CHAPTER 7 SIMULTANEOUS ASSAY ESTIMATION OF BUTENAFINE HYDROCHLORIDE AND BETAMETHASONE FROM CREAM FORMULATION.
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1 CHAPTER 7 SIMULTANEOUS ASSAY ESTIMATION OF BUTENAFINE HYDROCHLORIDE AND BETAMETHASONE FROM CREAM FORMULATION. 7.1 OBJECTIVE To develop a simultaneous assay eatimation method for Butenafine hydrochloride and Betamethasone from cream formulation. 7.2 INTRODUCTION Butenafine hydrochloride [207,208] (n-4-ter-butyl-benzyl-n-methyl-1- naphthaline methylamine hydrochloride) is a derivative of benzylamine having same mode of action and chemical structure to allylamine antifungal agent. It has the molecular weight of and an empirical formula C23H27N.HCl. It leads to accumulation of squalene and converts squalene to lanosterol. It is primarily fungicidal against dermatophytes and inhibits squalene epoxidase an enzyme. The azoles have been used as antifungal to treat dermatophytosis since the 1970s. The most used topical agents are miconazole and clotrimazole. The cytochrome P450-dependent enzymes inhibit lanosterol 14-demethylase and these act as fungi-static agents, a component of fungal cell membrane, this is important for the biosynthesis of ergosterol, Butenafine hydrochloride is slightly soluble in water, freely soluble in ethanol, methanol and chloroform. 224
2 Betamethasone dipropionate [209, 210] is chemically 9-fluoro-11bhydroxy-16b-methyl-3, 20-dioxopregna-1, 4-diene-17, 21-diyl dipropionate. It has molecular weight of and the empirical formula C28H37FO7. Betamethasone dipropionate is almost white to white crystalline powder, practically insoluble in water, sparingly soluble in alcohol, freely soluble in acetone and in methylene chloride. The cream formulation contains 0.05% betamethasone as betamethasone dipropionate, a synthetic corticosteroid is member of the class of steroids and 1% butenafine hydrochloride, a synthetic antifungal agent. 7.3 LITERATURE REVIEW Yan-Yan L.B. et al were reported High performance liquid chromatography determination of the sample, after extraction with methanol under ultrasonic bath, was separated on HP Hypersil column C18 (150 mm x 4.6 mm 5u ), with a mobile phase, consisted of methanol and 0.05 mol/l ammonium acetate, a flow rate of 0.8 ml/min, a detecting wavelength at 223 nm, the column temperature at 40 C, within the range of μg, the calibration curve was linear(r=0.9997), the mean recovery rate was 9.89 mg/g, accorded label content with the 10 mg/g [211]. Chunying W. et al were reported HPLC determination of butenafine hydrochloride ointment content. Kromasil-C18 column with acetonitrile - 225
3 methanol-0.0 5mol/L ammonium acetate buffer ph 6.8, (66:19:15) for the mobile phase, detection wavelength of 282 nm. The sample size in the range of 4.1 ~ 20.5 μg with a good linear relationship, the average recovery was 98.17% and the relative standard deviation (RSD) was 0.13% [212]. Rui-Guo C, Liu YC et al were reported the objective HPLC determination of butenafine hydrochloride in gel formulation. Waters Symmetry C18 (3.9 mm 150mm, 5μm) column with methanol and sodium acetate buffer in the ratio 73:27, as mobile phase at a flow rate 1.0 ml/min, detection wavelength of 282nm, column temperature of 35 C. Butenafine hydrochloride was linear in the range of ~ μg/ml having good linear relationship, the minimum detection limit of 0.39 ng. The average recovery was 99.72% and RSD was 0.52% (n = 6) [213]. Kok-Khiang P. et al were reported a simple and selective HPLC method using UV detection for simultaneous determination of Fusidic acid and betamethasone dipropionate in a cream formulation. A supelcosil C18 column was used for chromatographic separation. The mobile phase used was mixture of acetonitrile and 0. 01M sodium hydrogen orthophosphate in the ratio of 70:30, %v/v and ph was adjusted to 6.0 with glacial acetic acid. Chromatographic system was operarted at a flow rate of 1.0 ml/ min with the UV detector set at
