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1 Prajapathi et al: Simultaneous Estimation of IIaprazole and Domperi Internatio idone in Pharm maceutical Dosage Form 3549 onal Journal of Pharmaceutical Sciences and Nanotechnology Volume 9 Issue 6 November December 2016 Research Paper MS ID: IJPSN PRAJAPATHI Simultaneous Estimation of Ilaprazole Pharmaceutical Dosage Form and Domperidone in Kiral M. Prajapati 1, Gopi Patel *, Bhavesh Prajapati and Dr. Samir K. Shah 2 1 Department of Quality Assurance, Department of Pharmacology, Sardar Patel College of Pharmacy, Bakrol, Anand, Gujarat, India. Received October 6, 2016; accepted November 3, 2016 ABSTRACT A simple, accurate and precise stability-indicating RPvalidated HPLC method was developed and subsequently The retention times of ilaprazole and domperidone were found to be 3.0 min, 5.4 min respectively. The mean for simultaneous determination of ilaprazole and recoveries obtained for ilaprazole and domperidone were domperidone in bulk and pharmaceutical dosage form. The found to be ± % and ± % proposed HPLC method utilizes (250 mm 4.6 mm 5 respectively. Stress testing which covered acid, alkali C18 μm) column with mobile phase comprising of 0.5 % glacial hydrolysis, and peroxide, photolytic, thermal degradation acetic acid in water ph 5.5 adjusted with ammonia was performed to prove the specificity of the proposed solution: methanol in the ratio 45:55 v/v at a flow rate of 1.0 ml/min. Quantitation was achieved with UV detection at 286 nm based on peak area with linear calibration curves at concentration ranges μg/ml for ilaprazole and μg/ml for domperidone with correlation coefficient of method and degradation was achieved. The proposed method was successfully applied for the stability indicating simultaneous estimation of ilaprazole and domperidonee in routine quality control analysis in bulk and pharmaceutical formulation. KEYWORDS: Ilaprazole, Domperidone, RP-HPLC, Forced Degradation, Method Validation. Introductionn Ilaprazole (ILA) is a proton pump inhibitor used in the treatment of dyspepsia, peptic ulcer disease, gastro esophageal reflux disease and duodenal ulcer (Rang et al., 2007). It is chemically known as [-[4-methoxy-3- methyl-pyridin-2-yl) methylsulfinyl] ]-6-pyrrol-1-yl-1H-ben- is a D2 zoimidazole (Figure 1). Domperidone (DOM) receptor antagonist. It increases gastrointestinal peri- and it stalsis and motility that prevent reflux esophagitis is used to prevent nausea and vomiting (Brunton and Parker 2005). Chemically Domperidone is 5-chloro-1-[1- [3-(2-oxo-2,3-dihydro-1H-benzimidazol-l-yl)propyl]-piperi- din-4-yl]-1,3-dihydro-2h benzimidazole-2-one (Figure 2). these drugs. Therefore, the present study was aimed to develop a simple, economical, accurate and precise stability indicating RP-HPLC method for simultaneous determination of Ilaprazole and Domperidone in bulk and its pharmaceutical dosage forms. Fig. 1. Structure of ilaprazole. Based on the literature survey, few analytical methods such as UV (Birajdar et al., 2010) ), HPLC (Kalirajan et al., 2008) and HPTLC (Patel et al., 2008) were reported for the estimation of ILA and DOM either individually or combined with other drugs. However, there was no stability indicating method reported for Fig. 2. Structure of domperidone. Materials and Methods Ilaprazole and domperidone were procured as a gift samples from Zydus Cadila Ltd, Ahmadabad, Gujarat, India. All chemicals and reagents used were of analytical or pharmaceutical grade. HPLC grade methanol and water (Merck), AR grade glacial acetic acid (Spectrochem) were used for the analysis. The Pharmaceutical 3549
