KEYWORDS: Forced degradation fusidic acid HPLC Method validation Topical formulation.

Transcription

1 STABILITY INDICATING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) METHOD WAS DEVELOPED FOR RELATED SUBSTANCES OF FUSIDIC ACID IN TOPICAL PHARMACEUTICAL FORMULATION *Sudhakar P 1, Dr. Pankaj Sharma 1 and Dr.Birendra Shrivastava 1 1 School of Pharmaceutical Sciences, Jaipur National University, Jaipur , India ABSTRACT This RP-HPLC method was successfully validated per ICH guidelines and proved to be suitable for routine quality control use. The chromatographic separation was achieved on Lichrospher 100 RP mm x 4.0mm, 5 µm column, Using a mobile phase consisting a mixture of Acetonitrile, Methanol and 0.05M Ortho-Phosphoric acid in the ratio of 50:5:45 (v/v), at a flow rate of 1.5 ml/min. Injection volume 10 µl, The column temperature was set at 25 C and wavelength at 235 nm.the precision of the method observed in RSD is 1.1 % for Total impurities. The Overall RSD of Method Precision and Intermediate Precision results are 2.0 % for Total impurities, Individual % recovery values observed in the range of % to % for Fusidic acid. Sample solution is stable for atleast 36 hours at room temperature for 3-ketofusidic acid. The method is found to be robust under the following variable conditions like flow +10%, column oven +5 C, organic content in mobile phase + 2%, and wave length +5 nm. The linearity of response was determined in the range of µg/ml to µg/ml for Fusidic acid and µg/ml to µg/ml for 3-ketofusidic acid. The Correlation coefficient is , and respectively. The LOQ of Fusidic acid and 3-ketofusidic acid was determined in the lowest concentration µg/ml and µg/ml respectively. Degradation was observed in drug product and placebo were exposed to hydrolysis Acid (3 ml, 1 N HCl), Alkali (2 ml, 1N NaOH), Peroxide (5mL, 30% w/w of Hydrogen peroxide) and thermal (105 C/36 hours 5 minutes), photolytic degradation ( Lux hours & Watt hours / sq. m at 25 C). KEYWORDS: Forced degradation fusidic acid HPLC Method validation Topical formulation. *Corresponding author: Sudhakar P, School of Pharmaceutical Sciences, Jaipur National University, Jaipur India, Tel.: address: sudhakarp250@gmail.com 1 P a g e

2 1. INTRODUCTION The present study was aimed at developing a simple, specific, accurate, and precise HPLC method in topical formulation in commercially available and for use in stability studies and quality control applications. The HPLC method for determination of related substances of Fusidic acid in Fucidin cream has been validated to show specificity, linearity, precision, intermediate precision/ruggedness, accuracy, stability in analytical solution, limit of quantitation and robustness. Fucidin Cream contains fusidic acid active pharmaceutical ingredients (API). Fusidic acid is an antibiotic medicine used to treat infections with bacteria. It works by entering bacterial cells and interfering with the production of proteins that the bacteria need to divide and multiply. It doesn't directly kill the bacteria, but leaves them unable to increase in numbers. The bacteria eventually die or are destroyed by the immune system. Fusidic acid is included in this preparation to treat the bacterial infection that can sometimes occur in eczema 1-5. Chemical Name and Structure is given in Table 1. Table 1. Chemical structure of Fusidic acid. 6-7 Name Chemical Name Chemical Structure Fusidic acid (2Z)-2-[(3R,4S,5S,8S,9S,10S,11R,13R,14S,16S)-16- acetyloxy-3,11-dihydroxy-4,8,10,14-tetramethyl- 2,3,4,5,6,7,9,11,12,13,15,16-dodecahydro-1H- cyclopenta[a]phenanthren-17-ylidene]-6- methylhept-5-enoic acid. 2. MATERIALS AND METHODS 2.1. Chemical Reagents Acetonitrile (HPLC grade), Methanol (HPLC grade), Acetic Acid (HPLC grade), Water (HPLC grade) and Ortho- Phosphoric acid (85% AR grade) Instrumentation Waters 2489 UV/Visible Detector, Waters 2998 UV/Visible Detector, Waters e2695, Separations Module (Alliance), Empower Software version 3, Analytical Balance (LC GC XA82/220/2X), HPLC Column: Lichrospher 100 RP-18, 5µm, (125 x 4) mm Liquid Chromatography The chromatographic separation was achieved on Lichrospher 100 RP mm x 4.0mm, 5 µm column,using a optimized mobile phase consisting a mixture of Acetonitrile, Methanol and 0.05M Ortho-Phosphoric acid in the ratio of 50:5:45 (v/v), at a flow rate of 1.5 ml/min. Injection volume 10 µl, The column compartment temperature was set at 25 C. The typical HPLC chromatograms were extracted at 235 nm. 2 P a g e

