Vol - 4, Issue - 3, Supl - 1 Apr-Jul 2013 ISSN: Patel et al PHARMA SCIENCE MONITOR
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1 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR ESTIMATION OF DICLOFENAC SODIUM AND EPERISONE HYDROCHLORIDE IN PHARMACEUTICAL DOSAGE FORM Piyush M. Patel*, Chaitany A. Dave, Shashank K. Tiwari, Kunal D. Brahmbhatt Department of Quality Assurance, Shivam Pharmaceutical Studies and Research Centre, Valasan, Anand (Gujarat) ABSTRACT A simple, accurate and precise RP-HPLC method was developed for Diclofenac sodium and Eperisone Hydrochloride. Phenomanex Luna C 18 Column, (250 x 4.6 mm i.d.), Particle size 5μm was used as stationary phase. The mobile phase used was Acetonitrile: 0.05M Phosphate buffer (65:35 v/v) at ph 3.5. adjusted by ortho-phosphoric acid.. The mobile phase was delivered at flow rate 1.0 ml/min. UV detection was set at 280nm. The retention time of Diclofenac sodium was min and Eperisone Hydrochloride was min.the Linearity was observed over the concentration range of 5-15µg/ml and 15-45µg/ml for Diclofenac sodium and Eperisone Hydrochloride respectively. The LOD was found to be μg/ml and μg/ml for Diclofenac sodium and Eperisone Hydrochloride respectively. Whereas, LOQ was found to be μg/ml for Diclofenac sodium and μg/ml for Eperisone Hydrochloride. Moreover, the % RSD for repeatability, inter and intraday precision was found to be less than 2%, which reveals that the method is precise. The % recovery was found to be to % for Diclofenac sodium and to % for Eperisone Hydrochloride.However, the change in ph, flow rate and organic phase ratio also did not show any significant variance. Assay of the combined dosage form finalized the applicability of this method for simultaneous estimation of Diclofenac sodium and Eperisone Hydrochloride in Pharmaceutical dosage forms. Keywords: Diclofenac sodium, Eperisone Hydrochloride, method validation, HPLC. INTRODUCTION Diclofenac Sodium (sodium 2-[(2,6-dichlorophenyl)-amino]phenyl acetate) (Figure-1) is Aminobenzoates and Phenylacetaes used in the treatment of signs and symptoms of osteoarthritis and rheumatoid arthritis.it gives inhibition of leukocyte migration and the enzyme cyclo-oxygenase (COX-1 and COX-2), leading to the peripheral inhibition of prostaglandin synthesis. As prostaglandins sensitize pain receptors, inhibition of their synthesis is responsible for the analgesic effects of Diclofenac Antipyretic effects may be due to action on the hypothalamus, resulting in peripheral dilation, increased blood flow, and subsequent heat dissipation. IC Value
2 Eperisone Hydrochloride [(2RS)-1-(4-ethylphenyl)-2-methyl-3-(1 piperidyl)propan-1- one] (Figure-2) is Anti- Spasmodic drug used in treatment of Low back pain syndrome, Painful reflex muscle spasm, Post-cerebral stroke spasticity. It gives Myonal exhibits both skeletal muscle relaxant and vasodilator properties due to its actions within the CNS and on vascular smooth muscle. This combination is use for the treatment of Patients with acute musculoskeletal spasm associated with low back pain, pain management in Arthritis, Rheumatoid arthritis, acute non specific low back pain, and in accidentally muscle spasm. Combined dosage form of Diclofenac Sodium and Eperisone HCl is CDSCO Approved on In lieterature survey,based on my current referencing work I have not come across any reported RP-HPLC Method for Simultaneous Estimation of both drugs in synthetic mixture. [1-2] DICLOFENAC SODIUM EPERISONE HCl Figure1:-Structure of Diclofenac Sodium and Eperisone HCl. [3-4] MATERIAL AND METHODS: [5-7] Materials Diclofenac Sodium and Eperisone HCl samples were provided by Molecule laboratory,ahmedabad. All Chemicals get from this laboratory.and all work had been done at Molecule laboratory,ahmedabad. Instruments & Apparatus Shimadzu HPLC with Phenomanex Luna C 18 Column, (250 x 4.6 mm i.d.), Particle size 5μm;Labindia Double beam UV Visible spectrophotometer; Soltec sonica Ultrasonicator; IC Value
