Determination of Mosapride and Pantoprazole in a Fixed-dose Combination by Uv Spectrophotometric Methods and Rp-hplc

An accurate and reproducible UV-spectrophotometric methods and liquid chromatographic assay method were developed and validated for the determination of mosapride and pantaprazole in capsule formulation. Two wavelengths were selected for each UV method, first simultaneous equation 274 nm, 288.2 nm and second Q value analysis method 274 nm, 302 nm was the isobestic point for both the drugs. The 30 mM ammonium sulphate buffer : acetonitrile (50:50, v/v) was used for reverse-phase liquid chromatography to determine the contents of mosapride and pantaprazole in combination-capsule dosage form. The UV and HPLC methods were validated by determining parameters such as specificity, linearity, LOD and LOQ, precision, accuracy, ruggedness and robustness. The methods were found to be specific against placebo interference. Linearity was evaluated over the concentration range of 5-50.0 μg/mL by UV and 0.5 to 5.0 μg/mL by HPLC method respectively, for mosapride and pantaprazole (the value of R 0.999 found were by both the methods for mosapride and pantaprazole). Both the intraday and interday precision values of the systems and methods were determined. The accuracy of the methods ranged from 99.99 to 102.24 % for mosapride and from 100.45 to 101.22 % for pantaprazole. The proposed methods were found to be robust when slight but deliberate changes were made in analytical conditions. The developed methods were found suitable for the simultenous estimation of mosapride and pantaprazole in capsule formulation as well in raw materials for quality control. INTRODUCTION The combined dosage form of any pharmaceuticals is for the synergistic effect or to give longer time effect. In present study Mosapride citrate and Pantoprazole combination is used as antacid to decrease acidity (excess secretion of acid in stomach). Mosapride citrate (MOSP), known as (±) 4-amino-5-chloro-2ethoxy-N-[[4-(4-fluorobenzyl)-2-morpholinyl] methyl] benzamide 2-hydroxy-1,2,3-propanetri carboxylate citrate, dihydrate is a novel gastroprokinetic agent plays an important role in conjunction with life-style modifications in short and long term management of gastroesophageal reflux disease and dyspepsia in many of the asian countries. Pantoprazole (PANT), 5(difluoromethoxy)2[(3, 4dimethoxy -2pyridyl)methylsulfinyl]1H-benzimidazole, is a selective and long-acting proton pump inhibitor (1) . The literature review reveals that only a few methods have been developed for the quantification of individual drug MOSP and PANT. Determination of related-substances of MOSP in bulk drugs and pharmaceuticals by HPLC (2-4) , identification of a polar impurity in MOSP by LC-/MS/MS and by LC-/MS in equine tissues (5,6) , structural identification and characterization of potential impurities of PANT (7) , determination of PANT by HPLC in human plasma (8) , HPLC separation of domperidone and pantoprazole (9) , enantiomeric determination of PANT in human plasma by HPLC (10) are the current analytical methods for analysis of MOSP and PANT. In proposed method carried out for the simultaneous determination of MOSP and PANT in capsule formulation by two UVspectrophotometric methods and RP-HPLC method. The validation of methods were carried out as per ICH guidelines (11-13) . The chemical structures of the drugs are as shown in Figure 1. Experimental: Instrumentation HPLC The chromatographic separation was performed on the Waters liquid chromatographic system equipped with (Waters-1515) isocratic solvent delivery system pump with (Waters-2487) dual wavelength absorbance UV-detector and Rheodyne 7725i injector with 50 μL loop volume. Breeze 3.3 data station was applied for data collecting and processing. A phenomenox C18 column (250mm x 4.6mm i. d., 5μm particle size) was used for the separation. International Journal of Pharmaceutical Studies and Research E-ISSN 2229-4619 IJPSR/Vol. II/ Issue II/April-June, 2011/29-36 Figure 1. Chemical Structures of Mosapride citrate, Hydrochlorothiazide (IS), Pantoprazole The mobile phase consisted of a mixture of acetonitrile-30 mM ammonium sulphate buffer (pH 5.5) in the ratio of (50:50, v/v) [without adjustment of pH by any other buffer, it avoids complications of pH adjustment is the advantage of this buffer] The mobile phase was prepared daily, filtered, sonicated before use and delivered at the flow rate of 1.0 mL/min. UV-Spectrophotometer A Shimadzu UV-Visible Spectrophotometer-160A with data processing system was used. The sample solution was recorded in 1cm quartz cells against solvent blank over the range 200-400 nm. The optimal conditions for recording the spectra to achieve good reproducibility included scan speed at 60nm/s, slit width at 2 nm. The detection wavelength fixed at 275 nm as isobestic point for both drugs as shown in Figure 2. Chemicals and reagents The pharmaceutical grade gift reference standard of Mosapride citrate (99.84 %) obtained from HAB Pharmaceutical & Research Ltd. Worli, Mumbai, India. Pantoprazole (99.18 %) and Hydrochlorothiazide (99.42 %) (Internal standard) were procured from M/S. Apex pharmaceuticals (Andrapradesh), India., MOSA PLUS Capsule formulations from market. Acetonitrile and methanol used were of HPLC grade; all analytical grade chemicals and solvents were obtained from E Merck (India) Ltd, Mumbai. Ammonium sulphate AR grade was procured from Qualigens fine chemicals, Mumbai. Water HPLC grade was obtained from a Milli-QRO water purification system. Fig. 2 Overlay UV-spectrum for Mosapride citrate and