Indian Journal of Chemical Technology Vol. 22, November 2015, pp. 333-337 Absorbance ratio and derivative spectroscopy methods for the simultaneous estimation of lornoxicam and eperisone in their synthetic mixture. Jawed Akhtar 2, Jatin Prajapati*,1 & Gamal Osman Elhassan 2 1 Department of Quality Assurance, Parul Institute of Pharmacy and Research, Limda, Ta. Waghodia, Dist. Vadodara 391 760, Gujarat, India. E-mail: jatinprajapati3@gmail.com 2 Department of Pharmaceutics, Uniazah College of Pharmacy, Qassim University, KSA Received 14 March 2013; accepted 7 January 2014 Two simple spectrophotometric methods have been developed for simultaneous estimation of lornoxicam (LXM) and eperisone (EPE) from their synthetic mixture. Method-I involves, formation of Q-absorbance equation at 265 nm (isoabsorptive point) and 290.5 nm (λ max of Lornoxicam) and Method-II is the first order derivative spectroscopy for the simultaneous estimation of LXM and EPE. The absorbances of the standard solutions have been taken at two wavelengths 264.5 nm (ZCP of Lornoxicam) for EPE and 254 nm (ZCP of Eperisone) for LXM in 0.1N methanolic NaOH. and the linearity ranges between 2-24 µg/ml for both drugs in both methods. The accuracy and precision of the methods are determined and validated statistically. Both the methods show good reproducibility and recovery with less than 2. Both methods are found to be rapid, specific, precise and accurate and can be successfully applied for the routine analysis of lornoxicam and eperisone in bulk and also their combined dosage form. Keywords:, Derivative spectroscopy method, Eperisone, Lornoxicam, Method Development, Validation. LXM is chemically (3E)-6-chloro-3-[hydroxyl (pyridin-2-ylamino)methylene]-2 methyl-2,3-dihydro- 4H-thieno[2,3-e][1,2]thiazin-4-one 1,1-dioxide. LXM is an NSAID of the oxicam class with analgesic, anti-inflammatory and antipyretic properties. It inhibits prostaglandin synthesis by inhibiting both cyclooxygenase enzyme (COX-1 and COX-2). EPE is chemically (2RS)-1-(4-ethylphenyl)-2-methyl-3- (1-piperidyl) propane-1-one 1. EPE is an antispasmodic drug. It acts by relaxing both skeletal muscles and vascular smooth muscles, and demonstrates a variety of effects such as reduction of myotonia, improvement of circulation, and suppression of the pain reflex. The review of literature revealed that various involving spectrophotometry have been reported for LXM in single form and in combination with other drugs. LXM is estimated by UV 4-12, HPLC 13-16 and HPTLC 17 method. According to literature review, Eperisone is estimated by UV 18,19, GC-MS 20 and LC-EI-MS 21 methods. There is no any method reported for simultaneous estimation of LXM and EPE in a combination by UV and HPLC, but individually available for each drug and in combination with other drug. The present work describes the development of a simple, precise, accurate and reproducible spectrophotometric method for the simultaneous estimation of LXM and EPE in synthetic mixture. The developed method was validated in accordance with ICH Guidelines and successfully employed for the assay of LXM and EPE in synthetic mixture. Experimental Section Reagents and chemicals Analytically pure LXM and EPE were kindly provided by Cirex Pharmaceuticals Limited (Hetero Drugs Limited), Hyderabad. Analytical grade methanol was purchased from Merck PVT LTD, India. Instrument and apparatus Shimadzu-1800 UV-Visible spectrophotometer was used for spectral measurements with spectral band width 1 nm; wavelength accuracy is 0.5 nm and 1 cm matched quartz cells. Software used was UV Probe (version 2.34). An electronic analytical balance (Shimadzu) was used for weighing. Glassware used in each procedure were soaked overnight in a mixture of chromic acid and sulphuric acid rinsed thoroughly with double distilled water and dried in hot air oven.
