Bendre S.D. & Ghule P.J. Int. Res. J. Pharm. 2016, 7 (12) INTERNATIONAL RESEARCH JOURNAL OF PHARMACY

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1 INTERNATIONAL RESEARCH JOURNAL OF PHARMACY ISSN Research Article ANALYTICAL METHOD DEVELOPMENT, VALIDATION, AND ASSAY OF BETAMETASONE DIPROPIONATE CREAM BY HPLC METHOD Bendre S.D. *, Ghule P.J. Mula Rural Institute of Pharmacy, Sonai, Tal. Newasa, Dist. Ahmednagar, India *Corresponding Author Article Received on: 23/10/16 Revised on: 23/11/16 Approved for publication: 03/12/16 DOI: / ABSTRACT The efficient development and validation of analytical methods are critical elements in the development of pharmaceuticals. The scope of developing and validating a method is to ensure a suitable strategy for a particular analyte which is more specific, accurate, and precise. Here, the main focus is drawn to achieve improvement in conditions and standard operating procedures to be followed. The principal objective of this study was, therefore, to develop a new, simple, economical, selective, precise, reproducible, and stability-indicating high-performance liquid chromatographic (HPLC) method with a wide linear range and good sensitivity for assay of Betametasone Dipropionate and related substance study in the bulk and pharmaceutical formulations. The related substance study and assay is carried by In-house method. So, as In-house method we have selected the HPLC method. Keywords: RP-HPLC, Assay Development and validation, Betamethasone Dipropionate INTRODUCTION A survey of literature reveals that good analytical methods are not available for the drugs like Betamethasone Dipropionate. No RP-HPLC method has been reported so far for Assay and Related Substances identification by using gradient mode i.e. Mobile phase A- (Water: IPA) (70:30) and Mobile phase B- (ACN: THF) (80:20) Even though very few methods of determination of above drug is available, many of them suffer from one disadvantage or the other, such as low sensitivity, lack of selectivity and simplicity etc. The existing physicochemical methods are inadequate to meet the requirements; hence it is proposed to improve the existing methods and to develop new methods for the assay and related substance of the drugs in pharmaceutical dosage forms and different available analytical techniques like HPLC. The objective of the research work are; 1. To optimize the chromatographic condition for assay of Betametasone Dipropionate cream. 2. To optimize the chromatographic condition of estimation of related substances of Betametasone Dipropionate in Betametasone Dipropionate cream. Method is optimized according to different parameters by taking different individual conditions to avoid error, unreliability and contamination. Moreover, it provides adequate sensitivity and specificity. The validation of this method is carried out according to the ICH (Q1A, Q2B) guidelines. 4,6,7,8 This method is one of the useful methods owing to its ruggedness and robustness and found to be selective, specific, sensitive, linear, precise and economic. 1,2,3,4,9,10,11 MATERIALS AND METHODS Table 1: List of Material Sr. No. Name Supplier 1. Betamethasone Dipropionate (BD) Sample from R and D Laboratory, Glenmark 2. Chlorocresol Research Center, Mahepe, Navi Mumbai. Table 2: List of Chemical and Reagent Sr. No. Name Grade Supplier 1. Acetonitrile(ACN) HPLC Rankem Ltd,Mumbai 2 Methanol(CH 3OH) HPLC Rankem Ltd,Mumbai 3. Glacial Acetic Acid AR Merck Specialities Ltd,Mumbai. (CH 3COOH) 4. IPA HPLC Rankem Ltd,Mumbai. 5. THF HPLC Rankem Ltd,Mumbai. 6. Sodium Hydroxide(NaOH) LR Rankem Ltd,Mumbai. 7. Hydro Chloric Acid (Hcl) LR Qualigens Fine Chemicals,Mumbai. 8. Milli Q Water NA In-House 74

