Novus International Journal of Chemistry 2012, Vol. 1, No. 1
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1 Novus International Journal of Chemistry 2012, Vol. 1, No. 1 Development and validation of derivative spectrophotometric method for simultaneous estimation of Eperisone hydrochloride and Paracetamol in combined tablet dosage form Nirav Uchadadiya *, Falgun Mehta, Pinak Sanchaniya Department of Pharmaceutical Chemistry and Analysis, Indukaka Ipcowala College of Pharmacy, New Vallabh Vidyanagar, Gujarat , India ABSTRACT Simple, accurate, precise and specific spectrophotometric method has been developed for simultaneous determination of Eperisone Hydrochloride (EPE) and Paracetamol (PCM) in its combined tablet dosage form by using methanol as a solvent. The method is second order derivative method in which absorption at nm (ZCP of PCM) was used for quantification of EPE and nm (ZCP of EPE) for quantification of PCM. Method follows Beer s linearity in the range of 2-10μg/ml for EPE and 10-50μg/ml for PCM. The mean percentage recoveries were found to be in the range of % and % for EPE and PCM respectively. The proposed method was validated as per ICH guidelines and successfully applied to the estimation of EPE and PCM in combined Tablet dosage form. KEYWORDS: Eperisone Hydrochloride, Paracetamol, Second order derivative, Analytical Method validation. INTRODUCTION *Corresponding author: Nirav Uchadadiya Indukaka Ipcowala College of Pharmacy, Beyond GIDC Phase IV, P.B. NO. 53, P.O. Vithal Udyog Nagar, New Vallabh Vidyanagar, Gujarat , India nikup982@gmail.com EPE is chemically (2RS)1-Propanone,1-(4-ethylphenyl)-2-methyl-3-(1-piperidinyl)-, hydrochloride (1:1) (Figure 1a) 1,2, EPE is a centrally acting muscle relaxant, it acts at the level of spinal cord by blocking sodium channels and calcium channels. EPE exerts its spinal reflex inhibitory action predominantly via a pre-synaptic inhibition of the transmitter release from the primary afferent endings via a combined action on voltage-gated sodium and calcium channels. EPE increases the blood supply to skeletal muscles; this action is noteworthy since a muscle contracture may compress the small blood vessels and induce an ischemia leading to release of pain stimulating compounds 3. EPE is official in Japanese pharmacopoeia 1. Chemically PCM is, N-(4-hydroxyphenyl) acetamide (Figure 1b), PCM is a weak inhibitor of PG synthesis of COX-1 and COX-2. Cyclo-oxygenase serves as a pain activator, is responsible for the biosynthesis of prostoglandins. Used for the relief of headaches and other minor aches and pains and is a major ingredient in numerous cold and flu remedies 4. PCM is official in Japanese Pharmacopoeia 1, British Pharmacopoeia 5, United States Pharmacopoeia 6 and Indian Pharmacopoeia 7. Novus International Journal of Chemistry 2012, 1(1) 33
2 The review of literature revealed that analytical methods involving spectrophotometry 8, LC- ESI-MS 9, have been reported for EPE in single form and in combination with other drugs. Several analytical methods have been reported for PCM in single form and in combination with other drugs including spectrophotometry 10-14, HPLC 15-17, HPTLC 18,19. To the best of our knowledge, there is no published spectrophotometric method for this combination. So, the present paper describes a simple, accurate and precise method for simultaneous estimation of EPE and PCM in combined tablet dosage form by second order derivative method. The developed method were validated in accordance with ICH Guidelines 21 and successfully employed for the assay of EPE and PCM in combined dosage form. (a) (b) Figure 1: Chemical structure of (a) EPE and (b) PCM MATERIALS AND METHODS Reagents and chemicals Analytically pure EPE and PCM were kindly provided by Macleods Pharmaceuticals Ltd, Mumbai, Maharastra, India and Elysium Pharmacutical Ltd, Vadodara, Gujarat, India