DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR ESTIMATION OF FLUTICASONE PROPIONATE AND MUPIROCIN IN A COMBINED TOPICAL DOSAGE FORMHTML Full Text
DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR ESTIMATION OF FLUTICASONE PROPIONATE AND MUPIROCIN IN A COMBINED TOPICAL DOSAGE FORM
A. Khristi * and N. Prajapati
Parul Institute of Pharmacy, Limda, Waghodiya, Vadodara - 391760, Gujarat, India.
ABSTRACT: The objective of the present study was to develop and validate a precise and accurate reversed-phase high-performance liquid chromate-graphy for simultaneous estimation of for Fluticasone Propionate (FUP) and Mupirocin (MUP) in combined topical dosage form as per ICH guidelines. Chromatographic separation was achieved using HPLC Shimadzu, Japan, with column syncronis C18 (250 × 4.6 mm, 5 μm). The mobile phase was comprised of 0.01% OPA: Acetonitrile (30:70v/v) (pH: 5) pumped at a rate of 1.0 mL/min. About 20 μL of sample solutions were injected and monitored at 232 nm. Column temperature and sample compartment were maintained at 25° and 5°, respectively. Repeatability, intra, and inter-day precision results were well within the tolerable limits. The linearity was found for range 2.5μg/mL - 7.5μg/mL and 100μg/mL - 300 μg/mL for FUP and MUP respectively. The correlation coefficient of linearity was found to be 0.998 and 0.994 for FUP and MUP, respectively. The limit of detection was found to be 0.3527, and 0.5077and limit of quantification was found to be 1.0690 and 1.538 for FUP for MUP, respectively. This method appeared to be rapid, easy, accurate, specific, and robust. Therefore, the method could be applied for regular examination.
Fluticasone Propionate, Mupirocin, RP-HPLC, Validation
INTRODUCTION: 1-5 Atopic Dermatitis (AD), also known as atopic eczema, is a type of inflammation of the skin (dermatitis). It results in itchy, red, swollen, and cracked skin. Clear fluid may come from the affected areas, which often thicken over time. The condition typically starts in childhood with changing severity over the years. In children under one year of age, much of the body may be affected. Treatment involves avoiding things that make the condition worse, daily bathing with the application of a moisturizing cream afterward, applying steroid creams when flares.
Flutibact Skin Ointment (10gm) s manufactured by Glaxo Smith Kine is used in Atopic dermatitis, containing fluticasone 0.05% w/w and Mupirocin 2% w/w. Fluticasone Propionate Fig. 1, a medium potency synthetic corticosteroid, is used topically to relieve inflammatory and pruritic symptoms of dermatoses and psoriasis, intranasally to manage symptoms of allergic and non-allergic rhinitis while Mupirocin Fig. 2 used to treat bacterial infection of impetigo due to: Staphylococcus aureus and Streptococcus pyogenes. Mixed anti-inflammatory glucocorticoids and antibacterial agents, MUP, which inhibits synthesis of protein of the bacteria by binding to iso-leucyl tRNA- synthetase.
It is active against gram-positive and some gram-negative bacteria. Fluticasone Propionate indicates that it is in the medium range of potency as compared with other topical corticosteroids.
FIG. 1: CHEMICAL STRUCTURE OF FLUTICASONE PROPIONATE
FIG. 2: CHEMICAL STRUCTURE OF MUPIROCIN
MATERIALS AND METHOD:
Materials: 7-9 Fluticasone Propionate (API) has been procured from Vamsi Labs Pvt. Ltd.And Mupirocin (API)has been received as gift samples from Glenmark pharmaceuticals, Mumbai. HPLC grade Acetonitrile, Methanol, and Water has been procured from Fischer scientific, Mumbai. HPLC grade orthophosphoric acid has been procured from RFCL limited. AR grade Potassium dihydrogen phosphate has been procured from Merck, and Sodium hydroxide has been procured from SD fine chem.