Pantoprazole International Journal of Pharmaceutical Studies and Research E-ISSN 2229-4619 IJPSR/Vol. II/ Issue II/April-June, 2011/29-36 Figure 3. Linearity curve of Mosapride and Pantaprazole by UV spectrophotometric method. Figure 4. Linearity curve of Mosapride and Pantoprazole by HPLC method. Standard solutions and calibration curves For HPLC Standard stock solutions were prepared at concentration of 1 mg/mL of MOSP and PANT separately using a mixture of water and acetonitrile (1:1, v/v) for HPLC method. The working standard solutions were prepared of different concentrations ranging from 0.5 to 5.0 μg/mL for MOSP and PANT respectively, by maintaining the concentration of hydrochlorothiazide (IS) at a constant level of 10μg/mL. From the above each mixture 50μL was injected in triplicate for the estimation of each standard drugs, under the optimized chromatographic conditions, a steady baseline was recorded; the typical chromatogram was recorded for internal standard, pantoprazole and mosapride standards as shown in Figure 5. The retention times of internal standard, pantoprazole and mosapride were found to be 3.58, 5.68 and 8.85 min, respectively. The calibration curve was obtained by simple linear regression of concentration of drug to the response factor. For UV-Spectrophotmetry Standard stock solutions were prepared at concentration of 1 mg/mL of MOSP and PANT separately using a mixture of water and methanol (1:1, v/v) for spectrophotometric measurements. From stock solutions, the working standard solutions were prepared of different concentrations ranging from 5.0 to 50.0 μg/mL for MOSP and PANT respectively and scanned in the wavelength range of 200-400 nm. Two wavelengths selected for simultaneous equation were 274, 288.2 nm respectively as shown in Figure 2. Є (A 1% , 1cm) is calculated for each standard drug by measuring absorbance of 1% solution at 1cm path length. Similarly, mixed International Journal of Pharmaceutical Studies and Research E-ISSN 2229-4619 IJPSR/Vol. II/ Issue II/April-June, 2011/29-36 standard solutions were used for UVspectrometric analysis by simultaneous equation and Q-analysis method. Analysis of formulation by HPLC Twenty capsules were weighed and a quantity of powder equivalent to one capsule was weighed and transferred to 50 ml of mixture of acetonitrile and water (1:1, v/v) dissolved and filtered. The combined extracts were made up to 100 mL by adding required amount of IS with same mixture. The required amount of solution was centrifuged and further dilutions were made with mobile phase to get a concentration of MOSP, PANT and hydrochlorothiazide (IS) 2 μg/mL, 4 μg/mL and 10 μg/mL respectively. From the above mixture 50μL was injected in triplicate for the estimation of drugs in sample, under the optimized chromatographic conditions, a steady baseline was recorded; the typical chromatogram for sample was recorded as shown in Figure 6. The retention times of sample hydrochlorothiazide (IS), pantaprazole and mosapride were found to be 3.56, 5.67 and 8.84 min respectively. The detection wavelength was fixed at 275 nm. Figure 5. Typical chromatogram of standard solution with IS: 1) Peak of Hydrochlorothiazide (IS) at 3.58 min 2) Peak of Pantoprazole at 5.68 min 3) Peak of Mosapride citrate at 8.85 min. Figure 6. Typical chromatogram of sample solution with IS: 1) Peak of Hydrochlorothiazide at 3.56 min 2) Peak of Pantoprazole at 5.67 min 3) Peak of Mosapride citrate at 8.84 min. International Journal of Pharmaceutical Studies and Research E-ISSN 2229-4619 IJPSR/Vol. II/ Issue II/April-June, 2011/29-36 Analysis of formulation by UV Twenty capsules were weighed and a quantity of powder equivalent to one capsule was weighed and transferred to 50 ml of mixture of methanol and water (1:1, v/v) dissolved and filtered. The combined extracts were made up to 100 mL with same mixture. Further dilutions were made to get sample solution containing 15 μg/mL, 20 μg/mL MOSP, PANT respectively and scanned in the wavelength range of 200-400 nm. Two wavelengths selected for simultaneous equation method were 274 nm, 288.2 nm and for Q value analysis 274 nm and 302 nm was the isobestic point for both the standard drugs. Method 1: Simultaneous Equation method For simultaneous equation method the absorbance of standard individual solutions were measured at two wavelengths 274 nm, 288.2 nm and from that calculated absorptivity of MOSP and PANT respectively. The sample solution was measured at two wavelengths 274 nm, 288.2 nm as A1 and A2 respectively and concentration of the individual two drugs in sample was calculated using simultaneous equation. The method employs solving of simultaneous equations using Cramer’s rule and matrices. The simultaneous equations were A1 = Є1m × C1 + Є1p× C2 (1) A2 = Є2m× C1 + Є2p × C2 (2) Є1m and Є2 m absorptivity values of MOSP 274 nm wavelength Є1pand Є2 p absorptivity values of PANT 288.2 nm wavelength C1 and C2 are concentrations of MOSP and PANT respectively in sample solution. Method 2: Q value analysis method The overlay spectrum of standard solutions observed for MOSP and PANT were as shown in Figure 2. The two wavelengths were selected for measure absorbance, first at 302 nm as an isoabsorptive point for both the drugs and second at 274 nm wavelength of MOSP. The dilutions of the standard and sample solutions were carried out as reported in simultaneous equation method. The absorptivity values for both drugs at the selected wavelength were calculated and employed for Q analysis, the concentration of drugs in sample solution were determined by using the following formula. For MOSP