respectively as gratis samples. Analytical grade methanol was purchased from SRL limited, Mumbai, India. Tablet of EPE and PCM in combined dosage form, Myosone Plus, was procured from local market. Instruments A Shimadzu UV/Vis 1800 double beam spectrophotometer with a wavelength accuracy (± 0.3 nm), 1 cm matched quartz cells and UV probe 2.34 software was used for all the spectral measurements. Calibrated analytical balance Shimadzu BL-220H (Shimadzu Corporation Japan) was used for weighing purpose. Spectrophotometric condition All zero order spectrums (D 0 ) were converted to second derivative spectrum (D 2 ) using delta lambda 8. Preparation standard stock solutions Accurately weighed 100 mg of EPE and PCM standard were transferred to a separate 100 ml volumetric flask and dissolved in 50 ml methanol. The flasks were sonicated and volume was made up to the mark with methanol to give solutions containing 1000 µg/ml EPE and 1000 µg/ml PCM. From this solution 10 ml was transferred to volumetric flask of 100 ml capacity. Novus International Journal of Chemistry 2012, 1(1) 34
3 Volume was made up to the mark to give a solution containing 100 µg/ml of EPE and 100 µg/ml PCM. Second order Derivative method 2-10 µg/ml solution of EPE and µg/ml solution of PCM were prepared in methanol by appropriate dilution and spectrum was recorded between nm and first derivative spectrums were obtained using above condition. The overlain second derivative spectrums of EPE and PCM at different concentration were recorded. The zero crossing point (ZCP) of EPE was found to be nm and ZCP of PCM was found to be nm. 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 (Coefficient of Variation - C.V.) was expressed 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) were spiked into a pre weighed quantity of drugs. The D 2 spectrum was recorded by appropriate dilutions and the quantities of drugs were determined. Linearity Appropriate volume of aliquot from EPE and PCM 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 2-10 µg/ml EPE and µg/ml PCM. All D 0 and D 2 Spectrum were recorded using above spectrophotometric condition. D 2 absorbance at nm and nm were recorded for EPE and PCM 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 50, 100 and 150 %, 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 inter-day precision study of EPE and PCM was carried out by estimating different concentrations of EPE (4, 6, 8 µg/ml) and PCM (20, 30, 40 µg/ml), 3 times on the same day and on 3 different days (first, second, fifth) and the results are reported in terms of C.V. Detection limit and Quantization limit ICH guideline describes several approaches to determine the detection and quantization limits. 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. Novus International Journal of Chemistry 2012, 1(1) 35
4 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. Determination of EPE and PCM in their Combined Dosage Form Twenty tablets were weighed and powdered. A powder quantity equivalent to 50 mg EPE and 325 mg PCM was accurately weighed and transferred to volumetric flask of 100 ml capacity. 60 ml of methanol was transferred to this volumetric flask and sonicated for 15 min. The above solution was filtered through whatman filter paper (0.45µ).The flask was shaken and volume was made up to the mark with methanol. From this solution 20 ml was transferred to volumetric flask of 100 ml capacity and Volume was made up to the mark to give 100 µg/ml EPE and 650 µg/ml PCM. From this solution 4 ml was transferred to volumetric flask of 100 ml capacity and Volume was made up to the mark to give a solution containing 4 µg/ml of EPE and 26 µg/ml of PCM. The resulting solution was analysed by proposed methods. The quantization was carried out by keeping these values to the straight line equation of calibration curve. RESULTS AND DISCUSSION In Second