334 INDIAN J. CHEM. TECHNOL., NOVEMBER 2015 Method uses the ratio of absorbances at two selected wavelengths, one which is an isoabsorptive point and other being the λ max of one of the two components. From the overlay spectra of two drugs, it is evident that LXM and EPE showed an isoabsorptive point at 265 nm. The second wavelength used was 290.5 nm, which was the λ max of LXM. The concentration of two drugs in the mixture can be calculated using following equations. C X = [(QM QY)/(QX QY)] A1/aX1 (1) C Y = [(QX QM)/(QX QY) A1/aY1 (2) where, A 1 and A 2 were absorbances of mixture at 265 nm and 290.5 nm; ax1 and ay1 are absorptivities of LXM and EPE at 265 nm; ax2 and ay 2 are absorptivities of LXM and EPE respectively at 290.5 nm; QM = A 2 / A 1, QX = ax2 / ax1 and QY = ay2 / ay1. First order derivative spectroscopy The absorbance of resulting solutions was measured at 265 and 254 nm for LXM and EPE respectively, the calibration curves were plotted at these wavelengths. The overlain zero order spectra of LXM and EPE showed that the absorption maxima of LXM and EPE lie in close proximity and at absorption maxima of one, another exhibits substantial absorbance. This clearly indicates the existence of spectral interference in estimation of LXM and EPE. To overcome this, spectra of these two drugs were derivatised to first order between 200-400 nm. The overlain first derivative spectra of LXM and EPE reveal that LXM concentration can be estimated at 254.5 nm (ZCP for EPE) and EPE can be estimated at 264.5 nm (ZCP for LXM). Preparation standard stock solutions Accurately weighed 10 mg of LXM and EPE standard were transferred to separate 100 ml volumetric flask and dissolved in 50 ml methanolic NaOH (0.1N). The flasks were shaken and volume was made up to the mark with the same solvent to give solutions containing 100 μg/ml LXM and 100 μg/ml EPE. Selection of Analytical Wavelength Solutions of both LXM and EPE (2-12 μg/ml) were prepared in 0.1N methanolic NaOH by appropriate dilution and spectrum were recorded between 200-500 nm and first derivative spectrums were obtained using above condition. The overlain first derivative spectrums of LXM and EPE at different concentration were recorded. The zero crossing point (ZCP) of LXM was found to be 264.5 nm and ZCP of EPE was found to be 254 nm. Sample preparation LXM (0.8 mg) and EPE (10 mg) of API (Active Pharmaceutical Ingradients) were accurately weighed and transferred to volumetric flask of 100 ml capacity. Methanolic NaOH (50 ml, 0.1N) was transferred to this volumetric flask and sonicated for 15 min. The flask was shaken and volume was made up to the mark with the same solvent. From this solution 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 ml was transferred to volumetric flask of 100 ml capacity. Volume was made up to the mark to give a solution containing 0.16-0.96 μg/ml of LXM and 2-12 μg/ml of EPE. The resulting solution was analyzed by proposed method. The quantitation was carried out by keeping these values to the straight line equation of calibration curve. Method validation The proposed method has been extensively validated in terms of specificity, linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ), robustness and reproducibility. The accuracy was expressed in terms of percent recovery of the known amount of the standard drugs added to the known amount of the pharmaceutical dosage forms. The precision study was expressed as value with respect to the repeatability, intra-day and inter-day variation in the expected drug concentrations. After validation, the developed methods have been applied to pharmaceutical dosage form. Specificity Commonly used excipients (starch, microcrystalline cellulose and magnesium stearate) were spiked into a pre weighed quantity of drugs. The zero order and D1 spectrum was recorded by appropriate dilutions and the quantities of drugs were determined. Linearity Appropriate volume of aliquot from LXM and EPE standard stock solution was transferred to volumetric flask of 10 ml capacity. The volume was adjusted to the mark with methanol to give solutions containing 2-24 μg/ml of both LXM and EPE. All zero order and D1 Spectrum were recorded using above
