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1 Impact factor: 3.958/ICV: 4.10 ISSN: Pharma Science Monitor 9(2), Apr-Jun 2018 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES Journal home page: STABILITY INDICATING HPLC METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF ACECLOFENAC AND CYCLOBENZAPRINE HCl IN ITS PHARMACEUTICAL DOSAGE FORM Parekh Mukti P.*, Pragnesh Patani, Seju Patel Department of Quality Assurance, A-One college of Pharmacy, Enasan, India. ABSTRACT A simple, rapid, economical, precise and accurate stability indicating RP-HPLC method was developed for the simultaneous estimation of Cyclobenzaprine HCl and Aceclofenac in their combined dosage form has been developed. The separation was achieved by LC- 20 AT C18 (250mm x 4.6 mm x 2.6 µm) column and Buffer (ph 5.0): Methanol (60:40) as mobile phase, at a flow rate of 1 ml/min. Detection was carried out at 237 nm. Retention time of Cyclobenzaprine HCl and Aceclofenac were found to be min and min, respectively. The method has been validated for linearity, accuracy and precision. Linearity observed for Cyclobenzaprine HCl μg/ml and for Aceclofenac20-60 μg/ml. Developed method was found to be accurate, precise and rapid for simultaneous estimation of Cyclobenzaprine HCl and Aceclofenac in their combined dosage form. The drug was subjected to stress condition of hydrolysis, oxidation, photolysis and Thermal degradation, Considerable Degradation was found in alkaline degradation. The proposed method was successfully applied for the simultaneous estimation of both the drugs in commercial combined dosage form. KEYWORDS: Cyclobenzaprine HCl, Aceclofenac, Stability indicating RP-HPLC Method, Validation. INTRODUCTION 1-5 Aceclofenac down regulates the production of various inflammatory mediators including prostaglandin E2 (PGE2), IL-1β, and TNF from the arachidonic acid (AA) pathway. Inhibition of IL-6 is thought to be mediated by diclofenac converted from Aceclofenac. Aceclofenac is an Analgesic and NSAIDs, They used together with rest and physical therapy to treat skeletal muscle conditions such as pain or injury. Cyclobenzaprine exhibits anticholinergic activity, potentiation of norepinephrine, and antagonism of reserpine. Cyclobenzaprine does not directly act on the neuromuscular junction or the muscle but relieves muscle spasms through a central action, possibly at the brain stem level. Cyclobenzaprine binds to the serotonin receptor and is considered a 5-HT2 receptor antagonist that reduces muscle tone by decreasing the activity of descending serotonergic neurons.it works by blocking nerve impulses (or pain sensations) that are sent to your brain. Various methods are
2 Impact factor: 3.958/ICV: 4.10 ISSN: reported for the analysis of individual drug and in combination with other drugs for Aceclofenac and Cyclobenzaprine HCl but no Stability indicating HPLC method reported for these drugs in combined dosage form. Therefore, I proposed worthwhile to develop stability indicating RP- HPLC Method for the Simultaneous Estimation of Aceclofenac and Cyclobenzaprine HClin their Combined Dosage form. Fig: 1 Structure of Aceclofenac Fig: 2 Structure of Cyclobenzaprine HCl MATERIAL AND METHODS: Materials and Reagents: Aceclofenacand Cyclobenzaprine HClwere procured as generous gift sample by supplied by Provizer Pharma. Acetonitrile, water, methanol of HPLC grade were Purchased from (Aventor Performance Material, India) Preparation of standard solutions: (A) Aceclofenac standard stock solution: (400μg/mL) A 40 mg of Aceclofenacwas weighed and transferred to a 100mL volumetric flask. Volume was made up to the mark with methanol. (B) CyclobenzaprineHClstandard stock solution: (30μg/mL) A 30 mg of Cyclobenzaprine HClwas weighed and transferred to a 100 ml volumetric flask.and volume was made up to the mark with methanol and take 10 ml from this solution in 100 ml volumetric flask and make up with methanol. (C) Preparation of standard solution of binary mixtures ofaceclofenac (40 μg/ml) and Cyclobenzaprine HCl (3 μg/ml) Take 1 ml from the Aceclofenacstock solution and 1mL from Cyclobenzaprine HClstock solution and transferred to 10 ml volumetric flask and volume made up to the mark by mobile phase which was used in particular trials.
