is a non - selective β - adrenergic blocking agent with α 1 - blocking activity indicated for the treatment of mild to moderate congestive heart failure (CHF). It is the first β-blocker labeled in United States especially for the treatment of heart failure of ischemic or cardiomyopathic origin with significant antioxidant acitivity 1-3. Relative to other β-blockers, carvedilol (CAR) has minimal inverse agonism indicating a reduced negative chronotropic and inotropic effect, which decreases its potential to worsen symptoms of heart failure 4. At high dosages, it exerts calcium channel blocking activity 5. The benefits of using CAR in patients with CHF in both single-center and multicenter trials have been reported in the literature 6-8. It prevents vitamin E, gluthation and SH protein depletion induced by oxidative stress, the main defense mechanisms against tissue injury caused by free radicals 9. Several analytical methods such as spectrophotometry 10,11, HPLC 12-16, capillary electrophoresis 17,18, fluorometry 19,20, synchronous fluorimetry 21, HPLC-MS/MS 22, differential pulse voltammetry 23, GC-MS 24 have been widely used for the determination of CAR. Recently, a second polymorph 25 of CAR has also been reported. It is worthwhile to mention here that the two spectrophotometric methods reported recently 10,11 are in the UV region and thus are not a selective methods, since excipients present in the dosage form of the drug can interfere with the estimation method. In view of the importance of CAR in the treatment of CHF, simple methods for the determination of CAR have been developed. The proposed methods when compared with the reported spectrophotometric methods take the advantage over the UVspectrophotometric method in terms of selectivity (Table 1). The proposed methods are simple, accurate and easy to apply in routine analysis of the drug. Experimental Procedure A Shimadzu UV-2550 UV-VIS Spectrophotometer with 1 cm matched quartz cells was used for absorbance measurements. All reagents used were of analytical reagent grade and distilled water is used for the preparation of all solutions. Ninhydrin (0.3%), NaOH (1%), Na 2 CO 3 (2%), sodium nitroprusside (1%) and CH 3 CHO of A R grade were used. Preparation of carvedilol (CAR) solution A 1000 µg ml -1 standard drug solution was prepared by dissolving 0.1 g of CAR in 50 ml ethanol and diluting to the mark in a 100 ml calibrated flask. This stock solution was diluted appropriately to get the working concentration.

2 SREEVIDYA & NARAYANA: DETERMINATION OF CARVEDILOL IN PHARMACEUTICALS 75 Determination of CAR Using ninhydrin as reagent Aliquots containing ( ml) 10 µg ml -1 of CAR were transferred into a series of 10 ml standard flasks using a micro burette. To this 2 ml of 0.3% ninhydrin was added followed by 0.5 ml of 1% NaOH solution. The contents were shaken well and were set aside for 5 min and diluted up to the mark with distilled water and mixed well. The absorbance of each solution was measured at 402 nm against the corresponding reagent blank. Using acetaldehyde as reagent Aliquots containing ( ml) 10 µg ml -1 of CAR were transferred into a series of 10 ml standard flasks using a micro burette. To this 1 ml of freshly prepared acetaldehyde solution (10%) and 1 ml of sodium nitroprusside (1%) were added followed by 1 ml of Na 2 CO 3 (2%) solution. The contents were shaken well and were set aside for 15 min and was heated on a water bath for about 5 min and diluted up to the mark with distilled water and mixed well. The absorbance of each solution was measured at 558 nm against the corresponding reagent blank. Assay of formulations Two different CAR dietary supplement products were purchased and a sample stock solution of each was prepared by grinding an amount of powdered tablets equivalent to 0.1 g of CAR to a fine powder using a mortar and pestle and transferring into a 100 ml volumetric flask by washing with methanol and made upto the mark with methanol. A convenient aliquot was then subjected to analysis by each method. Table 1 Comparison table for reported method and proposed method Reagents Range λ max (nm) Molar absorptivity Reaction Ref (L mol -1 cm -1 ) conditions Methanol (as solvent) Methanol (as solvent) PEG-400+ Water (as solvent) 15 Proposed method Ninhydrin NaOH medium - Acetaldehyde + SNP Na 2 CO 3 medium - PEG Polyethylene glycol SNP Sodium nitroprusside CAR with ninhydrin in presence of a base to form yellowish-orange coloured iminium salt, which has an absorption maximum at 402 nm (Scheme 1). Method II is based on the reaction of CAR with acetaldehyde in presence of sodium nitroprusside in basic medium to form an adduct, which has an absorption maximum at 558 nm (Scheme 2). Optimization of experimental conditions Preliminary experiments were performed to fix the concentration of the reagents that could be measured spectrophotometrically and were found to be 0.3 % (2 ml), 10 % (1 ml), and 1% (1 ml) for ninhydrin, acetaldehyde and sodium nitroprusside respectively. For method I, NaOH medium was found to be ideal and 0.5 ml of 10 % NaOH in a total volume of 10 ml was adequate for the reaction to take place and Results and Discussion The determination of CAR is achieved by two simple methods. Method I involves the reaction of Scheme 1 Reaction of CAR with ninhydrin in presence of base