3 2.4. Standard and Sample preparation Preparation of 0.05 M Phosphoric Acid Add slowly while swirling 70 ml of phosphoric acid 85 % m/m to 1000 ml of water. Further dilute 50 ml to 1000 ml with water. Preparation of mobile phase Prepare a suitable quantity of a mixture of acetonitrile, methanol and 0.05 M phosphoric acid in the ratio of 50:5:45. Preparation of system suitability solution Dissolve 5 mg of 3-ketofusidic acid and 6 mg of diethanolamine fusidate reference substance in 50 ml of mobile phase and mix. Store the solution in a deep freezer. Preparation of standard solution Accurately weigh and transfer about 36 mg of Diethanolamine fusidate reference substance to a 50 ml volumetric flask. Dissolve in mobile phase and make up the volume to 50 ml with mobile phase and mix. Further dilute 1 ml to 100 ml with mobile phase and mix. Filter through glass fiber paper, discarding the first few ml of the filtrate. Preparation of performance check solution Dilute 3 ml of the standard solution to 100 ml with mobile phase. Preparation of placebo solution Disperse evenly g of the placebo in 25 ml of mobile phase. Heat this mixture until the placebo is melted and shake vigorously for 15 minutes. Cool the placebo mixture to a temperature less than 10 C and filter through glass fiber paper, discarding the first few ml of the filtrate. Heat the filtrate to room temperature before injection into the chromatograph. Preparation of sample solution Disperse evenly 0.75 g of the sample in 25 ml of mobile phase. Heat this mixture until the cream is melted and shake vigorously for 15 minutes. Cool the mixture to a temperature less than 10 C and filter through glass fiber paper, discarding the first few ml of the filtrate. Heat the filtrate to room temperature before injection into the chromatograph. Instrumental conditions Column : Lichrospher 100 RP-18, 5µm, (125 x 4) mm Column oven temperature : 25 C Flow rate : 1.5 ml/min. Detector : UV at 235 nm Injection volume:10 µl Run time : 20 min. Evaluation of system suitability a. Inject the system suitability solution into the chromatograph and record the chromatograms. The resolution between the peaks corresponding to 3-ketofusidic acid and fusidic acid is not less than If this resolution is not achieved, the mobile phase may be changed to a more aqueous solution. Repeat the test. b. Inject the performance check solution into the chromatograph and record the chromatograms. The signal to noise ratio of Fusidic acid is not less than 3. c. Inject the standard solution into the chromatograph and record the chromatograms. 3 P a g e

4 The relative standard deviation of six replicate injections for the area counts of Fusidic acid peak is not more than 5.0 %. Procedure Inject placebo and sample solution (single injection each) into the chromatograph and record the chromatograms. Measure the area counts of the peaks with retention times relative to fusidic acid between 0.32 and 3.5 Examine chromatogram of the placebo against sample chromatogram and disregard peaks observed in chromatogram of sample that are corresponding to peaks in the chromatogram of placebo. Retention Times Relative RT Excipients: < 0.5 min Sorbic acid approx 0.6 min 0.1 Butylhydroxyanisole 1.6 min Ketofusidic acid 3.9 min 0.7 Fusidic acid 5.3 min 1.0 S. No. Calculation: (Method I): Calculate % w/w of Fusidic acid related substances. = (T - F) X 100 T where, T = The sum of the area counts of all the peaks with retention times relative to fusidic acid between 0.32 and 3.5. F = The area counts of the fusidic acid peak. Disregard any peak with area less than 0.03% of the Fusidic acid area (F). Leave out of account the peak from butylhydroxyanisole (Rt.rel. 0.30).Method I gives results which are systematically too high, as most major related Substance show higher detector response than fusidic acid (Table: 1.A). Table: 1.A Retention times and Response Factors Relative to Fusidic Acid. Name of Impurity Relative RT Relative response factor (R) 1 24,25-Dihydroxyfusidic acid 21,25 lactone ,25-Dihydroxyfusidic acid 21,24-lactone Oxofusidic acid Ketofusidic acid Epideacetylfusidic acid Fusidic acid (Rt = 4.7 Min.) Fusidic acid (RT = 5.3 min) Epideacetylfusidic acid 16,21- lactone Deacetylfusidic acid 16,21-lactone Deoxyfusidic acid P a g e