2 A. Preliminary Tests / Qualitative Tests 2,11-15 Physical State of Drugs: Solid and white or almost white crystalline powder. Solubility Practically insoluble in water. Freely soluble in acetone and in methylene chloride. Sparingly soluble in alcohol. Melting Point: C Identification of Drugs by IR- Spectrum Table 3: Optimised Chromatographic Condition for Assay Method Parameter Condition Stationary Phase Discovery HS, C18, (150 x 4.6mm), 3µ or equivalent. Mobile Phase Mobile phase A-Water: IPA(80:20) Mobile phase B-ACN: THF(70:30) Flow Rate 1.0 ml/min Detection 240nm Pump Mode Gradient Injection Volume 20 µl Run Time 35min Column 50 0 C Temperature Retention Time About 8.5 minutes for Chlorocresol About 21.0 minutes for Betamethasone. Needle wash Methanol Gradient programme Table 4: Composition of Mobile Phase Figure 1: Infrared spectrum of Betamethasone Dipropionate API UV Spectrum Figure 2: UV Spectrum of Betamethasone Dipropionate API in Methnol (10ppm) B. Assay method development and optimization 2,11-15 Selection of chromatographic method Important information s about the sample composition and properties that should be collected are as follows; Number of compounds present Chemical structure (functionality) of the compounds Molecular weight of the compounds pka values of the compounds UV-spectra of the compounds Concentration range of the compounds in the sample. Sample solubility Method development was optimised by considering the various system suitable parameter such as Number of theoretical plates (efficiency) Capacity factor, Separation (relative retention) Resolution, Tailing factor Time (min) Mobile phase A Mobile phase B C. Assay method validation The optimized HPLC method has been developed for the determination of the assay of Betametasone Dipropionate in Betametasone Dipropionate cream (0.05%) w/w. This protocol is intended for the validation of assay of Betametasone Dipropionate in Betametasone Dipropionate cream (0.05%) w/w. 2,7,9-15 Methodology followed Reagents Methanol (HPLC grade) Glacial acetic acid (AR Grade) Acetonitrile (HPLC grade) Isopropyl Alcohol (HPLC grade, Rankem) Tetrahydrofuran (HPLC grade, Rankem) Water (Milli Q or equivalent) Preparation of Mobile phase Mobile phase A: Mixture of Water and Isopropyl Alcohol in the ratio 80:20 was prepared. Mobile phase B: Mixture of Acetonitrile and Tetrahydrofuran in the ratio 70:30 was prepared. Preparation of diluent: (0.1 % Glacial Acetic acid in Methanol) 1 ml of Glacial acetic acid was diluted in 1000 ml of Methanol. Chromatographic Conditions Column :Discovery HS, C18, (150 x 4.6mm), 3µ or equivalent. Flow Rate:1.0 ml / min. Detection :240 nm. Column Temp:50 C. Injection Volume:20µL. Run Time:35 minutes. Retention Times: About 8.5 minutes for Chlorocresol About 21.0 minutes for Betamethasone. 75

3 Gradient programme Table 5: Composition of Mobile Phase Time (min) Mobile phase A Mobile phase B Preparation of standard solution 40 mg of Betamethasone Dipropionate (equivalent to mg of Betamethasone) and 60 mg of Chlorocresol working standard was accurately weighed and transferred into a 100 ml volumetric flask and diluted with 60 ml of diluent and sonicated to dissolve, then diluted the above solution to volume with diluent and mix. 5.0 ml of the above solution was pipetted out into a 100 ml volumetric flask and diluted to volume with diluents to obtained the standard concentration of Betamethasone Dipropionate 20ppm and Standard concentration of Chlorocresol 30ppm. Preparation of sample solution: (For 0.05% w/w Cream) 3.11g of sample (equivalent to 2mg of Betamethasone Dipropionate) was accurately weighed and transferred into 100ml volumetric flask. 60 ml of diluent was added and warmed on a water bath at 60 C for 30 minutes with intermittent vigorous shaking and diluted up the volume with diluent. Then filtered through teflon membrane 0.45µ filter to obtained the sample preparation 20ppm. Evaluation of system suitability Standard solution was injected five times into the HPLC and the chromatograms were recorded. The area counts of Betamethasone and Chlorocresol peaks were measured. The relative standard deviation of five replicate injections for both the peaks should not be more than 2.0%. Procedure Equal volumes of the blank (diluents) and sample solution (in duplicate) were injected into the HPLC and the chromatograms were recorded. The area counts of Betamethasone and Chlorocresol peaks were measured. A. Specificity Preparation of standard test solution 40 mg of Betamethasone Dipropionate (equivalent to mg of Betamethasone) and 60 mg of Chlorocresol working standard was accurately weighed and transferred into a 100 ml volumetric flask and diluted with 60 ml of diluent and sonicated to dissolve, then diluted the above solution to volume with diluent and mix. 5.0 ml of the above solution was pipetted out into a 100 ml volumetric flask and diluted to volume with diluent. Preparation of sample solution 3.11g of sample (equivalent to 2mg of Betamethasone Dipropionate) was accurately weighed and transferred into 100ml volumetric flask. 