3 Impact factor: 3.958/ICV: 4.10 ISSN: Preparation of Mobile Phase: Composition: Potassium dihydrogen phosphate buffer 0.05M (ph 5.0): Methanol (60:40 v/v) Preparation of Buffer: Take 6.8 gm of Potassium dihydrogen phosphate buffer and add into the 800 ml of water and shake well to dissolve the KH2PO4 and then add 200 ml of water to make 1000 ml of Buffer Solution. Adjust the ph of Buffer with the 0.1 NNaOH to make the ph of Buffer 5.0 Chromatographic separation: Standard solutions of 20-60μg/ml of Aceclofenac and μg/ml Cyclobenzaprine HCl were injected in column with 20 μl micro-syringe. The chromatogram was run for appropriate minutes with mobile phase Buffer (ph 5): Methanol(60:40). The detection was carried out at wavelength 237nm. The chromatogram was stopped after separation achieved completely. Data related to peak like area, height, retention time, resolution etc were recorded using software. Chromatographic conditions: Column: C18 (25 cm 0.46 cm) Hypersil BDS Mobile Phase: Buffer (ph 5): Methanol (60:40) Flow Rate: 1.0 ml/min Detection Wavelength: 237 nm Run time: 8 min Injection volume: 20.0 μl STABILITY INDICATING METHOD: 6-8 (A)Acid degradation Acid decomposition studies were performed by Transferring 1 ml of stocksolution in to 10 ml of volumetric flask. 2 ml of 0.1 N HCl solutions was added and mixed well and put for 7 hrs. After time period the volume was adjusted with diluent to get 3μg/ml for Cyclobenzaprine HCl and 40μg/ml for Aceclofenac. (B)Base degradation Basic decomposition studies were performed by Transferring 1ml of stock solution in to 10 ml of volumetric flask. 2 ml of 0.1 N NaOH solutions was added and mixed well and put for 4 hrs. After time period the volume was adjusted with diluents to get 3μg/ml for Cyclobenzaprine HCl and 40μg/ml for Aceclofenac. (C)Oxidative degradation Oxidative decomposition studies were performed by Transferring One ml of stock solution in to 10
4 Impact factor: 3.958/ICV: 4.10 ISSN: ml of volumetric flask. Two ml of 3% H2O2 solutions was added and mixed well and put for 4 hrs. After time period the volume was adjusted with diluent to get 3μg/ml for Cyclobenzaprine HCl and 40μg/ml for Aceclofenac. (D) Photo Degradation Photo Degradation studies were performed by Transferring 1 ml of stock solution in to 10 ml of volumetric flask. The volumetric flask was kept under UV light in UV Chamber for 12hrs. Then the volume was adjusted with diluent to get 3μg/ml for Cyclobenzaprine HCl and 40μg/ml for Aceclofenac. (E)Thermal degradation Thermal Degradation studies were performed by Transferring 1 ml of stock solution in to 10 ml of volumetric flask. The volumetric flask was stored in oven at 80 C for 10 hrs. Then the volume was adjusted with diluent to get 3μg/ml for Cyclobenzaprine HCl and 40μg/ml for Aceclofenac. METHOD VALIDATION 9 Linearity The linearity for Aceclofenac and Cyclobenzaprine HCl were assessed by analysis of combined standard solution in range of 20-60μg/ml and μg/ml respectively, 5, 7.5, 10, 12.5,15 ml solutions were pipette out from the Stock solution of Aceclofenac (400μg/ml) and Cyclobenzaprine HCl (30μg/ml) and transfer to 100 ml volumetric flask and make up with mobile phase to obtain 20,30,40,50 and 60μg/ml and 1.5,2.25,33.75 and 4.5μg/ml for Aceclofenac and Cyclobenzaprine HCl respectively In term of slope, intercept and correlation co-efficient value, the graph of peak area obtained verses respective concentration was plotted. Precision Results should be expressed as Relative standard deviation (RSD) or coefficient of variance. (A) Repeatability Standard solution containing Aceclofenac (40µg/ml) and Cyclobenzaprine HCl (3µg/ml) was injected six times and areas of peaks were measured and % R.S.D. was calculated. (B) Intra-day precision Standard solution containing (20,40,60µg/ml) of Aceclofenac and (1.5,3,4.5µg/ml) of Cyclobenzaprine HCl were analyzed three times on the same day and % R.S.D was calculated. (C) Inter-day precision