2 3550 Int J Pharm Sci Nanotech Vol 9; Issue 6 November December 2016 formulation used in this study was Lupida D capsule procured from the local market and labeled to contain 10mg ILA and 30mg DOM per capsule. Diluent: Methanol: Water (50:50) v/v Instruments: RP- HPLC System (Analytical Technology, Alchrome A 2000 Software, C18 Column), ph meter (Systronic model no. 335), Weighing balance (Swisser), Sonicator Chromatographic Condition Preliminary studies were conducted and trails were made for the method development. The optimized mobile phase consisting of 0.5 % glacial acetic acid in water ph 5.5 adjusted with ammonia solution: Methanol in the ratio of 45:55 v/v. Flow rates were maintained at 1.0 ml/min. The detection response was measured at 286 nm at ambient temperature. Preparation of Standard Solution Accurately weighed about 100 mg of ilaprazole and 300 mg of domperidone in 100 ml of volumetric flask. Dilute it up to 100 ml using diluent to get concentration 1000 µg/ml solution of Ilaprazole and 3000 µg/ml of domperidone. Take 10 ml sample solution in 100 ml of volumetric flask diluted with diluent to get concentration 100 µg/ml of Ilaprazole and 300 µg/ml of domperidone. Preparation of Sample Solution 20 capsules were weighed and calculated average weight. Take 50 mg of ILA and 150 mg DOM in to 50 ml volumetric flask, added about 35ml of diluent into it, sonicated for 30 minutes, cooled to attain room temperature and made up to volume with diluent and mixed well. It was filtered through 0.45µ syringe filter. Further diluted 5ml of filtrate to 50ml with diluent and injected. To obtained final concentration 100µg/ml for ILA and 300 µg/ml for DOM. Method Validation Specificity: Specificity is ability to measure specifically the analyte of interest without any interference from excipient and mobile phase component. For the determination of specificity 100 μg/ml solution of the standard ILA and 300 μg/ml solution of the standard DOM was injected. Marketed formulation of same concentration was also injected. Both chromatograms were compared (Figure 3 and 4). Fig. 4. Specificity of sample. Linearity: Linearity was evaluated by analysis of working standard solutions of Ilaprazole and Domperidone of five different concentrations. Linearity was evaluated by analysis of working standards of ilaprazole and domperidone of five different concentrations. The ranges of linearity were from 80µg/ml to 120 µg/ml for ilaprazole and 240 µg/ml to 360 µg/ml for domperidone. The peak area and concentration of each drug was subjected to regression analysis to calculate the calibration equations and correlation coefficients (Figure 5 and 6). Fig. 5. Linearity of ILA. Fig. 6. Linearity of DOM. Fig. 3. Specificity of standard.
3 Prajapathi et al: Simultaneous Estimation of IIaprazole and Domperidone in Pharmaceutical Dosage Form 3551 Accuracy: The Standard was spiked with formulation at these concentration levels of 50%, 100%, 150% and the mixture were analyzed by the proposed method. The experiment was conducted in triplicate (Table 1). TABLE 1 Accuracy Data. Sample Level Mean % Recovery ± SD % Ilaprazole 50% ± % ± % ± Domperidone 50% ± % ± % ± Precision: Pure samples of ILA and DOM were analyzed over different days to obtain inter-day (intermediate precision, n = 3) and within the same day to obtain intra-day precision (repeatability, n = 3), then the % values were calculated (Table 2). TABLE 2 Precision Data. Intraday Conc µg/ml ILA Mean area ± SD % Conc µg/ml DOM Mean area ± SD % ± ± ± ± ± ± Interday ± ± ± ± ± ± Robustness: Method robustness was evaluated by changing the flow rate, wavelength and mobile phase composition to evaluate the impact on the performance of the method and the results will be expressed in terms of % (Table 3). TABLE 3 Robustness Data. Flow rate Mobile Phase ph Mobile Phase Ratio Parameter Variation ILA DOM % % Mean area ± SD Mean area ± SD ± ± ml/ min 1.1 ml/ ± ± min ± ± ± ± : ± ± : ± ± System suitability: System suitability is the checking of a system to ensure system performance before or during the analysis of unknown. Parameters such as Theoretical Plates, Tailing factors, Resolution (%, retention time and area for six repetitions) were determined and compared against the specifications set for the method (Table 4). TABLE 4 System Suitability Parameters. Parameters Ilaprazole (100 μg/ml) Domperidone (300 μg/ml) Retention Time (min) ± SD ± ± Tailing Factor (Tf) ± SD 1.1 ± ± Number of Theoretical Plates (N) ± SD ± ± Resolution (Rs) 7.8 Solution stability: Standard and sample solutions were prepared as per the proposed method and injected in to the chromatographic system at initial and 48 hrs. by storing solutions at room temperature. Calculated the % difference in at regular intervals (Table 5). TABLE 5 Solution Stability. For Standard ILAPRAZOLE DOMPERIDONE Time Mean Area % Difference Mean Area % Difference 0 hrs NA NA 24 hrs hrs For Sample ILAPRAZOLE DOMPERIDONE Time Mean Area %Difference Mean Area % Difference 0 hrs NA NA 24 hrs hrs Forced Degradation Acid degradation: Take 5 ml filtrate of above stock solution in to 50ml of volumetric flask, added 5ml of 1N HCl to it and it was kept for 3 hours at room temperature. Then added 5ml of 1N NaOH to neutralize it and volume was made up to mark with diluents and mixed well and injected (Chromatogram for blank, DOM, ILA and marketed formulation in Figure 7, 8, 9 and 10 respectively). Alkali degradation: Take 5 ml filtrate of above stock solution into 50 ml of volumetric flask, added 5 ml of 1 N NaOH to it and it was kept for 5 hours at room temperature. Then added 5 ml of 1 N HCl to neutralizeit and volume was made up to mark with diluents and mixed well and injected (Chromatogram for blank, DOM, ILA and marketed formulation in Figure 11, 12, 13 and 14 respectively). Fig. 7. Force degradation study: Blank chromatogram of acid
4 3552 Int J Pharm Sci Nanotech Vol 9; Issue 6 November December 2016 Fig. 8. Standard chromatogram of ILA for acid Fig.12. Chromatogram of ILA after basic Fig. 9. Chromatogram of DOM after acid Fig.13. Chromatogram of DOM after basic Fig.10. Chromatogram of Marketed (ILA and DOM) after acid Fig. 14. Chromatogram of marketed (ILA and DOM) after base Fig. 11. Chromatogram of blank after base Fig. 15. Chromatogram of blank after peroxide
5 Prajapathi et al: Simultaneous Estimation of IIaprazole and Domperidone in Pharmaceutical Dosage Form 3553 Peroxide degradation: Take 5 ml filtrates of above stock solution in to 50ml of volumetric flask; added 5ml of 3% H2O2 to it and it was kept for 3 hours at room temperature. Then volume was made up to mark with diluents and mixed well and injected (Chromatogram for blank, DOM, ILA and marketed formulation in Figure 15, 16, 17 and 18 respectively). Thermal degradation: Take 5 ml filtrate of above stock solution in to 50 ml of volumetric flask; it was kept for 3 hours at 80 C temperature. Then volume was made up to mark with diluents and mixed well and injected (Chromatogram for blank, DOM, ILA and marketed formulation in Figure 19, 20, 21 and 22 respectively). Fig. 19. Chromatogram of blank after thermal Fig. 16. Chromatogram of ILA after peroxide Fig. 20. Chromatogram of ILA after thermal Fig.17. Chromatogram of DOM after peroxide Fig.18. Chromatogram of marketed (ILA and DOM) after peroxide. Fig. 21. Standard chromatogram of DOM after thermal Sun light degradation: Take 5 ml filtrate of above stock solution in to 50 ml of volumetric flask; it was kept for 12 hours in sunlight. Then volume was made up to mark with diluents and mixed well and injected (Chromatogram for blank, DOM, ILA and marketed formulation in Figure 23, 24, 25 and 26 respectively).