5 3. RESULTS AND DISCUSSION 3.1. Method Development The chromatographic separation was achieved with column Lichrospher RP-18 (125 mm x 4.0mm, 5 µm), mobile phase mixture of Acetonitrile, Methanol and 0.05M Ortho-Phosphoric acid in the ratio of 50:5:45 (v/v), at a flow rate of 1.5 ml/min at 25 C (column oven) temperature, detection wavelength 235 nm with 10 μl injection volume Method Validation 8-27 This purpose of method validation was to demonstrate the suitability for routine application of the developed methodology in accordance with the ICH guidelines. Method validation was treated as a final verification of the method performance. An important part is system suitability test which covers in the detail number of resolution of individual components, Signal to noise ratio for Fusidic acid and repeatability of injection that was evaluated by retention time and peak area Specificity a. Blank solution was analysed, no interference from blank at the Table 2: Specificity retention time of Fusidic acid peak was observed. b. Placebo solution was injected, no interference from placebo at the retention time of Fusidic acid peak was observed. c. 3-ketofusidic acid was prepared and injected. d. Sample solution and spiked sample solution were injected and related substances of Fusidic acid are separated from each other and from the Fusidic acid peak in spiked sample solution. The peak purity plots of 3- ketofusidic acid in spiked sample solution indicates as purity angle is less than purity threshold that the peak was homogeneous and has no co eluting peaks and the peak purity of Fusidic acid peak in sample solution and spiked sample solution indicates as purity angle is less than purity threshold that the peak were homogeneous and has no co eluting peaks, indicating specificity of the method (Fig. 1).The purity angle and threshold are given in Table 2. S. No. Name of solution Name of the peak Purity Angle Purity Threshold 1 Sample solution Fusidic acid Spiked sample solution Fusidic acid Spiked sample solution 3-ketofusidic acid Figure 1: Specificity 5 P a g e

6 Degradation studies A forced degradation study was carried out on Fucidin cream and its placebo under the following conditions: Hydrolytic and oxidative degradation Sample and placebo were separately treated with 1 N hydrochloric acid, 1 N sodium hydroxide and 30 % w/w hydrogen peroxide solutions. Solutions of these samples were prepared as per the conditions given in Table 3 and analysed by the proposed method. Thermal degradation Sample and placebo were subjected to thermal degradation by keeping at 105 C for 36 hours 5 minutes followed by analysis by the proposed method. Table 3: Summary of Forced Degradation Results Mode of degradation Condition Photolytic degradation Photolytic degradation study was carried out by exposing the sample and placebo to light in a photolytic chamber at lux hours Watts hr/sq.m, followed by analysis by the proposed method. Using peak purity test, the purity of Fusidic acid peak was checked at every stage of the above mentioned study. The peak purity plot shows that Fusidic acid peak is homogeneous and has no coeluting peaks (as purity angle is less than purity threshold) indicating that the method is stability indicating and specific (Fig. 2-6). Data is summarized in Table 3. % Area % Degradatio n Purity Angle Purity Threshold Control No treatment Acid degradation 1 N HCl/3 ml Alkali degradation 1 N NaOH/2 ml Peroxide degradation 30 % w/w, H2O2/5 ml Thermal degradation Photolytic degradation 80 C / 2 h C / 2 h C / 2 h C / 36 hours 5 minutes lux hours Watts hr/sq.m P a g e