order derivative method the overlain D 2 spectrum of EPE and PCM at different concentrations revealed that at nm (ZCP of PCM) EPE possesses significant D 2 absorbance and at nm (ZCP of EPE) PCM possesses significant D 2 absorbance. Considering above facts, wavelength nm and nm were selected for the estimation of EPE and PCM, respectively (Figure 3) Linearity was assessed for EPE and PCM by plotting calibration curves of the D 2 absorbance versus the concentration over the concentration range 2-10 µg/ml for EPE (Figure 4a) and µg/ml for PCM ( Figure 4b). The correlation coefficients (r 2 ) for EPE and PCM were found to be and 0.999, respectively (Table 1.2). The following equations for straight line were obtained for EPE and PCM. Linear equation for EPE, y = 0.021x Linear equation for PCM, y = 0.013x The % recoveries were found to be in the range of for EPE and % for PCM (Table 1.3). The precision of method was determined by repeatability, intraday and inter-day precision and was expressed as the C.V. (Table ), which indicate good method precision. The Limit of detection for EPE and PCM was found to be 0.625μg/ml and 0.197μg/ml respectively. Limit of quantification for EPE and PCM was found to be 1.895µg/ml and 0.596μg/ml at nm and at nm respectively (Table 1.1). Method were 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 hours of preparation of solution in methanol. The proposed spectrophotometric method was successfully applied to EPE and PCM in combined dosage form. Novus International Journal of Chemistry 2012, 1(1) 36
5 Second order Derivative spectrophotometric Method Figure2: Overlain zero order absorbance spectra of EPE and PCM in methanol Figure 3. Overlain D 2 spectrum of EPE (2-10µg/ml) and PCM (10-50 µg/ml) in methanol EPE ABS PCM ABS y = 0.021x R² = EPE ABS Linear (EPE ABS) y = 0.013x R² = PCM ABS Linear (PCM ABS) (a) Figure 4: Calibration Curve of (a) EPE and (b) PCM (b) Table 1.1: Summary of Validation Parameters Parameters EPE PCM Recovery % Repeatability(C.V.) ( n=6) Precision Intra-day(n=3) Inter-day(n=3) Limit of Detection (μg/ml) Limit of Quantitaion (μg/ml) Specificity Specific Specific Robustness Robust Robust Solvent suitability Suitable for 24 hrs. Suitable for 24 hrs. Novus International Journal of Chemistry 2012, 1(1) 37
6 Table 1.2: Statistical data EPE and PCM by Second order derivative method Parameter EPE PCM Analytical Wavelength nm nm Range 2-10 µg/ml µg/ml Slope 0.021x 0.013x Intercept Regression Coefficient (r2) Standard deviation of Slope Standard deviation of Intercept % Level Table 1.3: Accuracy data for EPE and PCM by second order derivative method Total Amount drug(formulation) +added (μg/ml) Amount recovered (μg/ml) % Recovery EPE PCM EPE PCM % EPE % PCM (μg/ml) (μg/ml) (μg/ml) (μg/ml) 50 % % % Table 1.4: Precision data for EPE at nm Conc. Intraday C.V. Inter-day C.V. (μg/ml) (Abs. ± S.D) (Abs. ± S.D) ± ± ± ± ± ± (n = 3 determination) Conc. (μg/ml) Table 1.5: Precision data for PCM at nm Intraday (Abs. ± S.D) C.V. Inter-day (Abs. ± S.D) C.V ± ± ± ± ± ± (n = 3 determination) Table 1.6: Assay Results of Marketed Formulation Formulation Drug Amount Taken (μg/ml) Amount Found (μg/ml) (n = 3) Labelled Claim (mg) Amount found per Tablet (mg) % Label claim ±SD MYOSONE PLUS (Tablet) EPE ± 1.6 PCM ± 0.48 Novus International Journal of Chemistry 2012, 1(1) 38