60 ml of diluent was added and warmed on a water bath at 60 C for 30 minutes with intermittent vigorous shaking. Allowed to attain room temperature and diluted up the volume with diluent. Then filtered through Teflon membrane 0.45µ filter to obtained the sample preparation 20ppm. Preparation of placebo solution 3.11g of placebo was accurately weighed into 100ml volumetric flask. 60 ml of diluent was added and warmed on a water bath at 60 C for 30 minutes with intermittent vigorous shaking. Allowed to attain room temperature and diluted upto the volume with diluent. Centrifuge at 2000 rpm for 15 minutes. Then filtered through teflon membrane 0.45µ filter. Preparation of spike solution To the sample solution 1ml of impurity solution (1ppm) was spiked. Blank solution, Standard, Sample, spike sample and placebo of Betamethasone Dipropionate were injected as per the sequence details given in Table-5.2. Percent interference will be determined by comparing the response for any peak detected at the retention time for Betamethasone Dipropionate. Table 6: Sequence for Selectivity Sample Name No. of Inj. Inj. volume (µl) Run time (minutes) Blank Placebo Solution Betamethasone standard Betamethasone sample Spiked Solution Acceptance Criterion There should not any interference at the retention time of main peak i.e. the main peak should be pure. B. Linearity For establishing the linearity for Betamethasone Dipropionate andchlorocresol, a series of standard preparation of Betamethasone Dipropionate andchlorocresol were prepared to cover a range of 50 % to 150 % of sample concentration i.e 20ppm for Betamethasone Dipropionate, based on this the range proposed for Linearity determination is 50% to 150% test concentration (i.e. 10 ppm to 30 ppm) for Betamethasone Dipropionate and 30ppm for Chlorocresol, based on this the range proposed for Linearity determination is 50% to 150% test concentration (ie 15 ppm to 45 ppm) for Chlorocresol. A detail of dilutions is given in Table-5.5 and sequence for injection is given in Table-5.6. The Linearity graph should be plotted from50% to 150%. Preparation of linearity stock solution 40 mg of Betamethasone Dipropionate 60mg Chlorocresol working standard was accurately weighed and transferred into two different 100mL volumetric flask. Add 70mL of diluent to each and sonicate to dissolve, cool and make up volume with diluent and mix properly to get the stock solution of both. 76

4 Table 7: Dilutions for Linearity Betamethasone Dipropionate % Conc of Sample Amount of stock solution to be Final volume with diluent (ml) Concentration (ppm) transferred (ml) 50% % % % % % % Table 8: Dilutions for Linearity Chlorocresol % Conc of Sample Amount of stock solution to be Final volume with diluent Concentration (ppm) transferred (ml) (ml) 50% % % % % % % Table 9: Sequence for Linearity Betamethasone Dipropionate and Chlorocresol Sample Name No. of Inj. Inj. Volume (µl) Run time (minutes) Blank % % % % % % % Acceptance Criterion Correlation Coefficient should not less than C. Accuracy (Recovery) Recovery samples were prepared by spiking placebo preparations with known amounts of Betamethasone Dipropionate and Chlorocresol standard in triplicate at three levels (total nine determinations). Each test sample will be prepared as described below. Betamethasone Dipropionate placebo solution Placebo equivalent to 3110 mg of Betamethasone Dipropionate cream was accurately weighed and transferred into nine 100mL volumetric flasks. Betamethasone Dipropionate spiked solution Transferred accurate amount of API of both to above flasks to make the spiked solution in three levels i.e. 80%, 100% and 120%. About 60ml of diluents was added and sonicated for 30 min. Then cool and dilute up the volume with diluent to get the spiked solution of Betamethasone Dipropionate and Chlorocresol at different levels as described below in the Table 10. Table 10: Sample preparation for Accuracy Accuracy Level Conc. Of spiked sample of Betamethasone Dipropionate Conc. Of spiked sample of Chlorocresol 80%_sample_1 16 ppm 24 ppm 80%_ sample _2 16 ppm 24 ppm 80%_ sample _3 16 ppm 24 ppm 100%_ sample _1 20 ppm 30 ppm 100%_ sample _2 20 ppm 30 ppm 100%_ sample _3 20 ppm 30 ppm 120%_ sample _1 24 ppm 36 ppm 120%_ sample _2 24 ppm 36 ppm 120%_ sample _3 24 ppm 36 ppm 77