4 nm. The method was found satisfactory with respect to all validation parameters [214]. Kedor-Hackmann E.R. et al were reported 27the simultaneous determination of betamethasone dipropionate and salicylic acid in both ointment and topical solution using HPLC. The method was optimized using a Licrosphere 100 RP -18 (125 x 4 mm, 5 microns) column, tetrahydrofuran- acetonitrile- acetic acid 1% (20:25:55 v/v), ph 3.3, as mobile phase, and UV detection was carried at 254 nm. The method was found satisfactory with respect to linearity and range, recory and precission [215] Dyderski S. et al were reported were reported A reverded- phase HPLC method was developed for the determination of betamethasone dipropionate in lipophylic bases. Analysis was performed using a PLRP column with a mobile phase of water- methanol- acetonitrile and ultraviolet detection at 254 nm. The calibration curve was constructed for concentration (0-50 μg/ml the method is simple, accurate and precise [216]. Ankam R. et al were reported RP-HPLC for the simultaneous determination of betamethasone and butenafine hydrochloride in cream formulation. The determination was carried out on lichrocart lichrosphere RP-select B (250Χ4.6 mm, 5 μ) column in isocratic mode, the mobile phase used was mixture of 50 mm ammonium acetate buffer 227
5 and acetonitrile in the ratio of 60:40, adjusted to ph 4.5 ± 0.1 with glacial acetic acid. The flow rate was maintained at 2.0 ml/min and elution was monitored at 254 nm [217]. Minshan S et al were reported RP-HPLC method was developed for simultaneous determination of betamethasone, dipropionate salicylic acid and their related compounds in Diprosalic Lotion. A YMC Jsphere ODS (150 mm x 4.6 mm id.) column at 35 C and UV detection at 240 nm was used. A gradient elution was employed using acetonitrile and 0.05% v/v methanesulfonic acid solution as mobile phases. All compounds were evaluated in 38 min. All impurities and degradation products were well resolved, this indicating capacity of this method [218]. 7.4 THEORETICAL ANALYSIS Butenafine hydrochloride chemical structure and mode of action is similar to allylamine antifungals. It has the empirical formula C23H27N.HCl and a molecular weight of
6 Figure 7.01 Butenafine hydrochloride Butenafine Hydrochloride is Structural formula reveals that the molecule is ionic and somewhat non polar. Systematic name is [(4-tertbutylphenyl) methyl](methyl)(naphthalen-1-ylmethyl)amine. It require buffer to elute smoothly, hence 0.1% ortho-phosphoric acid can be selected as buffer as the acidic ph is more robust than the neutral. Figure 7.02 Betamethasone Structural formula Betamethasone dipropionate is [(8S,9R,10S,11S,13S,14S,16S,17R)-9- fluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(2-propanoyloxyacetyl)-6, 7, 8, 11, 12, 14, 15, 16-octahydrocyclopenta[a]phenanthren-17-yl] propanoate, chemical formula is C28H37FO7 and molecular mass is g/mol. Structural formula reveals that the molecule have no 229
7 acidic or basic functional groups, hence it will not demand for buffer, as it is nonpolar hence it require solvent strength for its elution. Hence methanol can be selected as initial solvent because of its low cost even if is more viscous with aqueous mobile phase than acetonitrile. As the analyte is combination of BH and BP, initial trials are selected with 0.1% OPA and methanol. Simple C-18 column was selected for initial trials as it is stable and rugged. Both the molecules having UV absorption hence simple UV detector selected, Betamethasone has lowest amount in the formulation and hence priority given to betamethasone, selectively 254 nm wavelength was selected for monitoring the both analytes. Initial trials were done on 25 C and flow rate at 1.5ml/minutes to get faster elution. Diluent selected based on the solubility of both analytes. Water and methanol in equal volume were used for initial trials. 7.5 EXPERIMENTAL INVESTIGATIONS The reference standard of Butenafine hydrochloride was procured from Hetero Drugs Limited, (Hyderabad, India) betamethasone dipropionate and cream sample were prepared in In-house facility. Creams are claimed to contain 1% w/w of Butenafine hydrochloride, 0.05% w/w synthetic corticosteroid betamethasone as betamethasone dipropionate and the following inactive ingredients: benzyl alcohol, ethyl alcohol, cetyl alcohol, and sodium benzoate, emulsifying wax, disodium edentate, polyethylene glycol 40 stearate, liquid petrolatum, polysorbate 230
