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Research Article
Volume 1 Issue 2 - 2015
Analytical Study of Rabeprazole Sodium in Formulation by Using HPLC
Asha Ranjani V1*, Chandrasekhar B1, Bharath Kumar KS2, Padmanaba Reddy Y2 and Prabhakar T1
1Department of Pharmaceutical Analysis, MLR Institute of Pharmacy, Jawaharlal Nehru Technological University-Hyderabad, India
2Department of Pharmaceutical Analysis, Jawaharlal Nehru Technological University-Ananthapur, India
3Department of Pharmaceutical Analysis, University College of Pharmaceutical Sciences, Andhra University, India
*Corresponding Author: Asha Ranjani V, Department of Pharmaceutical Analysis, MLR Institute of Pharmacy, Dundigal, Quthbullapur, Hyderabad, India.
Received: January 07, 2015; Published: March 27, 2015
Citation: Asha Ranjani V., et al. “Analytical Study of Rabeprazole Sodium in Formulation by Using HPLC”. EC Pharmaceutical Science 1.2 (2015): 86-94.
Abstract
The objective of this present work is to develop a simple, sensitive, precise method for the determination of Rabeprazole sodium in raw material and tablet dosage form were validated using RP-HPLC. The optimum wave length for detection was 284 nm at which better detector response for drug was obtained. The system with buffer, methanol (30:70% v/v) with 0.9 ml/min flow rate is quite robust. To ascertain its effectiveness, system suitability tests were carried out on freshly prepared stock solutions. The correlation coefficient was found to be 0.998. The mean recoveries were found in the range of 98.0-102.0% The LOD and LOQ were found to be 2.96 and 10.1 respectively. The proposed method was validated in accordance with ICH parameter and the results of all methods were very close to label value of commercial pharmaceutical formulation.
Keywords: Method development; System suitability; Rabeprazole; RP-HPLC; Method validation
Introduction
Rabeprazole is an antiulcer drug in the class of proton pump inhibitors. It is a prodrug in the acid of the parietal cells; it turns in to active sulphenamide form. Rabeprazole inhibits the H+, K+ and ATPase of the coating gastric cells. Rabeprazole sodium chemically known as 2-[{4-(3-methoxy propoxy) -3-methoxy pyridine-2-yl} methyl sulfinyl]-H-bezimidazole. It has an empirical formula of C18H20N3O3S and a molecular weight of 359.4 Rabeprazole which is structurally related omeprazole, is a substituted benzmidazole, and acts as a proton pump inhibitor (PPI) that suppresses gastric acid secretion through an interaction with(H+/K+)-ATPase in gastric parietal cells. Like other PPIs (omeprazole, lansoprazole and pantoprazole), rabeprazole is effective in the treatment of various peptic diseases, including gastric and duodenal ulcer, gastroesophageal reflux disease and Zollinger-Ellison syndrome. Rabeprazole may also be used with antibiotics to get rid of bacteria that are associated with some ulcers. It inhibits the final transport of hydrogen ions in to the gastric lumen. Literature studies reported various methods for the determination of Rabeprazole in tablet dosage form. These studies revealed few methods and validated a dissolution test for Rabeprazole sodium in coated tablets using RP-LC method. Majority of these were stated I the determination of Rabeprazole and its metabolites using buffer solution and biological fluids for therapeutic monitoring of rabeprazole. An attempt was done to develop a new, accurate, precise method in accordance to ICH guidelines [1,2].
Chemical structure of Rabeprazole sodium
Experimental
Instrumentation
Analytical weighing balance
HPLC system: Waters 2695 High performance liquid chromatography equipped with auto sampler and dual absorbance detector with Empower soft ware.
Column: C18 (4.6* 150 MM, 3.5 µm, XTerra)
UV Spectrophotometer: Lab India 3000
Sonicator: (Sonica 2200 MH)
Vacuum filter pump (model X15522050 of Millipore)
pH meter (Metler, Toledo)
Millipore filtration kit
Reagents used for the study
Methanol: HPLC grade
Water: Milli-Q-grade
Potassium di hydrogen phosphate: AR grade
Sodium hydroxide: AR grade
Assay method development: The objective of this work is to optimize the assay method for the estimation of Rabeprazole based on the literature survey. Few trials are done to make the optimization. Following conditions are selected for method optimization [3].
Preparation of Buffer: Weigh accurately 1.56 g of potassium di hydrogen phosphate and dissolve it in 500 ml of Milli-Q-water. Adjust the pH to 5.5 with sodium hydroxide, filter through 0.45 µ membrane filter and de gas.
Mobile phase: pH 5 buffer: Methanol (30:70)
Chromatographic conditions:
Flow rate: 0.9 ml/min
Column: C18 (X Terra 4.6* 150 mm, 3.5 µm)
Detector wave length: 284 nm
Column temperature: Ambient
Injection volume: 20 µl
Run time: 5 minutes
Diluents: mobile phase
Preparation of Rabeprazole standard and sample solution
Standard Solution Preparation:
Accurately weigh and transfer 10 mg of Rabeprazole Working standard into a 10 ml volumetric flask add about 7 ml of Diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent (Stock solution). Further pipette 0.4 ml of the above stock solution into a 10 ml volumetric flask and dilute up to the mark with diluent. Mix well and filter through 0.45 µm filter.
Sample Solution Preparation
Weigh 5 Rabeprazole Tablets and calculate the average weight. Accurately weigh and transfer the sample equivalent to 10 mg of Rabeprazole into a 10 ml volumetric flask. Add about 7 ml of diluent and sonicate to dissolve it completely and make volume up to the mark with diluent. Mix well and filter through 0.45 µm filter. Further pipette 0.4 ml of the above stock solution into a 10 ml volumetric flask and dilute up to the mark with diluent. Mix well and filter through 0.45 µm filter. Inject 20 µl of the standard, sample into chromatographic system and measure the area for the Rabeprazole peak and calculate the % assay by using the formula. Values are mentioned in table1 and chromatograms represented in figure 2.
Calculation:

Where: AT = Peak Area of Rabeprazole obtained with test preparation
AS = Peak Area of Rabeprazole obtained with standard preparation
WS = Weight of working standard taken in mg
WT = Weight of sample taken in mg
DS = Dilution of Standard solution
DT = Dilution of sample solution
P = Percentage purity of working standard
Method Validation
The method was validated for the parameters like linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy ,precision, ruggedness, robustness, system suitability parameters were also calculated. To evaluate the linearity different concentrations of sample solutions were prepared from stock solution and correlation coefficient was calculated. The samples were injected (20 μl) and signals from the samples were recorded at 2.65 min which were compared with those of blank. LOD and LOQ values were calculated as signal-to-noise ratio of 3:1 and 10:1 respectively. To determine accuracy of the method, sample solution of rabeprazole sodium at three different concentration levels were prepared and analyzed [4-7].
Linearity
Preparation of stock solution: Accurately weigh and transfer 10 mg of rabeprazole API sample in to a 10 ml volumetric flask add about 7 ml of diluent and sonicate to dissolve it completely to make up the volume up to mark with solvent. From this 20, 30, 40, 50, 60 μg/ml concentrated solutions are prepared. Linearity plot was represented in figure 2.
Figure 1: Linearity plot for Rabeprazole.
Accuracy
Preparation of stock solution: Accurately weigh and transfer 10 mg of Rabeprazole working standard in to a 10 ml volumetric flask add about 7 ml of diluent and sonicate to dissolve it completely and make up to volume with the same solvent. Preparation of 40ug/ml solution: Further pipette 0.4 ml of the above stock solution in to a 10 ml volumetric flask and dilute up to the mark with the diluent. Mix well and filter through 0.45um filter.
Figure 2: Chromatogram of standard.
Preparation of sample solutions: For preparation of 50% solution: Accurately weigh and transfer 5.0 mg of Rabeprazole API sample in to a 10 ml volumetric flask add about 7 ml of diluent and sonicate to dissolve it completely and make up the volume with the same solvent. Further pipette 0.4 ml of the above stock solution in to a 10 ml volumetric flask and dilute up to the mark with diluent. Mix well and filter through 0.45 um filter.
For preparation of 100% solution: Accurately weigh and transfer 10 mg of Rabeprazole API sample in to a 10 ml volumetric flask add about 7 ml of diluent and sonicate to dissolve it completely and make up to volume with the solvent. Further pipette 0.4 ml of the above stock solution in to a 10 ml volumetric flask and dilute up to the mark with diluent. Mix well and filter through 0.45 μm filter.
Figure 3: Chromatogram of sample.
For preparation of 150% solution: Accurately weigh and transfer 15 mg of Rabeprazole API sample in to a 10 ml volumetric flask add about 7 ml of diluent and sonicate to dissolve it completely and make up to volume with the solvent. Further pipette 0.4 ml of the above stock solution in to a 10 ml volumetric flask and dilute up to the mark with diluent. Mix well and filter through 0.45 μm filter [8-11].
Precision
Preparation of stock solution: Accurately weigh and transfer 10 mg of Rabeprazole working standard into a 10 ml volumetric flask add about 7 ml of diluent and sonicate to dissolve it completely and make up to volume with same solvent.
Preparation of 40 μg/ml solution: Further pipette 0.4 ml of the above stock solution in to a 10 ml volumetric flask and dilute up to the mark with the diluent. Mix well and filter through 0.45 um filter.
Procedure: The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits.
Intermediate Precision/Ruggedness: To evaluate the intermediate precision of the method, precision was performed on different day by using different make column of same dimensions. Preparation of stock solution: Accurately weigh and transfer 10 mg of Rabeprazole working standard in to a 10 ml volumetric flask and dilute up to the mark with the diluent. Mix well and filter through 0.45 μm filter.
Procedure: The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits [12-15].
Limit of Detection
Preparation of 40 μg/ml solutions: Accurately weigh and transfer 10 mg Rabeprazole working standard in to a 10 ml volumetric flasks add about 7 ml of diluent and sonicate to dissolve it completely and make up to the mark with solvent. Further pipette 0.4 ml of the above stock solution in to a 10 ml volumetric flask and dilute up to the mark with the diluent. Mix well and filter through 0.45 μm filter.
Figure 4: Chromatogram of LOD.
Preparation of 0.7% solution at specification level (0.28 μg/ml solution): Pipette 1 ml of 10 μg/ml solutions in to a 10 ml of volumetric flask and dilute up to the mark with the diluent.
Limit of Quantification
Preparation of 40 μg/ml solutions: Accurately weigh and transfer 10 mg Rabeprazole working standard in to a 10 ml volumetric flasks add about 7 ml of diluent and sonicate to dissolve it completely and make up to the mark with solvent. Further pipette 0.4 ml of the above stock solution in to a 10 ml volumetric flask and dilute up to the mark with the diluent. Mix well and filter through 0.45 μm filter.
Figure 5: Chromatogram of LOQ.
Preparation of 0.23% solution at specification level (0.092 μg/ml solution): Pipette 1 ml of 10 μg/ml solution in to a 10 ml of volumetric flask and dilute up to the mark with diluent.
Results and Discussions
Assay
Results are within the acceptance limits of 95-105%
Results were represented in following table 1
S No Rabeprazole
1 Sample Area 1884270
2 Standard Area 1807375
3 Standard Weight 10 mg
4 Sample Weight 10.1 mg
5 LC 20 mg
6 Average Weight 0.0204 mg
7 Standard Purity 99.7
8 Assay % 99.5
Linearity
Acceptance criteria: Correlation coefficient should not be less than 0.99
Results were represented in following table 2
S No Linearity Level Concentration Area
1 I 20 µg/ml 939926
2 II 30 µg/ml 1390971
3 III 40 µg/ml 1860230
4 IV 50 µg/ml 2285771
5 V 60 µg/ml 2779976
Correlation Coefficient 1.000
Accuracy
Results are within the acceptance limits 0f 98-102.0%
% Concentration (At Specification Level) Area Amount Added (mg) Amount Found (mg) % Recovery Mean Recovery
50% 951730 5.0 5.03 100.7% 99.8%
100% 1881869 10.0 9.95 99.5%
150% 2815614 15.0 14.8 99.3%
Precision
Results are within the acceptance limits and area of five standard injections results should not be more than 2%
S No Injection number (80 mcg/ml) Retention Time of Rabeprazole Area of Rabeprazole
1 Injection-1 2.669 1724358
2 Injection-2 2.668 1777933
3 Injection-3 2.655 1767353
4 Injection-4 2.666 1729271
5 Injection-5 2.662 1782024
  Average - 1749729
  Standard Deviation - 27398.33
  %RSD - 1.5
Robustness
Results are within the acceptance limits and area of five standard injections results should not be more than 2%
Proposed Variations USP Plate Count USP Tailing