5 Table 11: Sequence for Accuracy Sample Name No. Of Injections Injection Volume (µl) Run time (minutes) Blank Standard Standard Accuracy-80%/ Accuracy-80%/ Accuracy-80%/ Standard Accuracy-100%/ Accuracy-100%/ Accuracy-100%/ Standard Accuracy-120%/ Accuracy-120%/ Accuracy-120%/ Standard D. Precision System Precision Six replicate of standard solution were injected and the response was recorded. Acceptance criterion The Relative standard deviations (%RSD) of the six replicate injections of standard solution will NMT 2.0%. Method precision One analyst was independently prepared six sample preparations of Betamethasone Dipropionate and analyze as per the method. Details of injection sequence are given Table 12. Table 12: Sequence for System and Method Precision Sample Name No. of Injections Injection Volume Run time (minutes) (µl) Blank Standard solution Standard solution Standard solution Standard solution Standard solution Standard solution Sample Sample Sample Sample Sample Sample Intermediate precision (Ruggedness) Six standard solutions and six sample solutions of Betamethasone Dipropionate of the same lot using a different HPLC system, a different column on a different day will be analyzed. The mean and percent RSD values for area will be calculated. Acceptance criterion The Relative standard deviation (%RSD) of the twelve replicate injections ie the fresh six sample preparation and that of method precesion of sample solution will NMT 2.0%. E. Robustness Prepare Three sample preparations of the same lot of Betamethasone Dipropionate were prepared. The following modifications to the Chromatographic conditions will be evaluated: Change in column Temperature (±5 C) Change in wavelength (±5 nm) Change in Flow rate (± 0.1 ml\min)10% change For each set of change in the experimental condition, following sequence given in Table 13 will be followed. Table 13: Sequence for Change in Experimental Condition Sample Name No. of Injections Injection Volume (µl) Run time (minutes) Blank Standard Standard Sample Sample Sample

6 F. Change in column temperature Normal experimental condition for column temperature is 50 C. Change in column temperature was studied for actual ±5 C. G. Change in wavelength Normal experimental condition for detection is 240 nm. Change in wavelength was studied for actual ±5 nm. H. Change in flow rate Normal experimental condition for flow rate is 1.0 ml/minute. Change in flow rate was studied for actual ±0.1ml/minute. RESULT AND DISCUSSION 1,7,8,9 A. Linearity and Range Table 14: Linearity for Betamethasone Dipropionate % Concentration Concentration (µg per ml) Response (Area) Statistical analysis 50% Slope % % Intercept % % Correlation % Coefficient 150% Table 15: Linearity for Chlorocresol % Concentration Concentration Response (Area) Statistical analysis (µg per ml) 50% Slope % % Intercept % % Correlation % Coefficient 150% Figure 3: Linearity Assay Graph of Betamethasone dipropionate Figure 4: Linearity Assay Graph of Chlorocresol Acceptance criteria: Correlation coefficient should not be less than Conclusion: Correlation coefficient for Betamethasone dipropionate is and for chlorocresol is Therefore, the HPLC method for the determination of Chlorocresol and Betamethasone dipropionate in Betamethasone dipropionate augmented cream is linear. B. Precision System Precision: Six replicate injections of the Standard preparation were injected into the HPLC system using the method as described under Methodology. Table 16: Data sheet for system precision Injection Betamethasone Area Chlorocresol Area Mean SD %RSD