8 60, propylene glycol, simethicone, white petrolatum, sodium hydroxide and water. All reagents were analytical or HPLC grade ammonium acetate, acetonitrile, and glacial acetic acid were procured from Merck India Limited (Mumbai, India. Purified water was obtained by a Millipore Dire-Q 3 UV with pump (Molsheim, France) The HPLC system (Jasco, Tokyo, Japan) consisted of an AS-2057 intelligent sampler, PU-2080 quaternary pump, UV-2075 intelligent UV/Vis detector, and CO-2065 intelligent column oven and detector seted at 254nm. The analytical column, a lichrocart lichrosphere RPselect B (250 mm x 4.6 mm i.d., 5 μmparticle size) (Merck, Germany) was operated in ambient temperature (25 C) Experiment No.1 The mobile phase was acetonitrile and a solution of 0.1% phosphoric acid buffer ph 3.0 adjusted with 10% solution of phosphoric acid, (30:70; v/v). Mobile phase was filtered through 0.45 μ Teflon membrane filter. The mobile phase flow rate was maintained at 1.0 ml/min. Standard solutions of butenafine hydrochloride 200 μg/ml and betamethasone 10 μg/ml were prepared in mobile phase. These standard solutions were injected two times and detector response measured at 254 nm. Both the peaks were not eluted until 30 minutes, therefore next experiment carriedout with increased organic modifier in mobile phase. 231
9 7.5.2 Experiment No. 2. The mobile phase was acetonitrile and a solution of 50 mm potassium dihydrogen phosphate buffer adjusted ph to 6.8 with 10% solution of phosphoric acid, (40:60; v/v). Mobile phase was filtered through 0.45 μ Teflon membrane filter. The mobile phase flow rate was maintained at 1.0 ml/min. Standard solutions of butenafine hydrochloride 200 μg/ml and betamethasone 10 μg/ml were prepared in mobile phase. These standard solutions were injected two times and average detector response measured at 254 nm. Both butenafine and betamethasone peaks were eluted in 30 minutes. Since the run time was more and peak shape found was not satisfactory, next experiment carried with changing mobile phase composition Experiment No. 3. The final selected mobile phase was acetonitrile and a solution of 50mM ammonium acetate buffer adjusted ph to 4.5 with 10% solution of acetic acid (80:20; v/v). Mobile phase was filtered through 0.45 μ membrane filter. The mobile phase flow rate was maintained at 1.0 ml/min. Standard solutions of butenafine hydrochloride 200 μg/ml and betamethasone 10 μg/ml were prepared in mobile phase. These standard solutions were injected two tomes and detector response measured at 254 nm. 232
10 All the actives and impurities were not separated in dilute standard and sample spiked impurities. Therefore experiment carried with different mobile phase composition Experiment No. 4. The final selected mobile phase was acetonitrile and a solution of 50mM ammonium acetate buffer adjusted ph to 4.5with 10% solution of acetic acid (20:80; v/v). Mobile phase was filtered through 0.45 μ membrane filter. The mobile phase flow rate was maintained at 1.0 ml/min. Standard solutions of butenafine hydrochloride 200 μg/ml and betamethasone 10 μg/ml were prepared in mobile phase. These standard solutions were injected two times and average detector response measured at 254 nm. All the actives and impurities were separated in dilute standard while in sample spiked with impurities in 50 min. since run time is more, next experiment carried with fine tuning of mobile phase Experiment No
11 The final selected mobile phase was acetonitrile and a solution of 50mM ammonium acetate buffer adjusted ph to 4.5with ten percent solution of acetic acid (60:40; v/v). Mobile phase was filtered through 0.45 μ membrane filter. The mobile phase flow rate was maintained at 1.0 ml/min. Standard solutions of butenafine hydrochloride 200 μg/ml and betamethasone 10 μg/ml were prepared in mobile phase. These standard solutions were injected two times and detector response measured at 254 nm. All the actives and impurities were separated in dilute standard as well as sample spiked with impurities in 30 min. Since the run time was more, next experiment carried with increased flow rate Experiment No. 6. The final selected mobile phase was acetonitrile and a solution of 50mM ammonium acetate buffer adjusted ph to 4.5with 10% solution of acetic acid (6:4; v/v). Mobile phase was filtered through 0.45 μ membrane filter. The mobile phase flow rate was maintained at 2.0 ml/min. Standard solutions of butenafine hydrochloride 200 μg/ml and betamethasone 10 μg/ml were prepared in mobile phase. These standard solutions were injected two times and detector response measured at 254 nm. Both Butenafine hydrochloride and Betamethasone peaks were eluted in 15 minutes. Since the run time peak shapes found were satisfactory. 234