7 Fig. 2: Chromatogram of Acid degradation Sample Fig. 5: Chromatogram of Photolytic degradation Sample Fig. 3: Chromatogram of Alkali degradation Sample Fig. 6: Chromatogram of Thermal degradation Sample Fig. 4: Chromatogram of Peroxide degradation Sample Linearity of Response The linearity of response for Fusidic acid and 3-ketofusidic acid was determined in the range of µg/ml to µg/ml for Fusidic acid and µg/ml to µg/ml for 3-ketofusidic acid. Data shown in Table 4 and represented graphically in Figure: 7and 8. Indicate that the response is linear over the specified range. Fig. 7: Linearity plot of Fusidic acid A v e r a g e A r e a r a t i o Linearity plot of Fusidic acid Concentration Fig. 8: Linearity plots of 3-ketofusidic acid Linearity plot of Betamethasone-17 valerate Average Concentration P a g e

8 Table 4: Summary of linearity Data Area counts of Conc. Fusidic acid Conc. Area counts of 3- ketofusidic acid (µg/ml) (µv*sec.) (µg/ml) (µv*sec.) Slope 4894 Slope 5658 Intercept 532 Intercept 611 CC CC Precision System Precision the method has an acceptable level of precision. Six replicate injections of standard Intermediate Precision/Ruggedness solution were made into the HPLC system. Ruggedness of the method was verified by Data shown in Table 5 indicate an analyzing six samples of a single batch of acceptable level of precision for the analytical system. Method Precision Six sample solutions of a single batch of Fucidin cream was prepared and analysed by the proposed method. Data is shown in Table 5. The % RSD values indicate that Fucibet cream by two different analysts using different instrument and columns on different days. The mean, standard deviation and % RSD for the two sets of data are shown in Table 5. Ruggedness of the method is indicated by % RSD & overall % RSD values between the two sets of data. Table 5: System Precision, Method precision and Intermediate Precision Results Substance System Precision Area Method precision for Total impurities (% w/w) Intermediate Precision for Total impurities (% w/w) % % % Mean SD Mean SD Mean SD RSD RSD RSD Fusidic acid P a g e

9 Table 6: Summary of Recovery Results Amount Spiked Amount added-mean of triplicate (% w/w) Amount recovered- mean of triplicate (% w/w) % Recoverymean of triplicate 50% % % Accuracy Known amount of 3-ketofusidic acid was spiked with sample at three different levels, each in triplicate. The samples were analysed by the proposed method and the amount of 3-ketofusidic acid recovered after making corrections for the amount already present were calculated. Data shown in Table 6 indicate that the method has an acceptable level of accuracy Stability in Analytical Solution A fresh Sample solution of Fucidin cream was prepared and kept at room temperature. Solution was analyzed initially and at different time intervals. As the absolute % difference up to 2217 min. meets the acceptance criteria it is concluded that sample was stable in analytical solution for atleast 36 hours at room temperature Robustness Robustness of the method was investigated by checking the system suitability parameters by deliberately varying the instrumental conditions such as flow rate by ± 10 %, column oven temperature by ± 5 C, organic content in mobile phase by ± 2 % absolute and wavelength of detection by ± 5 nm. The system suitability parameters are presented in Table 7. The system suitability requirement was met under each variable condition. Under the condition of organic content in mobile phase (Methanol ratio constant and Acetonitrile content + 2 % absolute, Acetonitrile ratio constant and Methanol content + 2 % absolute and 0.05 M phosphoric acid content constant and Acetonitrile content + 2% absolute) significant changes in retention time of Fusidic acid peak. Hence, it is recommended that the organic content in mobile phase should be strictly adhered to as per method. Table 7: Summary of Robustness Results of the HPLC Method Experiment Resolution between 3- ketofucidic acid and Fusidic acid (NLT 3.50) Signal to noise ratio for Fusidic acid (NLT 3) %RSD (NMT 5.0) Flow plus Flow minus Column Temperature plus Column Temperature minus P a g e