3 76 INDIAN J. CHEM. TECHNOL., JANUARY 2009 at five levels was determined each day for a week, preparing all solutions fresh. The relative standard deviation values were less than 2 % which represent the best appraisal of the procedures in daily use. Table 2 Analytical parameters for the determination of CAR Parameters CAR-NIN CAR-AA λ max (nm) Scheme 2 Reaction of CAR with acetaldehyde and sodium nitroprusside in presence of base for method II, Na 2 CO 3 was found ideal and 1 ml of 2% Na 2 CO 3 was found to be optimum. A 5 min standing time was found necessary for the complete reaction to take place. Under these conditions, the system was stable for a period of over 8 h. Analytical data A linear correlation was found between absorbance and concentration of the drug. The optical parameters such as molar absorptivity, Beer s law limits and Sandell sensitivity values were calculated and are given in Table 2. The table also has values for correlation coefficients, intercepts and slopes. The limit of detection and quantification calculated according to ICH guidelines 26 are also incorporated. Method validation Accuracy and precision To evaluate the accuracy and precision of the methods, pure drug within the working limits was analyzed, each determination being repeated five times. The RE (%) and RSD (%) values were less than 1 which indicate the high accuracy and precision for the methods (Table 3). For a better analysis of reproducibility on a day-to-day basis, a series of experiments was run in which standard drug solution Beer s law limits Molar Absorptivity (L mol -1 cm -1 ) Sandell Sensitivity (µg cm -2 ) Limit of detection** Limit of quantification** Regression Equation* Y= bx + a Y= bx + a Slope (b) Intercept (a) Correlation Coefficient * Y is the absorbance and X is the concentration in µg ml -1 ** Calculated using ICH - Guidelines Table 3 Evaluation of accuracy and precision Using ninhydrin as reagent Amount taken Amount found* SD RE (%) RSD (%) Using acetaldehyde as reagent * Mean value of five determinations RE- Relative Error; SD- Standard Deviation; RSD-Relative Standard Deviation

4 SREEVIDYA & NARAYANA: DETERMINATION OF CARVEDILOL IN PHARMACEUTICALS 77 Interference study In the pharmaceutical analysis, it is important to test the selectivity towards the excipients and fillers added to the pharmaceutical preparations. Several species that can occur in the real samples together with drug were investigated. To investigate the effect of tablet fillers on the measurements involved in the methods, standard addition method was carried out. A mixture containing lactose, starch and glucose was extracted with water and filtered. The filtrate was then added to a known amount of pure drug solution and subjected to analysis by the proposed methods. An error of less than 1.5 % in the absorbance readings was considered tolerable. From this study it is apparent that the usual co-formulated substances seldom interfere in the proposed methods. Application The proposed methods were applied to the determination of CAR in two commercial dietary supplements (tablets). An aliquot containing 0.8 ml (10 µg ml -1 ) drug solution was taken and assayed according to the proposed methods. The content of the tablet formulation was calculated by applying suitable dilution factor. The accuracy of the proposed method was checked by a thorough analysis of each spiked sample (Table 4). The results were also compared statistically with those of the reference method at 95% confidence level. The calculated student s t- test and F-test values (Table 4) did not exceed the tabulated value, indicating that there was no significant difference between the proposed methods and the reference method in respect to accuracy and precision. Table 4 Result of assay of formulations by the proposed methods Brand name Labeled amount (mg) Found* ± SD Using NIN Found* ± SD Using AA Reference method 10 Carloc ± ± ± 0.14 a t = 2.22 b F = 3.14 a t = 2.63 b F = 1.47 Carvidas ± ± ± 0.07 a t = 2.25 b F = 1.44 a t = 2.69 b F = 1.77 * Mean value of five determinations a Tabulated t-value at 95% confidence level is 2.78 b Tabulated F-value at 95% confidence level is 6.39 Conclusion Simple spectrophotometric methods for the determination of CAR have been developed and validated according to ICH guidelines. The methods compared to other existing methods do not entail any rigorous experimental variables which affect the reliability of the results. The ingredients usually present in the pharmaceutical formulations of these drugs seldom interfere in the proposed methods. The proposed methods are simple, accurate and easy to perform and can be used for the routine determination of CAR in bulk and in dosage forms. Acknowledgement Authors thank Department of Science & Technology, Government of India, for financial support through FIST programme. References 1 Ruffolo R R (Jr), Gellai M, Heible J P, Willette R N & Nichols A J, Eur J Clin Pharmacol (Suppl 2), 38 (1990) Feuerstein G Z, Poste G & Ruffolo R R, Drugs Today, 31 (1995) Zeng H, Liu X & Zhao H, J Huazhong Univ Sci Technol Med Sci, 23 (2003) Vanderhoff B T, Ruppel H M & Amsterdam P B, Am Fam Physician, 58 (1998) Nichols A J, Gellai M & Ruffolo R R (Jr), Fundam Clin Pharmacol, 5 (1991) Olsen S L, Gilbert E M, Renlund D G, Taylor D O, Yanowitz F D & Bristow M R, J Am Coll Cardiol, 25 (1995) Metra M, Nardi M, Giubbini R & Dei Cas L, J Am Coll Cardiol, 24 (1994) Packer M, Bristow M R, Cohn J N, Colucci W S, Fowler M B, Gilbert E M & Shusterman N H, N Engl J Med, 334 (1996) Yue T L, McKenna P J, Gu J L, Cheng H Y, Ruffolo R R (Jr) & Feuerstein G Z, Cardiovasc Res, 28 (1994) Jain P S, Talele G S, Talele S G & Surana S J, Indian J Pharm Sci, 67 (2005) Gita C, Bhadra S & Jain N K, Indian J Pharm Sci, 67 (2005) Ptacek P, Macek J & Klima J, J Chromatogr B, 789 (2003) Hokama N, Hobara N, Ohshiro S, Kameya H & Sakanashi M, J Chromatogr B, 732 (1999) Behn F, Laer S & Scholz H, J Chromatogr Sci, 39 (2001) Eisenberg E J, Patterson W R & Kahn G C, J Chromatogr B, 493 (1989) Reiff K, J Chromatogr, 413 (1987) Clohs L & McErlane K M, J Pharm Biomed Anal, 24 (2001) Clohs L & McErlane K M, J Pharm Biomed Anal, 31 (2003) 407.

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