12 In-house developed R&D samples (Lot-1, 2 and 3) each two tubes about 5 g sample was taken in 100 ml beaker and mixed properly. Cream approximately 20 mg of butenafine hydrochloride and 1 mg of betamethasone equivalent sample was taken in 100 ml volumetric flask and dissolved in mobile phase and made volume up to the mark. Above sample was transferred to 200 ml volumetric flask and chilled in ice bath for 10 min. supernatant solution was filtered through Whatman filter paper No. 40. The further aqliquot solution was further diluted to get final concentration. The solution was evaluated at 254 nm. The chromatographic conditions utilized were proper for proper resolution of peaks Experiment No. 7(Method Validation) Specificity Two types of specificity experiments were performed. In the first one, specificity assessed by comparing the chromatograms obtained from the pharmaceutical preparation and the standard solution with those obtained from excipients which take part in the commercial cream and verifying the absence of interferences. In the second type, forced degradation performed in order to check the suitability of analytical conditions for stability study of Butenafine and Betamethasone. The accelerated degradation conditions applied were: light (UVC), acid, basic basic and oxidant media. Samples were analysed and compared with 235
13 control sample solution (with no degradation treatment) and under light protection. The peak purity was evaluated using the tools of the Waters software. Excipient solutions were submitted to the same degradation conditions in order to explain no interference. Specific details of the experiments conditions are described below: Effect of UV light: 1 ml of a solution containing 0.2 mg/ml of butenafine and 0.01 mg/ml of betamethasone in acetonitrile was placed in a closed 1 cm quartz cell. The cell was exposed to a UV chamber (100 x 18 x 17 cm) with internal mirrors and UV fluorescent lamp CRS F30W T8 emitting radiation at 254 nm for 15, 30, 60, 120 and 180 minutes. The same procedure was realized for preparation for LC analysis. Samples, protected in aluminum foil were submitted simultaneously to identical conditions and used as control. After the degradation treatment, the samples were diluted to 200 μg/ml with a mixture of water and acetonitrile, (60:40; v/v) and immediately analyzed. Effect of Oxidation: Butenafine and betamethasone standards were dissolved in acetonitrile (0.2 mg/ml of butenafine and 0.01 mg/ml of betamethasone), 5 ml of this solution was transferred to a volumetric flask, where hydrogen peroxide solution (30%) was added until the final concentration of 10 % and the volume was completed with acetonitrile. After 20 hours the solution was diluted with a mixture of water and acetonitrile, (60:40; v/v) to final concentration of 200 μg/ml of 236
14 butenafine hydrochloride and 10 μg/ml of betamethasone, filtered and analysed. Similar procedure was realized for the commercial cream, when 25 ml of the initial solution 0.2 mg/ml of butenafine and 0.01 mg/ml of betamethasone, obtained as described in sample preparation for LC analysis, were transferred to a volumetric flask and submitted to degradation. A control solution containing the excipients was prepared under the same circumstances of the commercial cream. Effect of Acid Hydrolysis: 5 ml of 0.2 mg/ml of butenafine and 0.01 mg/ml of betamethasone reference standard solution was transferred to a volumetric flask and HCl (acid degradation) was added until the final concentration of 1M HCl. After 5 hours and 1 and 6 days, one aliquot of the solution was neutralized with NaOH 1M and diluted with acetonitrile and water (40:60, v/v) until the final concentration of 200 μg/ml of butenafine hydrochloride and 10 μg/ml of betamethasone for LC analysis. Similar procedure was realized with the cream, when 25 ml of the initial solution 0.2 mg/ml of butenafine and 0.01 mg/ml of betamethasone (obtained as described in sample preparation for LC analysis) were transferred to a volumetric flask and submitted to the degradation. A control solution containing the excipients was prepared under the same circumstances of the cream. Effect of Alkaline Hydrolysis: 237