5 Impact factor: 3.958/ICV: 4.10 ISSN: Standard solution containing (20,40,60µg/ml) of Aceclofenac and (1.5,3,4.5µg/ml) of Cyclobenzaprine HCl were analyzed three times on the different day and % R.S.D was calculated. Accuracy (A) For Aceclofenac 20µg/ml drug solution was taken in three different flask label A, B and C. Spiked 80%, 100%, 120% of standard solution in it and diluted up to 100ml. The area of each solution peak was measured at 237 nm. The amount of Aceclofenac was calculated at each level and % recoveries were computed. (B) For Cyclobenzaprine HCl 1.5µg/ml drug solution was taken in three different flask label A, B and C. Spiked 80%, 100%, 120% of standard solution in it and diluted up to 100ml. The area of each solution peak was measured at 237 nm. The amount of Cyclobenzaprine HCl was calculated at each level and % recoveries were computed. LOD and LOQ The LOD was estimated from the set of 3 calibration curves used to determination linearity. The LOD may be calculated as, LOD = 3.3 (SD/Slope) Where, SD= Standard deviation of Y-intercepts of 3 calibration curves. Slope = Mean slope of the 3 calibration curves. The LOQ was estimated from the set of 3 calibration curves used to determine linearity. The LOQ may be calculated as, LOQ = 10 (SD/Slope) Where, SD = Standard deviation of Y-intercepts of 3 calibration curves. Slope = Mean slope of the 3 calibration curves. Robustness Following parameters were changed one by one and their effect was observed on system suitability for standard preparation. 1. Flow rate of mobile phase was changed (± 0.2 ml/min) 0.8 ml/min and 1.2 ml/min. 2. ph of Mobile phase was changed ( ± 0.2 ) 4.8 and Ratio of Mobile phase was changed (±2) Buffer: Methanol (58:42) and Buffer: Methanol (62:38) RESULTS AND DISCUSSION Selection of Elution Mode
6 Impact factor: 3.958/ICV: 4.10 ISSN: Reverse phase chromatography was chosen because of its recommended use for ionic and moderate to non-polar compounds. Reverse phase chromatography is not only simple, convenient but also better performing in terms of efficiency, stability and reproducibility. C18 column is least polar compare to C4 and C8 columns. Here, A 250 x 4.6 mm column of 5.0μm particle packing was selected for separation of Aceclofenac and Cyclobenzaprine HCl. Isocratic mode was chosen due to simplicity in application and robustness with respect to longer column stability Selection Of Wavelength Both Aceclofenac and Cyclobenzaprine HCl show reasonably good response at 237 nm. Fig.3: Overlay UV Spectrum of Aceclofenac and Cyclobenzaprine HCl showing selection of wavelength detection. Mobile Phase Optimization After considering the varying combinations of various mobile phases, Buffer: Methanol (60:40), [Buffer (0.05 M KH2PO4, ph 5) Take 6.8 gm KH2PO4 into a 1000 ml beaker, add 800 ml water and dissolve, adjust ph 5 with 0.1 N NaOH, Make up Volume 1000 ml with water was finalized as it was showing good peak shapes and a significant amount of resolution. The mobile phase Buffer (ph 5.0): Methanol (60:40v/v) was selected because it was found to ideally resolve the peaks with retention time (RT) and min for Cyclobenzaprine HCl and Aceclofenac respectively Fig. 4: Chromatogram of Aceclofenac- Cyclobenzaprine HCl in Buffer(pH 5.0) : Methanol (60:40 v/v) (Final)
7 Impact factor: 3.958/ICV: 4.10 ISSN: Parameters Table 1: Results for system suitability test Data observed Cyclobenzaprine HCl Aceclofenac Theoretical plates per column Symmetry factor/tailing factor Resolution Stability-Indicating Method Fig. 5: Cyclobenzaprine HCl and Aceclofenac Standard for stability Acid degradation Fig. 6: Acid Degradation Blank