Selection of Solvent: Based on solubility studies Acetonitrile and water mixture in the ratio of 50:50 was selected for method development of both the drugs Fluticasone Propionate and Mupirocin.
Selection of Wavelength: Standard solutions of Fluticasone Propionate and Mupirocin 10 μg/mL of each were prepared in acetonitrile: water mixture (50:50) as a solvent. Each solution was scanned between 200-400 nm using Methanol as a blank. The point at which both drugs show common absorbance (isosbestic point) was selected as a wavelength for determination in overlay spectra of both drug as shown in Fig. 3.
FIG. 3: OVERLAY SPECTRA OF FLUTICASONE PROPIONATE AND MUPIROCIN
Selection of Mobile Phase and Optimization of Chromatographic Condition:
Selection of Mobile Phase: Depending upon the solubility of the drugs, various solvent were tried as mobile phase for separation of Fluticasone Propionate and Mupirocin.
Preparation of Stock Solution for Fluticasone Propionate and Mupirocin: Accurately weighed 5 mg of Fluticasone Propionate and 20 mg of Mupirocin was transferred separately into 100 mL and 10 volumetric flasks respectively, dissolved and diluted up to mark with diluent. It gives a stock solution having a concentration of 50μg/mL Fluticasone Propionate and 2000μg/mL of Mupirocin, respectively.
Preparation of Working Standard Solution for Fluticasone Propionate and Mupirocin: From the stock solution of Fluticasone Propionate (50 µg/mL) 0.5, 0.75, 1.0, 1.25, and 1.5mL of aliquots were transferred in five different 10 mL volumetric flask and from the stock solution of Mupirocin (2000 µg/mL) 2.5, 3.75, 5.0, 6.25, and 7.5mL of aliquots were transferred in five different 10 mL volumetric flask and volume was made up to mark with the methanol to prepare 2.5, 3.75, 5.0, 6.25, and 7.5mL µg/mL of the Fluticasone Propionate and 100, 150, 200, 250 and 300 µg/mL of the Mupirocin.
Preparation of Mobile Phase: 0.1% OPA buffer was prepared (1.0 mL of ortho-phosphoric acid was diluted to 1000mL with HPLC grade water) and pH adjusted to 5 with NaOH and sonicated for 20 min, and acetonitrile was added.
RESULTS AND DISCUSSION: Different trials have been taken during optimization of the method where the list has been shown in Table 1.
TABLE 1: SELECTION OF MOBILE PHASE OF MIXTURE
|S. no.||Mobile Phase||Ratio
|Retention Time (min)||Remark||Figure no.|
|1||Water : tetrahydrofuran : 1.05% ammonium acetate (pH5.7)||35:15:50||5.786||-||Mupirocin peak was observed but tailing was observed and Fluticasone Propionate peak was not observed||
|2||Methanol: Phosphate buffer||20:80||4.585||-||Mupirocin peak was observed but system suitability criteria not followed and Fluticasone Propionate peak was not observed.||
|3||Acetonitrile: 0.1M phosphate buffer
|28:80||4.528||-||Mupirocin peak was observed and Fluticasone Propionate peak was not observed.||
|4||Buffer 0.1% OPA : ACN: Methanol||35:50:15||1.861||4.794||Mupirocin and Fluticasone Propionate peak was observed. All system suitability parameters were not as per criteria.||
|5||0.1%OPA Buffer: CAN||65:35||2.454||12.592||Peak separation was good with high resolution. RT prolonged Theoretical plates and tailing factors for both drugs were as per system suitability criteria||
|6||0.1%OPA Buffer: CAN||30:70||2.969||6.793||Peak separation was good with high resolution. Theoretical plates and tailing factors for both drugs were as per system suitability criteria.||
Selection of Optimized Chromatographic Condition: Selection of optimized chromatographic conditions based on different trials.
Optimized Chromatographic Condition by Trial and Error Method:
Column: Syncronis C18, 250 mm × 4.6 mm, 5 μm.