7 Acceptance criteria: RSD should not be more than 2.0%. Conclusion: The RSD of system precision is 0.38% for Betamethasone dipropionate and 0.06% for Chlorocresol. Therefore, the HPLC method for the determination of Chlorocresol and Betamethasone dipropionate in Betamethasone dipropionate augmented cream is precise. Method precision: Six replicate injections of the Sample preparation were injected into the HPLC system using the method as described under Methodology. Table 17: Data sheet for method precision Sample % Label claim Betamethasone % Label claim Chlorocresol Mean SD %RSD Acceptance criteria: RSD should not be more than 2.0%. Conclusion: The RSD of method precision is 0.30% for Betamethasone dipropionate and 0.34% for Chlorocresol. Therefore, the HPLC method for the determination of Chlorocresol and Betamethasone dipropionate in Betamethasone dipropionate augmented cream is reproducible. C. Ruggedness (Intermediate Precession) Different HPLC system chosen for ruggedness study. Experiment: Six sample preparations of the same lot of Betamethasone dipropionate augmented cream 0.05% were made by a different analyst, using different column on a different day and injected in duplicate into a different HPLC using the method as described under Methodology, along with Standard preparation. Table 18: Ruggedness of Betamethasone dipropionate Sample Analyst -1 % Label claim Analyst -2 % Label claim Mean SD %RSD Overall Mean Overall SD Overall %RSD 0.89 Table 19: Ruggedness of Chlorocresol Sample Analyst -1 % Label claim Analyst -2 % Label claim Mean SD %RSD Overall Mean Overall SD Overall %RSD 0.43 Acceptance criteria: Overall RSD for twelve results should not be more than 2.0% Conclusion: The RSD of intermediate precision is 0.89% for Betamethasone dipropionate and 0.43% for Chlorocresol. Therefore, the HPLC method for the determination of Chlorocresol and Betamethasone dipropionate in Betamethasone dipropionate augmented cream is rugged. D. Accuracy (Recovery) Placebo of Betamethasone dipropionate augmented cream was spiked with Chlorocresol and Betamethasone dipropionate Drug Substance at three different levels: 80%, 100% and 120% of the label claim in triplicate (in total nine determinations) and then proceeded with Sample solution as described under Methodology. Each of the Sample solution was injected in duplicate and the average area count was taken for calculation. Table 20: Accuracy for Betamethasone dipropionate Sample No. Amount added (mg) Amount recovered (mg) % Recovery Accuracy 80% Accuracy 80% Accuracy 80% Accuracy 100% Accuracy 100% Accuracy 100% Accuracy 120% Accuracy 120% Accuracy 120% Mean SD % RSD

8 Table 21: Accuracy for Chlorocresol Sample No. Amount added (mg) Amount recovered (mg) % Recovery Accuracy. 80% Accuracy 80% Accuracy 80% Accuracy 100% Accuracy 100% Accuracy 100% Accuracy 120% Accuracy 120% Accuracy 120% Mean SD % RSD Acceptance criteria: Mean recovery should be in the range of 98.0% to 102.0%. The RSD should not be more than 2.0%. Conclusion: Mean recovery for Betamethasone dipropionate is 100.3% and RSD is 0.28%. Mean recovery for Chlorocresol is 100.3% and RSD is 0.44%. Therefore, the HPLC method for the determination of Chlorocresol and Betamethasone dipropionate in Betamethasone dipropionate augmented cream is accurate. F. Robustness Experiment: Three Sample preparations of the same lot (as used in precision study) of Betamethasone dipropionate augmented cream 0.05% were prepared as described under Methodology. The samples along with standard were injected in duplicate under different chromatographic condition as shown below. G. Change in Temperature (+ 5 C) Table 22: Change in Temperature for Betamethasone (+ 5 C) Control (+5 C) Control (-5 C) Cumulative Mean Cumulative Mean Cumulative SD Cumulative SD Cumulative %RSD 0.64 Cumulative %RSD 0.37 Table 23: Change in Temperature for Chlorocresol (+ 5 C) Control (+5 C) Control (-5 C) Cumulative Mean Cumulative Mean Cumulative SD Cumulative SD Cumulative %RSD 0.58 Cumulative %RSD 0.34 H. Change in Flow rate (+ 10%) Table 24: Change in Flow rate for Betamethasone (+ 10 %) Control (+10%) (-10%) Cumulative Mean Cumulative SD Cumulative %RSD Table 25: Change in Flow rate for Chlorocresol (+ 10 %) Control (+10%) (-10%) Cumulative Mean Cumulative SD Cumulative %RSD I. Change in wavelength (+ 5nm) Table 26: Change in wavelength for Betamethasone (+ 5 nm) Control (+ 5nm) (-5nm) Cumulative Mean Cumulative SD Cumulative %RSD Table 27: Change in wavelength for Chlorocresol (+ 5 nm) Control (+ 5nm) (-5nm) Cumulative Mean Cumulative SD Cumulative %RSD