3 Labindia ph Meter. Borosil Measuring cylinder 10 to 100 ml. Borosil Pipette 1-10ml. Borosil Volumetric flask 10 ml, 100 ml. (all apparatus were previously calibrated and made up of glass.) Chromatographic conditions The mobile phase, a mixture of Acetonitrile: Phosphate buffer (65:35v/v) pumped at a flow rate of 1.0 ml/min through the Phenomanex Luna C 18 Column (250 x 4.6 mm i.d.), Particle size 5μm at 37 0 C.The mobile phase was degassed prior to use under vacuum by filtration through a 0.45 μm filter paper.concentrations were measured at 280 nm by UV detector. METHODOLOGY Preparation of standard stock solution Diclofenac Sodium standard stock solution (100µg/ml): Stock solution was prepared by accurately weighing 10 mg standard powder of Diclofenac Sodium and transferring to 100ml volumetric flask containing a few ml of Mobile Phase.Volume was made up to the mark with Mobile Phase to yield a solution containing 100µg/ml of Diclofenac Sodium. Eperisone Hydrochloride standard stock solution (300µg/ml): Stock solution was prepared by accurately weighing 30 mg standard powder of Eperisone HCl and transferring to 100ml volumetric flask containing a few ml of Mobile Phase.Volume was made up to the mark with Mobile Phase to yield a solution containing 300µg/ml of Eperisone Hydrochloride. Calibration curve for Diclofenac Sodium and Eperisone Hydrochloride: Appropriate volume from standard Diclofenac Sodium and Eperisone HCl stock solution was transferred to 10 ml volumetric flask. The volume was then adjusted to the mark with mobile phase to give a solution containing 5.0,7.5,10.0,12.5 and 15.0μg/ml of Diclofenac Sodium 15, 22.5, 30, 37.5 and 45.0μg/ml of Eperisone HCl.The mixed standard solution was chromatographed using mobile phase at a flow rate of 1.0 ml/min.the calibration curve was plotted as peak area vs. concentration for both the drugs. Optimized Chromatographic conditions: IC Value
4 Stationary phase : Phenomanex Luna C 18 Column(250 x 4.6 mm i.d.), Particle size 5μm; Mobile phase: Acetonitrile:0.05M Phosphate buffer(65:35 v/v) at ph 3.5 adjusted by ortho-phosphoric acid; Detection wavelength: 280nm; Injection volume : 20 µl ; Temperature of column : Room temperature ; Flow rate : 1.0 ml/min. Figure 2:- Chromatogram of mixed standard solution containing 10μg/ml Diclofenac Sodium and 30μg/ml Eperisone HCl using mobile phase Acetonitrile:0.05 M Phosphate Buffer ph 3.5 Adjusted with Orthophosphoric Acid (65:35,v/v) at 280nm. System Suitability test: System suitability parameters mainly include Resolution,Column efficiency in terms of theoretical plates and tailing factor.this parameters are as follows: TABLE 1: RESULS OF SYSTEM SUITABILITY TEST PARAMETER SYSTEM SUITABILITY PARAMETERS Retention times (R T ) (min) Theoretical plates (N) DICLOFENAC SODIUM EPERISONE HCl R T > N > 2000 Resolution (R S ) R S > 2 Tailing factor (A S ) A S < 2 SPECIFICATIONS IC Value
5 VALIDATION OF RP-HPLC METHOD: [8-10] 1. Linearity and Range: The linearity range for Diclofenac Sodium was found to be in the range of 5-15µg/ml and for Eperisone Hydrochloride was 15-45µg/ml. Calibration curve data for Diclofenac Sodium and Dicyclomine Hydrochloride are presented in Table: 2-3.Calibration curve for Diclofenac Sodium and Dicyclomine Hydrochloride are depicted in figure:3-4. Figure 3:- Calibration curve of Omeprazole Figure 4:- Calibration curve of Dicyclomine HCl TABLE 2: RESULT OF CALIBRATION READINGS FOR DICLOFENAC SODIUM Concentrations (μg/ml) Area Mean ± S.D. (n=5) %RSD ± ± ± ± ± IC Value
6 TABLE 3: RESULT OF CALIBRATION READINGS FOR EPERISONE HCL Concentrations (μg/ml) Area Mean ± S.D. (n=5) %RSD ± ± ± ± ± Accuracy (% Recovery): The data for accuracy for Diclofenac Sodium and Eperisone HCl are presented in T TABLE 4: ACCURACY DATA OF DICLOFENAC SODIUM AND EPERISONE Drugs Diclofenac Sodium Eperisone HCl Accuracy level Amt. taken HCL Amt. added Recovered Conc. Mean % Recovery ±SD 80% ± % ± % ± % ± % ± % ± Precision: I. Intra-day precision: For intra-day, Diclofenac Sodium 10µg/ml and Eperisone HCl 10µg/ml were analyzed three times in a day and % relative standard deviation (%RSD) was calculated in table:5. TABLE 5: INTRA-DAY PRECISION DATA FOR DICLOFENAC SODIUM AND EPERISONE HCL Conc DICLOFENAC SODIUM Conc PERISONE HCL Mean Area± SD %RSD Mean Area± SD %RSD ± ± ± ± ± ± IC Value
