Spectrophotometric determination of meloxicam by sodium nitroprusside and 1,10-phenanthroline reagents in bulk and its pharmaceutical formulation

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1 Indian Journal of Chemical Technology Vol. 20, March 2013, pp Spectrophotometric determination of meloxicam by sodium nitroprusside and 1,10-phenanthroline reagents in bulk and its pharmaceutical formulation M Gurupadayya*, M N Trinath & K Shilpa Department of Pharmaceutical nalysis, JSS College of Pharmacy, JSS University, Mysore , India Received 26 December 2011; accepted 11 December 2012 Two simple and selective spectrophotometric methods have been developed for the quantitative determination of meloxicam in pure form and in pharmaceutical formulations. The first method is based on the electron transfer reaction of drug with sodium nitroprusside in presence of hydroxyl amine HCl and the absorption maxima is recorded at 363 nm ( ). Second method is based on the formation of a complex of drug with ferric chloride and followed by coupling with 1,10-phenanthroline, and the absorption maximum is recorded at 343 nm ( ). oth the methods follow eer s range in the concentration range 4-20 µg ml -1 and µg ml -1 for s and respectively. Good correlation coefficients ( ) are observed between absorbance and corresponding concentrations of the drug. The calculated apparent molar absorptivity values are found to be and mol -1 cm -1 for the s and respectively. The limits of detection and quantification are also reported for both spectrophotometric methods. Intra-day & inter-day precisions and the accuracy of the methods have also been evaluated. No interference is observed from common pharmaceutical adjutants. The reliability of the methods is further ascertained by performing recovery tests by standard addition method. Keywords: Ferric chloride, Meloxicam, 1,10-Phenanthroline, Sodium nitroprusside, Spectrophotometry Meloxicam, an oxicam derivative, and a member of the enolic acid group of nonsteroidal anti-inflammatory drugs (NSIDs) with analgesic and antipyretic properties. Prostaglandins are substances that contribute to inflammation of joints. Meloxicam inhibits prostaglandin synthetase (cylooxygenase 1 and 2) and leads to a decrease in synthesis of prostaglandins, thus reducing the inflammation. Literature survey reveals that some methods for the determination of meloxicam in biological fluid by HPLC method 1, LC-MS 2, stability indicating HPLC 3, pharmacokinetic method in plasma 4,5 and HPTLC 6 are developed. It was also found that meloxicam estimated by analytical methods such as spectrophotometric method using safranin T 7, ferric chloride 8, derivative method 9, ultra violet 10,11, electrophoresis 12, pulse polarography 13, electrochemical oxidation 14, electrochemical reduction 15 voltametry 16 and flow injection analysis 17 are reported for the determination of meloxicam in pharmaceuticals. *Corresponding author. bm_guru2004@yahoo.co.in These spectrophotometric methods have their relative merits but the majorities of them use relatively expensive reagents 5, involve extraction 6 and elevated temperature 18, use of buffer system 15 and are time consuming 18. In this study, two simple, sensitive, cost-effective and accurate methods have been developed for the determination of meloxicam using sodium nitroprusside and ferric chloride followed by coupling with 1, 10-phenanthroline reagents. Experimental Procedure pparatus Spectral and absorbance measurements were made on Shimadzu UV-1800 series by using 1cm quartz cells. Shimadzu electronic balance was used for weighing the samples. Chemicals and reagents Meloxicam, pharmaceutical grade, was obtained as a gift sample from Sun Pharmaceutical s Mumbai, India. ll the chemicals are of analytical reagent grade of Merck Pharmaceuticals. Double distilled water was used to prepare all solutions. Sodium nitroprusside solution (0.4% w/v) and hydroxyl amine