8 Impact factor: 3.958/ICV: 4.10 ISSN: Fig. 7: Cyclobenzaprine HCl Acid Degradation Standard Fig. 8: Aceclofenac Acid Degradation Standard Fig. 9: Cyclobenzaprine HCl and Aceclofenac Acid Degradation Sample
9 Impact factor: 3.958/ICV: 4.10 ISSN: Base degradation Fig. 10: Base Degradation Blank Fig. 11: Cyclobenzaprine HCl Base Degradation Fig. 12: Aceclofenac Base Degradation
10 Impact factor: 3.958/ICV: 4.10 ISSN: Fig. 13:Cyclobenzaprine HCl and Aceclofenac Base Degradation Sample Oxidative degradation Fig. 14: Oxidation Degradation Blank Fig. 15:Cyclobenzaprine HCl Oxidation Degradation
11 Impact factor: 3.958/ICV: 4.10 ISSN: Fig. 16:Aceclofenac Oxidation Degradation Fig. 17:Cyclobenzaprine HCl and Aceclofenac Oxidation Degradation sample Photo degradation Fig. 18: Photo Degradation Blank
12 Impact factor: 3.958/ICV: 4.10 ISSN: Fig. 19:Cyclobenzaprine HCl Photo Degradation Fig. 20:Aceclofenac Photo Degradation Fig. 21:Cyclobenzaprine HCl and Aceclofenac Photo Degradation sample
13 Impact factor: 3.958/ICV: 4.10 ISSN: Thermal degradation Fig. 22: Thermal Degradation Blank Fig. 23:Cyclobenzaprine HCl Thermal Degradation Fig. 24:Aceclofenac Thermal Degradation
14 Impact factor: 3.958/ICV: 4.10 ISSN: Fig. 25:Cyclobenzaprine HCl and Aceclofenac Thermal Degradation sample Calculation for Stability: Table 2:Cyclobenzaprine HCl and Aceclofenacstd for stability Drugs Area Cyclobenzaprine HCl Aceclofenac Table 3: Cyclobenzaprine HCl % Degradation Cyclobenzaprine HCl Parameter Standard Sample Area %Degradation Area %Degradation Acid Base Oxidation Photo Thermal Table 4: Aceclofenac % Degradation Aceclofenac Parameter Standard Sample Area %Degradation Area %Degradation Acid Base Oxidation Photo Thermal
15 Impact factor: 3.958/ICV: 4.10 ISSN: VALIDATION OF RP-HPLC METHOD Specificity: Fig. 26:- Chromatogram of Aceclofenac and Cyclobenzaprine HClstd Fig. 27:- Chromatogram of Aceclofenac and Cyclobenzaprine HCl sample Fig. 28:- Chromatogram of Blank The Chromatograms of Aceclofenac and Cyclobenzaprine HClstandards and Aceclofenac and Cyclobenzaprine HClsample show no interference with the Chromatogram of Aceclofenac and Cyclobenzaprine HClBlank, so the Developed method is Specific. Linearity and Range The regression line equation for Aceclofenac and Cyclobenzaprine HClare as following: For Aceclofenac: y = x and For Cyclobenzaprine HCl:y = 62.66x
16 Impact factor: 3.958/ICV: 4.10 ISSN: Table 5 : Linearity data for Aceclofenac. Sr. No Concentration (µg/ml) Area Table 6:Linearity data for Cyclobenzaprine HCl. Sr.No Concentration(µg/ml) Area Fig. 29: Overlay chromatogram of different concentrations of binary mixtures of Aceclofenac and Cyclobenzaprine HCl Fig. 30: Calibration Curve of Aceclofenac (20-60μg/ml).
17 Impact factor: 3.958/ICV: 4.10 ISSN: Fig. 31: Calibration Curve of Cyclobenzaprine HCl ( μg/ml). Precision (A) Repeatability Table 7: Repeatability data for Aceclofenac Aceclofenac Sr. No. Conc. (μg/ml) Area Mean ± S.D (n=6) % R.S.D ± Table 8: Repeatability data for Cyclobenzaprine HCl Cyclobenzaprine HCl Sr No. Conc (μg/ml) Area Mean ± S.D (n=6) % R.S.D ±
18 Impact factor: 3.958/ICV: 4.10 ISSN: (B) Intraday precision Table 9: Intraday precision data for estimation of Aceclofenac and Cyclobenzaprine HCl SR. NO Conc. (µg/ml) Aceclofenac Area Mean ± S.D. (n=3) % R.S.D Conc. (µg/ml) Cyclobenzaprine HCl Area Mean ± S.D. (n=3) % R.S.D ± ± ± ± ± ± (C) Inter-day precision Table 10: Interday precision data for estimation of Aceclofenac and Cyclobenzaprine HCl SR. NO Conc. (µg/ml) Aceclofenac Area Mean ± S.D. (n=3) % R.S.D Conc. (µg/ml) Cyclobenzaprine HCl Area Mean ± S.D. (n=3) % R.S.D ± ± ± ± ± ± Accuracy Table 11: Recovery data for Aceclofenac SR. NO. Conc. Level (%) Sample amount (μg/ml) Amount Added (μg/ml) Amount recovered (μg/ml) % Recovery % % % % Mean Recovery ± S.D ± ± ± 0.766