7 II. Inter-day precision For interday, Diclofenac Sodium 10µg/ml and Eperisone HCl 30µg/ml were analyzed on three different days and % releative standard deviation (%RSD) was calculated in table:6. TABLE 6: INTER-DAY PRECISION DATA FOR DICLOFENAC SODIUM AND EPERISONE HCL Conc DICLOFENAC SODIUM Conc EPERISONE HCl Mean Area± SD %RSD Mean Area± SD %RSD ± ± ± ± ± ± Limit of Detection: The limit of detection for for Diclofenac Sodium and Eperisone HCl was found to be μg/ml and μg/ml. 5. Limit of Quantification: The limit of quantification for Diclofenac Sodium and Eperisone HCl was found to be μg/ml and μg/ml. 6. Robustness: To evaluate robustness of the method few parameters were purposely varied. The parameters were included variation of flow rate,change in ph of the buffer, change in mobile phase ratio. The average value of % RSD for determination of Diclofenac Sodium and Eperisone HCl less than 2 % revealed the robustness of the method. IC Value
8 TABLE 7: ROBUSTNESS DATA FOR DICLOFENAC SODIUM AND EPERISONE HCL Paramete Variation Area %RSD r DIC EPR IC PR Flow rate +0.2ml/min ml/min ph Mobile phase Assay of Combined Dosage Form: The results are shown in Table 8. The data revealed % for Diclofenac Sodium and for. Eperisone HCl. These data confirmed the applicability of this method for combined dosage forms. TABLE 8: ASSAY OF COMBINED DOSAGE FORM Formulation Drug Labeled Claim (mg/tab) Tablets Diclofenac Sodium Amount Found (mg/tab) Eperisone HCl %Assay IC Value
9 TABLE 9: RESULT OF VALIDATION PARAMETERS PARAMETERS DATA OBTAINED DICLOFENAC SODIUM EPERISONE HCL Linearity Linearity Range Correlation Co-efficient Precision Intraday (%RSD) Interday Accuracy (% Recovery) LOD(μg/ml) LOQ(μg/ml) Robustness Robust Robust % Assay RESULTS AND DISCUSSION RP-HPLC method was developed for the estimation of DIC and EPR in Pharmaceutical dosage form. The chromatogram for DIC and EPR obtained by the proposed method showed the retention time to be min and min correspondingly. The drugs existing a good linearity at the range of 05 to 15μg/ml for DIC and 15 to 45μg/ml EPR respectively which assures that the proposed chromatographic conditions and the mobile phase were suitable for the opinion. The chromatographic conditions were optimized using mobile phase of varied polarity. Finally a mobile phase consisting of Acetonitrile:0.05M phosphate buffer (ph- 3.5) in the ratio of 65:35 gave good linearity of response and the shape of chromatogram was also perfect. The regression equation of the drugs concentration over peak area was calculated. The system suitability parameters are presented in table 1. The number of theoretical plates for DIC and EPR was found to be 7269 and 2640 respectively which confirms the good efficiency of the column for the drugs and nature of mobile phase. The low value of LOD and LOQ indicates the sensitivity of the method. This method reduces the total run time for HPLC, leads to low solvent consumption and makes the analysis more efficient. The method is rapid, precise,simple and accurate for simultaneous estimation of Diclofenac Sodium and Eperisone Hydrochloride in their combined dosage forms. IC Value
10 ACKNOWLEDGEMENT The author are thankful to Molecule Laboratory,Ahmedabad.Also Thanks my Friends Chaitany A.Dave, Mr.Shashank Tiwari,Mr.kunal Brahmbhatt,Mr.Shrey Shah, Mr.Arshad Hala for successful conduct & completion of this research work. REFERENCES 1. Tripathi K.D, Essential of Medical Pharmacology, 6 th Edition, Jaypee brothers, 2005, pp Goyal R.K. et al, Elements of pharmacology, 16th Edition, B.S.Shah Prakashan, , pp pharmacological & pharmacokinetic properties of diclofenac sodium December 2012, 4. pharmacological properties of Eperisone hydrochloride December 2012, 5. Sharma B.K, Instrumental Methods of Chemical Analysis, GOEL Publication House, Meerut, pp Ahuja S, Sephen S, Modern Pharmaceutical Analysis Separation Science and Technology, 3 rd Volume, 1 st Indian Reprint, Academic Press, 2005, pp Skoog D.A. et al, Fundamentals of Analytical Chemistry, 8 th Edition, Thomson Asia Pvt Ltd, Singapore, 2004, pp ICH guidelines,validation of analytical procedure Methodology Q2B, I.C.H. Harmonized Tripartite Guidelines, ICH, Q2A Text on validation of analytical procedures, Oct 1994; International Conference on Harmonization. 10. ICH, Q3B validation of analytical procedures, methodology Nov 1996; International conference on Harmonization. For Correspondence: Piyush M. Patel pusptl363738@gmail.com IC Value
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