2 112 INDIN J. CHEM. TECHNOL., MRCH 2013 hydrochloride solution (0.4% w/v) are prepared in distilled water. Sodium carbonate solution (0.4% w/v) was prepared by dissolving 400 mg of sodium carbonate in 100 ml volumetric flask using distilled water. Ferric chloride and 1,10-phenanthroline solutions were prepared by dissolving 50 mg each into 50 ml volumetric flasks separately, then dissolved and diluted with 0.01 M sulphuric acid. The tablet 750 mg of M-CM (Unichem Laboratories Ltd, Mumbai, India) was procured from local market. Standard solution of drug Meloxicam stock solution was prepared by weighing 20 mg meloxicam in 40 ml of 0.1 M NaOH and it is made into 100mL with distilled water to get 200 µg ml -1. This solution was used for. stock solution of meloxicam was also prepared by dissolving 10mg in 100mL methanol (100 µg ml -1 ) and this solution was used for. Fig. 1 bsorption spectra of meloxicam with sodium nitroprusside Transfer ml (200 µgml -1 ) meloxicam stock solution to 5 different volumetric flask (10 ml). To this, 1 ml of sodium nitroprusside solution and 1 ml of hydroxyl amine hydrochloride solution, followed by 5 ml of distilled water were added and shaken for 2 min. Finally, 1 ml sodium carbonate solution was added and volume was made to 10 ml with distilled water (4-20 µgml -1 ). The absorbance was measured at 363 nm against blank solution (Fig. 1). Transfer 1-5 ml (100 µgml -1 ) meloxicam stock solution into 10 ml volumetric flask. To this 1 ml of ferric chloride solution and 1 ml of 1,10-phenanthroline solution were added and heated for 10 min. Then the volume was made upto 10 ml with distilled water (10-50 µgml -1 ). The absorbance of the solutions was measured at 343 nm against blank solution (Fig. 2). nalysis of formulation The contents of 10 tablets of meloxicam were weighed and powdered. The average weight was determined and the powder equivalent to 7.5 mg was transferred into a 100 ml volumetric flask. Then 40 ml of 0.1 M NaOH was added, shaken for 10 min and diluted up to 100 ml with distilled water to get 300 µgml -1. The contents of the flask were Fig. 2 bsorption spectra of meloxicam with 1,10- phenanthroline thoroughly mixed and filtered by using Whatmann s No. 41 filter paper. The sample solution was diluted and analyzed at 363 nm using blank. The contents of 10 tablets of of meloxicam were weighed and powdered. verage weight was determined and the powder equivalent to 7.5 mg was transferred to a 100 ml volumetric flask. Then 50 ml of methanol was added shaken for 10 min and diluted to 100 ml with same solvent. The contents of the flask were thoroughly mixed and filtered by using Whatmann s No. 41 filter paper. The sample solution was further diluted and analyzed at 343 nm against reagent blank.

3 GURUPDYY et al.: SPECTROPHOTOMETRIC DETERMINTION OF MELOXICM 113 Results and Discussion In, meloxicam functions as a donor due to the presence of cyclic tertiary nitrogen in benzothiazine ring of the molecule 18. Sodium nitroprusside in the presence of hydroxylamine and alkali exists as aquoferrocyanide [Fe(CN) 5 H 2 O] 3-. In a general way, it may be expected that the electron transfer depends upon the extent of delocalization of the donor and acceptor metal orbitals of the intervening ligands. From this stand point, ligands such as water and ammonia, which contain single bonds, are expected to be much less effective in conducting electrons between metal ions than unsaturated ligands such as CN-whose complexes are characterized by high degree of covalency and electron delocalization. The reaction mechanism is shown in Fig. 3. The optical characteristics of the methods such as absorption maxima, eer s law limits, molar absoptivity and Sandell s sensitivity and regression equation were reported in Table 1. In, meloxicam reacts with ferric chloride and then coupled with 1,10-phenonthroline reagent to form complex (Fig. 4). The colored complex resulting from meloxicam may be due to the fact that each of the two nitrogen atoms has an unshared pair of electrons that can be shared with Fe (II) ion formed by the reaction of meloxicam with Fe (III). The molecules of meloxicam attach themselves by means of dative bonds (ferroin complex) The proposed methods for quantification of meloxicam in tablets were simple, precise, accurate, rapid and sensitive. The methods are linear in the concentration range reported. The developed methods are free from interference due to the excipients present in the tablets and can be used for quantitative estimation of meloxicam in tablets. Statistical analysis was carried out and the result are found to be satisfactory. The optical characteristics of the methods such as absorption maxima, eer s law limits, molar absoptivity, Sandell s sensitivity and regression equation are reported in Table 1. validation Linearity The linearity of an analytical method is its ability to elicit test results that are directly or by a well-defined Table 1 Optical parameters for and Parameter λ max, nm eer s law limits, µgml Sandell s sensitivity, µg/cm 2 /0.001.U. Regression equation, y = mx + c y = x y = x Slope, m Intercept, c Limit of detection, µgml Limit of quantification, µgml -1 Molar extinction coefficient, L mol -1 cm -1 Correlation coefficient (r) y bsorbance; x Concentration in µgml -1. Fig. 3 Reaction mechanism of meloxicam and sodium nitroprusside Fig. 4 Reaction mechanism of meloxicam and 1,10- phenanthroline

4 114 INDIN J. CHEM. TECHNOL., MRCH 2013 mathematical transformation proportional to the concentration of analyte in samples within a given range. The range of analytical method is the interval between upper and lower level of analyte including levels that have been demonstrated to be determined with precision and accuracy using the method. The linearity ranges are 4-20 µgml -1 and µgml -1 for s and respectively. ccuracy The accuracy of the method is the closeness of the measured value to the true value for the sample. To determine the accuracy of the proposed method, different levels of drug concentrations [lower concentration (LC, 80%), intermediate concentration (IC, 100%) and higher concentration (HC, 120%)] were prepared from independent stock solutions and analysed. ccuracy was assessed as the percentage relative error and mean % recovery. To provide an additional support to the accuracy of the developed assay method, a standard addition method was employed, which involved the addition of different concentrations of pure drug to a known pre analyzed formulation sample and the total concentration was determined using the proposed methods. The % recovery of the added pure drug was calculated using the following relationship: % Recovery = [(Ct Cs)/Ca] 100 where Ct is the total drug concentration measured after standard addition; Cs, the drug concentration in the formulation sample; and Ca, the drug concentration added to formulation. The values for and are shown in Tables 2 and 3 respectively. Precision Repeatability was determined by using different levels of drug concentrations (same as in accuracy study), prepared from independent stock solutions and analyzed. Inter-day, intra-day and inter instrument variations were studied to determine intermediate precision of the proposed analytical methods. Different levels of drug concentrations (6 times) were prepared three different times in a day and studied for Table 2 ccuracy of Drug added Meloxicam, µgml -1 Total amount of Total amount of drug found % Recovery Mean % RSD % Taken dded drug, µgml -1 in triplicates, µgml Table 3 ccuracy of Drug Meloxicam, µgml -1 added, % Taken dded Total amount of drug µgml -1 Total amount of drug found % Recovery Mean % RSD in triplicates, µgml