AKHTAR et al.: ESTIMATION OF LORNOXICAM AND EPERISONE IN THEIR SYNTHETIC MIXTURE 335 spectrophotometric condition. Zero order absorbances were recorded at 265 nm (isoabsorptive point) and 290.5 nm (λ max of Lornoxicam) and D1absorbances at 254 and 264.5 nm were recorded for LXM and EPE, respectively (n=6). Calibration curves were constructed by plotting average absorbance versus concentrations for both drugs. Straight line equations were obtained from these calibration curves. Accuracy Accuracy was assessed by determination of the recovery of the method by addition of standard drug to the pre-quantified placebo preparation at 3 different concentration levels 80, 100 and 120%, taking into consideration percentage purity of added bulk drug samples. Each concentration was analyzed 3 times and average recoveries were measured. Precision The repeatability was evaluated by assaying 6 times of sample solution prepared for assay determination. The intraday and interday precision study of LXM and EPE was carried out by estimating different concentrations of LXM (0.16, 0.48, 0.96 μg/ml) and EPE (2, 6, 12 μg/ml), 3 times on the same day and on 3 different days (first, second, third) and the results are reported in terms of. Detection limit and Quantitation limit ICH guideline describes several approaches to determine the detection and quantitation limits. These include visual evaluation, signal-to-noise ratio and the use of standard deviation of the response and the slope of the calibration curve. In the present study, the LOD and LOQ were based on the third approach and were calculated according to the 3.3σ/S and 10σ/S criterions, respectively; where σ is the standard deviation of y-intercepts of regression lines and S is the slope of the calibration curve. Robustness The sample solution was prepared and then analyzed with change in the typical analytical conditions like stability of analytical solution. Reproducibility The absorbance readings were measured at different laboratory for sample solution using another spectrophotometer by analyst and the values obtained were evaluated using t- test to verify their reproducibility. Results and Discussion and first order derivative method were developed for the determination of LXM and EPE The proposed method has been extensively validated as per ICH guidelines. Considering above facts, wavelength 265 and 290.5 nm were selected in the absorbance ratio method for the estimation of LXM and EPE, respectively wavelength 254 and 264.5 nm were selected for the estimation of LXM and EPE, respectively Linearity was assessed for LXM and EPE by plotting calibration curves of the absorbance versus the concentration over the concentration range 2-24 μg/ml for both drugs in both methods. The correlation coefficients (r 2 ) for LXM and EPE were found to be 0.9984 and 0.9982, respectively in first method and 0.9968 and 0.9971 in derivative method (Table 1). The % recoveries were found to be in the range of 99.51 ± 1.09 for LXM and 99.57 ± 1.44 for EPE in first method and 99.69 ± 1.22 for LXM and 99.13 ± 0.64 for EPE in second method (Table 2). value for intraday and interday precision was less than 2 indicating good method precision (Table 3). The Limit of detection for LXM and EPE was found to be 0.0052 and 0.0042 μg/ml at 291.5 and 254.5 nm respectively and in derivative method 0.18 