15 5 ml of 0.2 mg/ml of butenafine and 0.01 mg/ml of betamethasone reference standard solution was transferred to a volumetricflask and NaOH (alkaline degradation) was added until the final concentration of 1M NaOH. After 5 hours and 1 and 6 days, one aliquot of the solution was neutralized with HCl 1M and diluted with acetonitrile and water (40:60, v/v) until the final concentration of 200 μg/ml of butenafine hydrochloride and 10 μg/ml of betamethasone for LC analysis. Similar procedure was realized with the cream, when 25 ml of the initial solution 0.2 mg/ml of butenafine and 0.01 mg/ml of betamethasone (obtained as described in sample preparation for LC analysis) were transferred to a volumetric flask and submitted to the degradation. A control solution containing the excipients was prepared under the same circumstances of the cream. Precission: The repeatability was verified from six independent sample preparations in the same day, obtained as described in Sample preparation for LC analysis. The intermediate precision was tested by assaying freshly prepared sample solutions at the concentration on two different days. Precision was reported as %RSD. Six replicate injections of the standard preparation were madeinto the HPLC used the methodology given in experimental result. 238
16 Six spiked sample preparations and one control sample preparation of Butenafine hydrochloride and betamethasone cream were prepared and injected into the HPLC using the method as described under experimental result. Accuracy: The accuracy was determined by the recovery of known amounts of Butenafine and betamethasone standards added to the placebo in the beginning of the preparative process. The added levels were 80, 100 and 120% of the nominal drug concentrations. The results were expressed as the percentage of Butenafine hydrochloride and Betamethasone reference standards recovered from the sample Ruggedness: Six spiked sample preparations and one control sample preparations of Butenafine hydrochloride and Betamethasone Cream were analysed by a different analyst, using different column, on different day and injected into a different HPLC using the method as described in experimental result, along with standard preparation. Robustness: Standard preparation, diluent, placebo preparation and sample preparation in triplicate of the sample of Butenafine hydrochloride and Betamethasone Cream were prepared as described in experimental 239
17 result. The samples along with standard and placebo were injected under different chromatographic conditions as shown below. Linearity and Range To test linearity, standard plots were constructed with six concentrations in the range of 100 to 300 μg/ml of Butenafine hydrochloride and 5 to 15 μg/ml Betamethasone prepared in triplicates. The linearity was evaluated by the least square regression Stability of analytical solution: Standard solution, Sample solution were analysed initially and at different time intervals at room temperature. The system suitability was verified through the evaluation of the obtained parameters for the standard elution, such as theoretical plates, peak asymmetry and retention factor, verified in different days of the method validation. The objective of validation of an analytical procedure is to demonstrate that the method suitable for its intended purpose. The method was validated for linearity, precision (repeatability and intermediate precision), accuracy, specificity, robustness and system suitability. 7.6 EXPERIMENTAL RESULTS 240
18 On the basis of butenafine Hydrochloride and betamethasone analytical method development experimental trials, RP-HPLC method was suitable for simultaneous determination of butenafine hydrochloride and betamethasone assay. Final experiment chromatographic conditions were applied Preparation of stock solutions: Prepare solution having the concentration of Butenafine hydrochloride 200 ppm and betamethasone 10 ppm in mobile phase. Sample preparation: 5 g cream smple was weighed and transferred into 100 ml beaker and mixed properly. An accurately weighed quantity of cream equivalent to 20 mg of butenafine hydrochloride and 1 mg of betamethasone was taken in 100 ml volumetric flask and dissolved in mobile phase. Whole solution was transferred in 250 ml volumetric flask and chilled it in ice bath for 10 min. The solution was filtered through Whatman filterpaper No.40. The aliquot portion of the filtrate was further diluted to get final concentration. Separately injected equal volumes of diluent, standard preparation in six replicates and sample twice in to equilibrated HPLC system and record chromatograms and measured the response in terms of peak area. System suitability parameters occurred during method validation were Theoretical plates mores than 5000, tailing factor less than 1.5, relative 241