Variation in mobile phase composition

10% less 2854.3 1.4
*Actual 2253.6 1.5
10% more 2111.0 1.4

Variation in flow rate

0.6 ml/min 2168.0 1.4
0.8 ml/min 2253.6 1.5
1.0 ml/min 2074.3 1.4
Ruggedness
The results are within the acceptance limit, the proposed method is found to be rugged.
  Retention Time of Rabeprazole Area of Rabeprazole
Standard (80 mcg) 2.669 1890004
Analyst (1) (80 mcg) 2.672 1885751
Analyst (2) (80 mcg) 2.672 1892673
Analyst (3) (80 mcg) 2.667 1861622
Analyst (4) (80 mcg) 2.668 1871563
Average - 1880323
Standard Deviation - 13249.5
%RSD - 0.70
Limit of Detection
Calculation of S/N Ratio:
Average Baseline Noise obtained from Blank: 51 µV
Signal Obtained from LOD solution (0.7% of target assay concentration): 151 µV
S/N = 151/51= 2.96
Acceptance Criteria:  S/N Ratio value shall be 3 for LOD solution.
Conclusion: The LOD for Rabeprazole was found to be 2.96.
Limit of Quantification:
Calculation of S/N Ratio:
Average Baseline Noise obtained from Blank: 51 µV
Signal Obtained from LOD solution (0.23% of target assay concentration): 516 µV
S/N = 516/51 = 10.1
Acceptance Criteria: S/N Ratio value shall be 10 for LOQ solution.
Conclusion: The LOQ for Rabeprazole was found to be 10.1
Conclusion
In the present work, an attempt was made to provide a newer, sensitive, simple, accurate and low cost RP-HPLC method. It is successfully applied for the determination of Rabeprazole in pharmaceutical preparation without the interferences of other constituents in the formulations.
Bibliography
  1. Beckett AH and Stenlake JB. “Practical Pharmaceutical Chemistry”. part B. 4th edn., CBS publishers and Distributors New Delhi (2002): 272-280.
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Copyright: © 2015 Asha Ranjani V., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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