5 GURUPDYY et al.: SPECTROPHOTOMETRIC DETERMINTION OF MELOXICM 115 Table 4 Intra-day and Inter-day precision values for and [mount of drug taken: 4 µgml -1 for and 10 µgml -1 for in both intra-day and inter-day cases] Intra-day precision Inter-day precision mount of drug found, µgml -1 Mean % RSD mount of drug found, µgml -1 Mean % RSD intra-day variation. The same procedure was followed for three different days to study inter-day variation. One set of different levels of the concentrations was reanalyzed using the Shimadzu 1800 UV-VIS spectrophotometer connected to computer with UV-PC software for studying the inter-instrument variation. The per cent relative standard deviation (% R.S.D.) of the predicted concentrations from the regression equation was taken as precision. Precision studies were also carried out using the real samples of meloxicam in a similar way to standard solution to prove the usefulness of the method. The values for and are shown in Table 4. Limit of detection (LOD) and limit of quantitation (LOQ) The LOD and LOQ for and by the proposed method were determined using calibration standards. LOD and LOQ are calculated as 3.3 σ/s and 10 σ/s, respectively, where s is the slope of the calibration curve and σ is the standard deviation of y-intercept of regression equation. LOD and LOQ are found to be µgml -1 for & µgml -1 for and µgml -1 and µgml -1 for s and respectively. Conclusion Two useful methods for the determination of meloxicam have been developed and validated as per ICH guidelines. The analysis of the drugs does not require any extraction procedure. The other active ingredients and excipients usually present in pharmaceutical dosage forms do not interfere. These methods can be used to monitor the content uniformity of tablets and purity of raw material. validation has been demonstrated by a variety of tests for specificity, sensitivity, linearity, precision, accuracy, recovery, and stability. The result of analysis suggests the applicability, reproducibility, and utility of the method for direct estimation of meloxicam in quality control laboratories. References 1 Velpandian T, Jaiswal J, hardwaj R K & Gupta S K, J Chromatogr [] iomed Sci ppl, 738 (2000) Wiesner J L, de Jager D, Sutherland F C, Hundt H K, Swart K J, Hundt F & Els J, J Chromatogr []: nal Technol iomed Life Sci, 25 (2003) Farzana S & Pradeep R, Trop J Pharm Res, 8(3) (2009) havesh Dasandi, Shivaprakash, Saroj H & hat K M, J Pharmaceut iomed nal, 28 (2002) Xia Yun-Yue, Jiang Kai-Yu & Xie Mei-Lin, Chinese Pharm, 22 (2002) Namita Desai & Purnima min, Indian J Pharm Sci, 63(3) (2001) Hassan E M, J Pharm iomed nal, 27 (2002) Dhandapani, Eswara Murali S, Susrutha N, Rama Swetha, Sonia Rani S K, Sarath abu T, Seetharamanjaneyulu G V & Celestin aboo R V, Int J Pharm Sci Res, 1 (2010) nanth V, Venkatamahesh R, Ulaganathan C, alaji M & Sachin Kumar Singh, Der Pharma Chemica, 3 (2011) You WW, Liu Y & Wang Z, Chinese J nal Chem, 27 (1999) Nemutlu E & Kir S, Hacettepe University, J Faculty Pharm, 24 (2004) Nemutlu E & Kir S, J Pharm iomed nal, 31(2) (2003) ltiokka G, tkosar Z & Tuncel M, Die Pharmazie, 56(2) (2000) Radi, El-Ries M, El-nwar F & El-Sherif Z, nal Lett, 34 (2001) eltagi M, Ghoneim M M & Radi, J Pharm iomed nal, 27 (2002) Radi E, Ghoneim M & eltagi, Chem Pharm ull, 49 (2001) l-momani I F, nalyt Sci, 22 (2006) Sudha Lakshmi P & Rambabu C, J Pharm Res, 4 (2011) Srinubabu G, Pundarika Rao D, Sridhar L & Seshagiri Rao J V L N, E-J Chem, 3 (2006) Sastry C S P, Yaramati S & Pulugurtha V S R, Mikrochim cta, 126 (1997) Sarsambi P S, bdul Faheem, bhay Sonawane & Malipatil S M, J Indian Council Chem, 27 (2010) 205.