19 standard deviation for six replicates of standard solution is less than 2.0%. 7.7 DISCUSSION OF RESULTS Linearity and range: the correlation coefficients are less than for Butenafine hydrochloride and Betamethasone. Precision: system precision RSD is less than 2% and method precision RSD is less than 2% for Butenafine hydrochloride and Betamethasone. Accuracy: the mean recoveries for Butenafine hydrochloride and Betamethasone are within %. Specificity: Retention time of Butenafine hydrochloride and Betamethasone peaks in sample preparation is comparable with respect to retention time of Butenafine hydrochloride and Betamethasone peaks in standard preparation. Peak purity passes for Butenafine hydrochloride and Betamethasone peaks in standard and sample preparations. No intereference was observed at the retention time of Butenafine hydrochloride and Betamethasone peaks. Peak purity passes for all degradation conditions. Ruggesness: the RSD of twelve results obtained from two different analysts are within 10 %. 242
20 Robustness: Butenafine hydrochloride and Betamethasone peaks were resolved with each other and system suitability complies for all variable conditions, the test method is robust for all variable conditions. Stability in analytical solution: Standard and sample solutions are stable for 12 h at room temperature System suitability: Theoretical plates are less than 2000, tailing factor is less than 2.0 and relative standard deviation is less than 5.0 for six standard replicate injections. Table 7.01 Peak Purity Data of Butenafine Hydrochloride and Betamethasone Sr. No. 1 Name Butenafine hydrochloride in standard solution Purity Criteria Pass 243
21 2 3 4 Butenafine hydrochloride sample solution Betamethasone in standard solution Betamethasone in sample solution Pass Pass Pass Table 7.02 Recovery Data of Butenafine hydrochloride Levels (%) Added conc. (μg/ml) Average Area Recovere d conc. (μg/ml) Recovery (%) Mean recover y (%) 80 Spl Spl Spl Spl Spl Spl Spl Spl Spl Mean (%) SD 1.96 RSD (%) 1.94 Table 7.03 Recovery Data of Betamethasone. Added levels (%) Added conc. (μg/ml) Average Area Recovere d conc. (μg/ml) Recovery (%) Mean recover y (%) 244
22 80 Spl Spl Spl Spl Spl Spl Spl Spl Spl Mean (%) 99.2 SD 1.98 RSD (%) 1.99 Table 7.04 Linearity Data of Butenafine HCl Levels (%) x a (μg/ml) y=mx+c b=a-y
23 Correlation Intercept (c) Slope (m) x=concentration, a=experimental area, y=predicted area and b=residuals. Table 7.05 Linearity Data of Betamethasone Levels (%) x a (μg/ml) y=mx+c b=a-y Correlation Intercept (c) Slope (m) Table 7.06 Method Precision Data of Butenafine HCl Sample Sample wt (mg) Average Area Mg/Unit % Assay Spl
24 Spl Spl Spl Spl Spl Average SD 1.19 RSD 1.17 Table 7.07 Method Precision Data of Betamethasone Sample Sample wt (mg) Average Area Mg/Unit % Assay Spl Spl Spl Spl Spl Spl Average SD 1.73 RSD 1.69 Table 7.08 Intermediate Precision Data of Butenafine HCl. Sample Sample wt (mg) Average Area Mg/Unit % Assay Spl
25 Spl Spl Spl Spl Spl Average SD 0.81 RSD 0.81 Table 7.09 Intermediate Precision Data of Betametasone. Sample Sample wt (mg) Average Area Mg/Unit % Assay Spl Spl Spl Spl Spl Spl Average SD 0.66 RSD 0.66 Figure 7.03 Sample chromatograph of Betamethasone and Butenafine HCl mv
26 Typical chromatogram of butenafine hydrochloride (200 µg/ml, t R =4.703 min) and betamethasone (10 µg/ml, t R =7.767 min) sample under optimized conditions. 7.8 SUMMARY, CONCLUSION AND RECOMMENDATIONS The reversed phase LC method proposed was found to be simple, fast, accurate, precise, linear, robust and specific and it is powerful tool to investigate chemical stability of butenafine hydrochloride and betamethasone in cream formulation. The robustness of the method was verified with small variation on ph, concentration of organic phase, detector wavelength, column manufacturer and analysis temperature. All the parameters meet the criteria of the ICH guidelines for method validation. Its chromatographic retention time 4.7 of Butenafine hydrochloride and 7.76 min of betamethasone allows the analysis of a large number of samples in an adequate period of time. Therefore the method could be recommended for routine quality control analysis commercial cream, as well as for routine quality control analysis of raw material and of cream. The method applicability to stability studies was proved through the evaluation of the main factors that affect the drug content in solution and through the butenafine and betamethasone 249
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