10 Wavelength Plus Wavelength Minus Acetonitrile constant Methanol plus Acetonitrile constant Methanol minus Methanol constant Acetonitrile plus Methanol constant Acetonitrile minus H3PO4 Constant Acetonitrile plus H3PO4 Constant Acetonitrile minus Limit of Quantitation The limit of quantitation of Fusidic acid and 3-ketofusidic acid was determined by showing precision by making six replicate injections of lower concentration solution. Data is shown in Table 8. Table 8: Limit of quantitation Conc. Fusidic acid 3-ketofusidic acid µg/ml % w/w Injection S/N value Area counts (µv*sec.) S/N value Area counts (µv*sec.) Mean SD RSD (%) CONCLUSION Based on the results given in summary report, it is concluded that Isocratic RP- HPLC method was successfully developed for the related substances of Fusidic acid in topical pharmaceutical formulation. The developed method is selective, precise, accurate, linear, and robust. The forced degradation data proved that the method is specific for the analytes and free from the interference of the placebo / known impurities / and 10 P a g e

11 degradation products. Moreover, it may be applied for the individual and simultaneous determination of fusidic acid compounds in a pharmaceutical drug product and substance. 5. ACKNOWLEDGEMENTS The authors wish to thank the management of APPL for supporting this work. We would also like to thank colleagues in US Pharma lab for providing chemicals and impurity standards for our research work. 6. REFERENCE 1. Schultz Larsen F, Hanifin JM. Epidemiology of atopic der- matitis. Immunol All Clin North America 2002; 22: Leung DYM, Bieber T. Atopic dermatitis. Lancet 2003; 361: Abeck D, Mempel M. Staphylococcus aureus colonisation in atopic dermatitis and its therapeutic implications. Br J Dermatol 1998; 139: Menday AP, Noble WC. Topical betamethasone/fusidic acid in eczema: efficacy against and emergence of resistance in Staph- ylococcus aureus. J Dermatol Treat 2000; 11: Finn schultz larsen, lene simonsen, An Efficient New Formulation of Fusidic Acid and Betamethasone 17-Valerate (Fucicort Lipid Cream) for Treatment of Clinically Infected Atopic Dermatitis, Acta Derm Venereol 2007; 87: S.P. Denyer and R.M. Baird. Registration and Regulatory Affairs Guide to Microbiological Control in Pharmaceuticals. Ellis Horwood, West Sussex, England, 1990, Pub med chem. National Center for Biotechnology Information, U.S. National Library of Medicine8600 Rockville Pike, BethesdaMD, 20894USA /product. 8. Guidance for Industry Analytical Procedures and Methods Validation for Drugs and Biologics, U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER) February International conference on Harmonization. Validation of Analytical Procedure, Text and Methodology Q2 (R1), IFPMA, Switzerland, International Conference on Harmonization- Q1A (R2) Stability Testing of New Drug Substances and Products (November 2003) 11. International Conference on Harmonization ICH- Q1B Stability Testing: Photo stability Testing of New Drug Substances and Products (May 1997) 12. International Conference on Harmonization ICH- Q1C Stability Testing for New Dosage Forms (May 1997) 13. International Conference on Harmonization ICH -Q3A (R2) Impurities in New Drug Substances (June 2008) 14. International Conference on Harmonization ICH -Q3B (R2) Impurities in New Drug Products (August 2006) 15. International Conference on Harmonization ICH- Q3C Impurities: Residual Solvents (December1997) 11 P a g e

12 16. International Conference on Harmonization ICH-Q3C Tables and List (February 2012) 17. International Conference on Harmonization ICH-Q5C Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products (July 1996) 18. International Conference on Harmonization ICH-Q6A Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances (December 2000) 19. International Conference on Harmonization ICH-Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products (August 1999) 20. International Conference on Harmonization ICH-Q7 Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients (August 2001) 21. United States Pharmacopeia/National Formulary General Chapter <621> Chromatography 22. United States Pharmacopeia/National Formulary General Chapter <1010> Analytical Data Interpretation and Treatment 23. United States Pharmacopeia/National Formulary General Chapter <1224> Transfer of Analytical Procedures 24. United States Pharmacopeia/National Formulary General Chapter <1225> Validation of Compendia Procedures 25. United States Pharmacopeia/National Formulary General Chapter <1226> Verification of Compendial Procedures 26. United States Pharmacopeia/National Formulary General Notices and Requirements, Applying to Standards, Tests, Assays, and Other Specifications of the United States Pharmacopeia. 27. ASTM Standard, E Standard Practice for Using Significant Digits in Test Data to Determine Conformance with Specifications, ASTM International, West Conshohocken, PA, ( 12 P a g e