Flow Rate: 1.0 mL/min.
Wavelength: 232 nm
Injection Volume: 10 μL
Run Time: 8 min
0.1% OPA Buffer B. Acetonitrile
Mobile Phase Ratio: 30: 70 % v/v
The results of the optimized chromatographic conditions have been shown in Table 2. Where both the drugs eluted, as shown in Fig. 4.
FIG. 4: CHROMATOGRAM OF MIXTURE IN 0.1% OPA BUFFER (pH 5): ACN (30:70) v/v AT 232 nm
TABLE 2: RESULT OF OPTIMIZED CHROMATOGRAPHIC CONDITION
|Drug||Retention time (min)||Area (mv)||Theoretical plates||Tailing Factor||Resolution|
Validation of RP-HPLC Method:
System Suitability Test: In this solution of Fluticasone Propionate and (5µg/mL) and Mupirocin (200µg/mL) was prepared. Parameters such as tailing factor, theoretical plate, resolution, reproducibility (% RSD, retention time, area) were determined, as shown in Fig. 5. The results of the system suitability have been shown in Table 3.
FIG. 5: CHROMATOGRAM OF SYSTEM SUITABILITY
TABLE 3: SYSTEM SUITABILITY DATA OF FLUTICASONE PROPIONATE (5μg/mL) AND MUPIROCIN (200μg/mL)
|No. of runs||Retention time (min)||Theoretical plates||Tailing factor||Resolution|
|Limit||< 2||>2000||< 2||> 2|
Specificity: There was no interference of the placebo in the formulation of the chromatogram, as shown in Fig. 6, 7, and 8.
FIG. 6: CHROMATOGRAM OF BLANK
FIG. 7: CHROMATOGRAM OF STANDARD FLUTICASONE PROPIONATE AND MUPIROCIN
FIG. 8: CHROMATOGRAM OF FORMULATION FLUTICASONE PROPIONATE (5µg/mL) AND MUPIROCIN (200µg/mL)
Linearity and Range: The linearity for Fluticasone Propionate and Mupirocin was found to be in the range of 2.5 to 7.5µg/mL and 100 to 300µg/mL, respectively Fig. 9, 10, and 11.
The peak areas for linearity of Fluticasone Propionate and Mupirocin have been shown in Table 4 and Table 5, respectively.
TABLE 4: LINEARITY DATA FOR FLUTICASONE PROPIONATE
|Concentration of Fluticasone Propionate (µg/mL)||Peak area|
FIG. 9: OVERLAY CHROMATOGRAM OF FLUTICASONE PROPIONATE AND MUPIROCIN
FIG. 10: CALIBRATION CURVE OF FLUTICASONE PROPIONATE
TABLE 5: LINEARITY DATA FOR MUPIROCIN
|Concentration of Mupirocin (µg/mL)||Peak area|
FIG. 11: CALIBRATION CURVE OF MUPIROCIN
Repeatability: The data for repeatability of area measurement for Fluticasone Propionate (5µg/mL) and Mupirocin (200µg/mL) based on six measurements of the same solution of Fluticasone Propionate and Mupirocin % RSD was calculated. The peak areas have been shown in Table 6.
TABLE 6: REPEATABILITY DATA OF FLUTICASONE PROPIONATE (5μg/mL) AND MUPIROCIN (200μg/mL)
|S. no.||Peak Area|
|Mean peak area||1953928.83||133593|
Intraday Precision: The data for intraday precision of area measurement for the standard solution of Fluticasone Propionate (3.75, 5 and 6.25 µg/mL) and Mupirocin (150, 200 and 250 µg/mL), total nine determination were analyzed at three consecutive times on same day and % RSD was calculated. The peak areas have been shown in Table 7.