and 0.046 μg/ml at 254 and 264.5 nm respectively. Limit of quantification for LXM and EPE was found to be 0.015 and 0.013 μg/ml at 291.5 and 254.5 nm Calibration curve of LXM Table 1 Calibration Curve of LXM and LXM Calibration curve of EPE Conc Abs at 265 Absorptivitya x1 nm a x2 (µg/ml) nm a y1 290.5 nm Abs at 290.5 Absorptivity Conc Abs at 265 Absorptivity- Abs at (µg/ml) nm Absorptivity- a y2 2 0.077 0.0385 0.084 0.0420 2 0.084 0.042 0.016 0.008 4 0.16 0.0400 0.162 0.0405 4 0.163 0.041 0.026 0.007 6 0.226 0.0377 0.246 0.0410 6 0.229 0.038 0.038 0.006 8 0.327 0.0409 0.353 0.0441 8 0.301 0.038 0.051 0.006 10 0.341 0.0341 0.371 0.0371 12 0.414 0.035 0.075 0.006 12 0.414 0.0345 0.453 0.0378 16 0.574 0.036 0.091 0.006 Mean (a x1 ) : 0.037 Mean (a x2 ): 0.039 Mean (a y1 ): 0.037 Mean (a y2 ): 0.006
336 INDIAN J. CHEM. TECHNOL., NOVEMBER 2015 Drug Amount of drug Amount of taken (µg/ml) drug added (%) LXM 0.32 EPE 4 respectively and 0.54 and 0.14 μg/ml at 254 and 264.5 nm respectively. The method was also found to be specific, as there was no interference observed when the drugs were estimated in presence of excipients and robust, as there was no significant change in absorbance up to 24 h of preparation of solution in methanol. All the results of validation parameters were shown in result summary (Table 4). The proposed spectrophotometric method was successfully applied to LXM and EPE synthetic mixture and its combined dosage form. Conclusion The proposed absorbance ratio method and first order derivative method provide simple, Table 2 Accuracy study for synthetic mixture Amount of drug found (µg/ml) % Recovery Amount of drug Amount of taken (µg/ml) drug added (%) Amount of drug found (µg/ml) % Recovery 80 0.54 98.43 0.8 80 1.42 100.44 100 0.61 100.92 100 1.50 98.21 120 0.68 99.48 120 1.67 100.44 80 6.99 98.06 10 80 17.29 98.47 100 7.89 100.95 100 19.36 99.75 120 8.64 99.71 120 21.36 99.18 Drug Conc. (μg/ml) Intraday LXM EPE Table 3 Precision study for synthetic mixture Interday Intraday Interday 0.16 0.0164 ± 1 10-4 0.61 0.0162 ± 1.1 10-4 0.71 0.00061± 5.7 10-6 0.94 0.00061± 5.7 10-6 0.94 0.48 0.0478 ± 1.53 10-4 0.32 0.0474 ± 2.5 10-4 0.53 0.00098± 1.1 10-5 1.17 0.00099± 1.5 10-5 1.16 0.96 0.0976 ± 1 10-4 0.10 0.0975 ± 2 10-4 0.21 0.00166± 2.1 10-5 1.25 0.00166± 2.1 10-5 1.25 2 0.1097 ± 1.5 10-5 0.14 0.1091 ± 2.5 10-4 0.23 0.01046± 5.7 10-5 0.55 0.01064± 5.7 10-4 0.54 6 0.3288 ± 3 10-4 0.09 0.3283 ± 3.5 10-4 0.11 0.0264 ± 1 10-4 1.16 0.02616± 5.7 10-5 0.22 12 0.6746 ± 2 10-4 0.03 0.6744 ± 3.5 10-4 0.05 0.05083± 5.7 10-5 0.11 0.0506 ± 1 10-4 0.19 Table 4 Result Summary Sr. No. Parameters LXM EPE Parameters LXM EPE 1 Wavelength 265 nm 290.5 nm Zero Crossing Point 264.5 nm 254 nm 2 Range (µg/ml) 2-24 2-24 Range (µg/ml) 2-24 2-24 3 Linearity R 2 = 0.9984 R 2 = 0.9982 Linearity R 2 = 0.9968 R 2 = 0.9971 4 Precision (%) 0.10-0.61 0.01-0.14 Precision (%) 0.93-1.25 0.11-0.55 Intraday Interday 0.21-0.71 0.05-0.23 IntradayInterday 0.93-1.25 0.19-0.53 5 Accuracy 99.51 ± 1.09 99.57 ± 1.44 Accuracy 99.61 ± 1.22 99.13 ± 0.64 6 LOD 0.0052 0.0042 LOD 0.18 0.046 7 LOQ 0.015 0.013 LOQ 0.54 0.14 8 Robustness Robust Robust Robustness Robust Robust 9 Specificity Specific Specific Specificity Specific Specific specific, precise, accurate and reproducible quantitative analysis for simultaneous determination of LXM and EPE in synthetic mixture. The method is validated as per ICH guidelines in terms of specificity, linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ), robustness and reproducibility. The proposed method can be used for routine analysis and quality control assay of LXM and EPE in combined dosage form. Acknowledgement Authors are thankful to Cirex Pharmaceuticals Limited (Hetero Drugs Limited), Hyderabad for providing gratis sample.