19 Impact factor: 3.958/ICV: 4.10 ISSN: Table 12: Recovery data for Cyclobenzaprine HCl SR. NO. Conc. Level (%) Sample Amount Amount(mc Added(mcg/ g/ml) ml) Amount recovered (mcg/ml) % Recovery % Mean Recovery ± S.D % % % ± ± ± LOD and LOQ Table 13: Limit of Detection data for Aceclofenac and Cyclobenzaprine HCl Aceclofenac Cyclobenzaprine HCl LOD = 3.3 x (SD/ Slope) LOD = 3.3 x (SD/ Slope) = 3.3 x (86.305/166.83) = 3.3 x (2.108/62.66) = 1.707µg/ml = 0.111µg/ml Table 14: Limit of Quantitation data for Aceclofenac and Cyclobenzaprine HCl Aceclofenac Cyclobenzaprine HCl LOQ = 10 x (SD/ Slope) LOQ = 10 x ( SD / Slope ) = 10 x (86.305/166.83) = 10 x (2.108/62.66) = µg/ml =0.336µg/ml
20 Impact factor: 3.958/ICV: 4.10 ISSN: Robustness Table 15: Robustness data for Aceclofenac SR NO. Area at Area at Area at Area at Area at Area at Flow rate Flow rate ph (-0.2) ph Mobile Mobile (- 0.2 (+ 0.2 (+0.2) phase(-2) phase(+2) ml/min) ml/min) % R.S.D Table 16: Robustness data for Cyclobenzaprine HCl SR NO. Area at Area at Area at Area at Area at Area at Flow rate Flow rate ph (- 0.2) ph (+ Mobile Mobile (- 0.2 ( ) phase(-2) phase(+2) ml/min) ml/min) % R.S.D Analysis Of Marketed Formulation By Developed Method Applicability of the proposed method was tested by analyzing the commercially available Tablet formulation Flexabenz plus Tablet Flexabenz plus Table 17: Analysis of marketed formulation Label claim Assay (% of label claim*) Mean ± S. D. Cyclobenzaprine Aceclofenac % Aceclofenac % Cyclobenzaprine HCl HCl 200mg 15mg ± ± 1.124
21 Impact factor: 3.958/ICV: 4.10 ISSN: CONCLUSION Stability indicating HPLC method was developed for simultaneous estimation of Aceclofenac and Cyclobenzaprine HCl. In developed method, good resolution and separation of two drugs was achieved M Sodium dihydrogen phosphate (ph 5.0): Methanol (60:40 v/v) was used as mobile phase. Retention time of Cyclobenzaprine HCl and Aceclofenac were found to be min and min respectively with a flow rate of 1 ml/min. The proposed method was accurate and precise. Therefore proposed method can be used for routine analysis of Aceclofenac and Cyclobenzaprine HCl in Cream. Forced degradation study of Cyclobenzaprine HCl and Aceclofenac was performed by RP-HPLC method which includes Acid, Base, Oxidative, Photo and Thermal degradation. Results of degradation were found within limit. It can be successfully adopted for routine quality control analysis of Aceclofenac and Cyclobenzaprine HCl in Combined dosage form without any interference from common excipients and impurity. REFERENCES 1. Introduction to Muscle Spasm, August-2017, 2. Drug profile for Aceclofenac, August-2017, 3. Government of india, ministry of health and family welfare 2014, Indian Pharmacopoiea-2014,1, pp Drug profile for Cyclobenzaprine, August-2017, 5. Drug profile for Cyclobenzaprine HCl, August-2017, 6. Kasture AV., Mahadik KR., Wododkar SG and More HN A Text Book of Pharmaceutical Analysis,NiraliPrakashan, 17 th Edition Pune, 2002, Shethi PD, HPLC-Quantitative Analysis of Pharmaceutical Formulations CBS Publishers & Distributers, New Delhi, 1996, Chatwal GR, Instrumental Method of Chemical Analysis, Part-1, Himalaya Publishing House 5 th Edition, 2002,2, FDA, "Guidance for Industry; Analytical Procedures and Methods Validation (Draft guidance), Food & Drug Administration, Rockville, US Department of Health and Human Services, ICH, Validation of Analytical Procedures; Methodology, Q2 (R1), International Conference on Harmonization, IFPMA, Geneva 1996.
Impact factor: 3.958/ICV: 4.10 ISSN:
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