9 Acceptance criteria: System suitability should meet as per the test method at each variable condition. Overall RSD should not be more than 2.0% for the results obtained at control and variable conditions. J. System suitability %RSD of five replicate injections, Retention time, USP Tailing and USP Tangent for Chlorocresol and Betamethasone dipropionate in Standard solution were maintained as per method on every day. Table 28: System Suitability for Betamethasone %RSD of Standard preparation Retention Time USP Tailing USP Tangent Specificity Forced Degradation Method precision Ruggedness Solution stability Robustness Control Flow +10% Flow -10% Wavelength +5nm Wavelength -5nm Temp +5 C Temp -5 C Table 29: System Suitability for Chlorocresol %RSD of Standard preparation Retention Time USP Tailing USP Tangent Specificity Forced Degradation Method precision Ruggedness Solution stability Robustness Control Flow +10% Flow -10% Wavelength +5nm Wavelength -5nm Temp +5 C Temp -5 C Acceptance Criteria: RSD should not be more than 2.0%. USP tangent should not be less than USP Tailing should not be more than 2.0. CONCLUSION The test method is validated for Specificity, Linearity and Range, Precision, Accuracy (Recovery), Ruggedness, Stability of Analytical solution and Robustness and found to be meeting the predetermined acceptance criteria. The validated method is Specific, Linear, Precise, Accurate, Rugged and Robust for Assay of Chlorocresol and Betamethasone dipropionate in Betamethasone dipropionate augmented cream. Hence this method can be introduced into routine use for the assay of Chlorocresol and Betamethasone dipropionate in Betamethasone dipropionate augmented cream. ACKNOWLEDGMENT The author is thankful to Glenmark Research Centre Mhape, Mumbai for providing all the necessary facility for completing the research work. REFERENCES 1. Becket AH, Stenlake JB, Practical Pharmaceutical Chemistry, CBS Publications and Distributors, New Delhi, 2005;2(4): Azim Md. Sabir, Mitra Moloy, Bhasin Parminder S. HPLC method development and validation: A Review. Int. Res. J. Pharm. 2013;4(4): DOI: / Udo Huber Quality Control of Pharmaceutical drugs Turning analysis and performance data into comprehensible charts- Pharmaceutical Application Note, 1999; 1: Watson DG, Pharmaceutical Analysis-A Text Book for Pharmacy Student and Pharmaceutical Chemist. Churchill Livingstone London, 1999; Jeffery GH, Bassett J, Mendham J, Denney RC, Vogel s Textbook of Quantitative Chemical Analysis, Longman Scientific and Technical, 1989; 5: Chatwal GR, Anand SK, Instrumental Methods of Chemical Analysis, Himalaya Publishing House, Mumbai, 2004;5: Kealey D, Haines PJ, Instant Notes in Analytical Chemistry, BIOS Scientific Publishers Limited, 2002; 3: Becket AH, Stenlake JB, Practical Pharmaceutical Chemistry, CBS Publications and Distributors, New Delhi, 2005;4(2): Skoog DA, Holler FG, Nieman TA. Principles of Instrumental Analysis, Thomson Brooks/Cole Asia Pvt. Ltd., Singapore. 2004;(5); Willard HH, Meritt LL, Dean JA, Settle FA. Instrumental Methods of Analysis, CBS Publishers and Distributors, New Delhi,2004;(7); Christian GD, Analytical Chemistry, John Wiley and Sons (Asia) Pte. Ltd., Singapore. 2003;(6); Braun RD, Introduction to Instrumental Analysis, Pharma Book Syndicate, Hyderabad. 2006;(1); Chatwal GR, Anand SK, Instrumental Methods of Chemical Analysis, Himalaya Publishing House, New Delhi. 2007;(5):

10 14. Ahuja S, Dong MW, Handbook of Pharmaceutical Analysis by HPLC, Elsevier Academic Press Publication, Oxford, U.K. 2005;(2); Kazakevich Y, Lobrutto R, HPLC for Pharmaceutical Scientists; John Wiley and Sons, Inc., A Wiley-interscience Publication, USA, 2007, Cite this article as: Bendre S.D., Ghule P.J. Analytical method development, validation, and assay of Betametasone dipropionate cream by HPLC method. Int. Res. J. Pharm. 2016;7(12): Source of support: Nil, Conflict of interest: None Declared Disclaimer: IRJP is solely owned by Moksha Publishing House - A non-profit publishing house, dedicated to publish quality research, while every effort has been taken to verify the accuracy of the content published in our Journal. IRJP cannot accept any responsibility or liability for the site content and articles published. The views expressed in articles by our contributing authors are not necessarily those of IRJP editor or editorial board members. 83