AKHTAR et al.: ESTIMATION OF LORNOXICAM AND EPERISONE IN THEIR SYNTHETIC MIXTURE 337 References 1 Japanese Pharmacopoiea, 16 th edn, (Government of Japan, Ministry of Health, Labour and Welfare, published by the Pharmaceuticals and Medical Devices Agency), 2011. 2 Beckett A H & Stenlake J B, Practical Pharmaceutical Chemistry, 4 th edn Part-II, (C B S Publishers and distributers, New Delhi), 2001. 3 ICH Q2(R1) - Validation of analytical procedures: Text and Methodology, International Conference on Harmonization of technical requirements for registration of pharmaceuticals for human use, Geneva, (2005). 4 Prajapati P, Vaghela V, Rawtani D, Patel H, Kubavat J & Baraiya D, J Pharm Anal, 2(4) (2012) 306. 5 Sahoo S K, Giri R K, Patil S V, Behera A R & Mohapatra R, Trop J Pharm Res, 11 (2) (2012) 269. 6 Patel A, Patel N, Patel M, Lodha A, Chaudhuri J, Jadia P, Joshi T & Dalal J, IOSR J Pharm, 2(3) (2012) 364. 7 Bendale A R, Makwana J J, Narkhede S P, Jadhav A J & Vidyasagar G, J Chem Pharm Res, 3 (2) (2011) 258. 8 Warkar C B & Rele R V, Int J Pharm Bio Sci, 2 (2) (2011) 124. 9 Singh B, Saini G, Sharma D N, Roy S D & Gautam N, Int J Pharm Sci Res, 2 (1) (2011) 102. 10 Lakshmi S, Lakshmi K S & Tintu T, Int J Pharm Pharma Sci, 2 (4) (2010) 166. 11 Jain N, Jain R, Sahu V, Sharma H, Jain S & Jain D, Der Pharma Chemica, 2 (6) (2010) 165. 12 Bhavsar K C, Gaikwad P D, Bankar V H & Pawar S P, Int J Pharm Tech, 2 (2) (2010) 429. 13 Mokale S N, Nirmal S S, Lahoti S R & Sangshetti J N, Der Pharma Sin, 2 (5) (2011) 138. 14 Shah D A, Patel N J, Baldania S L, Chhalotiya U K & Bhatt K K, Sci Pharm, 79 (2011) 113. 15 Kuchekar B S, Sahoo M, Syal P, Ingale S, Ingale K, Sindhe S, Sali M & Choudhari V P, Int J Res Pharm Sci, 2 (1) (2011) 1. 16 Attimarad M, J Basic Clin Pharma, 1 (2) (2010) 115. 17 Jain D, Dubey N & Sharma Y, Int J Pharm Biomed Sci, 2 (2) (2011) 55. 18 Patel U J, Patel P U & Patel S K, Int Res J Pharm, 3 (1) (2012) 203. 19 Sai P, Anupama B, Jagathi V & Rao G, Int J Res Pharm Sci, 1 (3) (2010), 317. 20 Takeshi S, TakeshiY, YukoY, Shota M, Akihiro N, Akira N & Sadaki I, J Health Sci, 56 (5) (2010), 598. 21 Ding L, Wei X, Zhang S, Sheng J & Zhang Y, J Chromatogr Sci, 42 (2004) 254.