7 CONCLUSION The proposed second order derivative method provide simple, specific, precise, accurate and reproducible quantitative analysis for simultaneous determination of EPE and PCM in combined dosage form. The methods were validated as per ICH guidelines in terms of specificity, linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ), robustness and reproducibility. The proposed methods can be used for routine analysis and quality control assay of EPE and PCM in combined dosage form. ACKNOWLEDGEMENT Authors are thankful to Macleods Pharmaceuticals Ltd. (Mumbai, India) and Elysium Pharmacutical Ltd (Vadodara, India) for providing gratis sample with the great pleasure. The authors also thankful to Indukaka Ipcowala College of Pharmacy (Newvallabh Vidyanagar, India) for providing the necessary facilities for research work and to all the staff members and friends for their guidance and help throughout the research work. REFERENCES 1. Japanese Pharmacopoeia, govt of Japan, the ministry of Health, Labour and Welfare 16 th edi, The Merck index, An encyclopedia of chemicals, drugs and biological, Merck Research Laboratories, USA2006, 13 th edi, 10, Beltrame A, Granngie S and Guerra L, Clinical Experience with eperisone in the treatment of acute low back pain. Minerva Medica. 2008, 99(4): Tripathi KD, Essentials of Medical Pharmacology, 5 th Edn, Jaypee Brothers Limited, 2008, British Pharmacopoeia,,The Stationery Office on behalf of the Medicines and Healthcare products Regulatory Agency (MHRA),London, 2011, vol-i, United States Pharmacopoeia and National Formulary, (24 th ) Asian Edition, The United States Pharmacopoeia Convention Inc., U.S.A., Indian pharmacopoeia, Govt. of India,Ministry of health & family welfare, the controller & publication, Delhi, 2010, (vol-iii), Patel PU, Patel SK and Patel UJ, Spectrophotometric Method for Simultaneous Estimation of Eperisone hcl and Diclofenac Sodium in Synthetic mixture. Int. res. J. of pharm., 2012, Zhang S, Ding L, Weix,Sheng J and Zhang Y, Rapid and Sensitive Liquid Chromatography, Electrospray Ionization, Mass Spectrometry method for the determination of Eperisone in Human plasma. J. of chrom. sci., 2004, 42, Kondawar MS, Shah RR, Waghmare JJ and Shah ND, UV Spectrophotometric estimation of Paracetamol and Lornoxicam in Bulk drug and Tablet dosage form using Multiwavelength method. Int. J. of Pharm. Tech. Res., 2011, 3: Sawant R,Bhangale L,Joshi R and Lanke P, Validated spectrophotometric methods for simultaneous estimation of Paracetamol, Domperidone and Tramadol HCl in pure and tablet dosage form. J. Chem. Metrol, 2010, Kalra K, Naik S, Jarmal G and Mishra N, Spectrophotometric Method for Simultaneous Estimation of Paracetamol and Domperidone in Tablet Formulation Asian J. Res. Chem., 2009, Novus International Journal of Chemistry 2012, 1(1) 39
8 13. Narajji C,Patel HR and Karvekar MD, Simultaneous Estimation of Aceclofenac, Paracetamol and Tizanidine in their Combined Dosage forms by Spectrophotometric and RP- HPLC Method. J. Anal Bioanal Techniques, 2011, Mahaparale S, Telekone RS and Raut RP, Simultaneous spectrophotometric determination of drotaverine HCl and Paracetamol in tablet. Indian J. of Pharm. Sci., 2010, Prasanna RB and Reddy MS RP-HPLC Method for Simultaneous Estimation of Paracetamol and Ibuprofen in Tablets. Asian J. Res. Chem., 2009, Lotfi MA and Frida D, simultaneous LC determination of paracetamol and related compounds in pharmaceutical formulations using a carbon-based column. J. of Pharm. And Biomedical Anal., 2002, Dudhane NP,Umekar MJ and Lohiya RT, Validated RP-HPLC Method for Estimation of Metoclopramide Hydrochloride and Paracetamol in solid dosage form J. of Pharm. Res., 2010, Yadav A, Singh MR, Mathur CS, Saini KP, and Singh NG, A Simple and Sensitive HPTLC Method for Simultaneous Analysis of Domperidone and Paracetamol in Tablet Dosage Forms. J. of Planar Chrom., 2009, Deshpanday PB, Gandhi VS and Bhangale SY, HPTLC Method for Simultaneous estimation of Etoricoxib and paracetamol in Combined Tablet Dosage. J. Of Pharm. And Bio. Anal., 2010, 03: Bechett A.H., Stenlake J.B., Practical Pharmaceutical Chemistry, CBS publishers and distributors,4th edi, 2002, part II, , ICH Harmonized Tripartite Guidelines, Validation of analytical procedures: Text and Methodology, Q2 (R1), Geneva, Novus International Journal of Chemistry 2012, 1(1) 40
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