6 3554 Int J Pharm Sci Nanotech Vol 9; Issue 6 November December 2016 Fig. 22. Chromatogram of marketed ( ILA and DOM) after thermal Fig. 26. Chromatogram of marketed (ILA and DOM) after sun light Results and Discussion Fig. 23. Chromatogram of blank after sun light Fig. 24. Chromatogram of ILA after sun light In RP-HPLC, chromatographic separation was achieved on reversed-phase ODS-BPH hyper chrome C18 column ( mm, 5 μm) in isocratic mode using 0.5% Glacial Acetic Acid in Water ph 5.5 adjusted with Ammonia solution: Methanol (45: 55v/v) as the mobile phase at a flow rate 1.0 ml/min and spectrophotometric UV detection at 286 nm. The method was validated for specificity, linearity, precision, accuracy and robustness. Linearity of ILA and DOM were in the range of μg/ml and μg/ml, respectively. The percentage recoveries obtained for both drugs were 99.00% % (ILA) and % (DOM), respectively. Percent was found to be less than 2.0 for all parameters for both drugs. LOD and LOQ for ILA in this method were found 4.62μg/ml and 14.01μg/ml respectively, and LOD and LOQ for DOM, were found 6.58μg/ml and 19.69μg/ml. (Table 6). Assay of marketed formulations of ILA and DOM were also done by this HPLC and they were found respectively % and % and other system suitability parameters were found in given limit. Ilaprazole and domperidone solutions were subjected to forced degradation by acid, alkali, oxidation, thermal and sunlight. The study was done on both standard and sample drug formulation. Similarities in results were seen in both studies. The study revealed that Ilaprazole was more degraded than domperidone. From study, Ilaprazole was degraded up to 9-17 % and domperidone was 12 14%. TABLE 6 Validation Parameters. Parameters Ilaprazole Domperidone Linearity range μg/ml μg/ml Accuracy ± % ± % Method precision (% ) Inter-day Intra day LOD LOQ Fig. 25. Chromatogram of DOM after sun light
7 Prajapathi et al: Simultaneous Estimation of IIaprazole and Domperidone in Pharmaceutical Dosage Form 3555 Conclusions From, this study it is concluded that the proposed stability-indicating RP-HPLC method was found to be simple, accurate, precise and useful for routine analysis of ilaprazole and domperidone in bulk and pharmaceutical dosage form. The obtained results were satisfactory as per ICH guidelines. Acknowledgements The author is grateful to Sardar Patel College of Pharmacy, Bakrol for providing research facilities. References Birajdar AS, Subramania N, Meyyanathan, and Bojraj S (2010). Application of UV-Spectrophotometry of Rabeprazole and Domperidone on Pharmaceutical Dosage Form. Plegia Research Library 1: Brunton LL, and Parker KL (2005). Goodman and Gilman s. In The Pharmacological Basis of Therapeutics; 11th Edn; McGraw-Hill Medical Publishing Division, New York, pp 986. Kalirajan R, Anandaraja K, and Mathew SM (2008). Simultaneous Determination of Rabeprazole and Domperidone in Dosage forms by RP HPLC. Rasayan Journal of Chemistry 1: Patel BH, Suhagia BN, Patel MM, and Patel JR (2008). HPTLC Determination of Rabeprazole and Domperidone in Capsule and its Validation. Journal of Chromatographic Science 46: Rang HP, Dale MM, Ritte JM, and Flower RJ (2007). In Pharmacology. 6th ed. Elsevier Science LTD, Churchill Livingstone, pp 393. Address correspondence to: Gopi Patel, Sardar Patel College of Pharmacy Bakrol, Anand, Gujarat, India. Ph: ; gopiptl@gmail.com
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