TABLE 7: INTRADAY PRECISION DATA OF FLUTICASONE PROPIONATE AND MUPIROCIN
Inter-day Precision: The data for inter-day precision of standard solution of Fluticasone Propionate (3.75, 5 and 6.25 µg/mL) and Mupirocin (150, 200 and 250 µg/mL), total nine determination were analyzed at three consecutive days and % RSD was calculated. The peak areas have been shown in Table 8.
TABLE 8: INTER DAY PRECISION DATA OF FLUTICASONE PROPIONATE AND MUPIROCIN
|Day 1||Day 2||Day 3|
Different Wavelength: Robustness carried out by changing the wavelength, flow rate, and mobile phase ratio % RSD was calculated for Fluticasone Propionate and Mupirocin. The peak areas have been shown in Tables 9, 10, and 11.
TABLE 9: DIFFERENT WAVELENGTH DATA FOR FLUTICASONE PROPIONATE AND MUPIROCIN
|231 nm||232 nm||233 nm|
TABLE 10: DIFFERENT FLOW RATE DATA FOR FLUTICASONE PROPIONATE AND MUPIROCIN
|0.9 mL/min||1.0 mL/min||1.1 mL/min|
TABLE 11: DIFFERENT MOBILE PHASE RATIO DATA FOR FLUTICASONE PROPIONATE AND MUPIROCIN
TABLE 12: DATA OF LOD AND LOQ
|SD of the Y-Intercepts of
5 Calibration curve
|Mean slope of 5
LOD and LOQ: LOD and LOQ were calculated, and peak areas are shown in Table 12 as follows:
Accuracy: Accuracy of the method was confirmed by the recovery study. % recovery of both Fluticasone Propionate and Mupirocin was found between 98% to 102%. The results have been reported in Table 13.
TABLE 13: ACCURACY DATA FOR FLUTICASONE PROPIONATE AND MUPIROCIN
|Amt. of Sample taken (µg/mL)||Amt. of Standard spiking (µg/mL)||Total
recovery (mean ±SD)
DISCUSSION: RP-HPLC method was developed using 0.01% OPA: Acetonitrile (30:70) pH 5 as a mobile phase flow rate 1.0mL/min and detection wavelength was 232 nm and retention time was found to be for 6.774 min and 2.995 min for Fluticasone Propionate and Mupirocin respectively. The linearity of the developed method was found to be nearer to 1, in the range 2.5-7.5µg/mL and 100-300µg/mL for Fluticasone Propionate and Mupirocin respectively. % RSD was found to be < 2 for repeatability, precision, and robustness. %assay was found to be 100.04-101.08% and 99.04-100.1% for Fluticasone Propionate and Mupirocin, respectively. The % recovery was found to be 99.06-99.9% for fluticasone propionate and 98.74-99.95% for Mupirocin, respectively. Validation of the developed method was done as per ICH guidelines, and these results show the validation parameters within the range according to ICH guidelines.
CONCLUSION: The developed method was simple, precise, accurate, and reliable for the simultaneous estimation of Fluticasone Propionate and Mupirocin in combined dosage form as per ICH guidelines. The % RSD of all results is less than 2% that shows a high degree. Hence, the proposed method was simple, easy, cost-effective, and can be used for routine analysis of Fluticasone Propionate and Mupirocin in the combined dosage form.
ACKNOWLEDGEMENT: The authors express their sincere thanks to the Dean, Faculty of Pharmacy, Parul University, and Director of Sotac Pharmaceuticals Inc. for providing the facilities and the Director of Glenmark pharmaceuticals, Mumbai for providing Mupirocin (API) as free gift samples of pure drug.
CONFLICTS OF INTEREST: There are no conflicts of interest of any author regarding any of the work done.
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How to cite this article:
Khristi A and Prajapati N: Development and validation of RP-HPLC method for estimation of fluticasone propionate and mupirocin in a combined topical dosage form. Int J Pharm Sci & Res 2020; 11(9): 4411-19. doi: 10.13040/IJPSR.0975-8232.11(9).4411-19.
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