International Journal of Drug Development and Research

Keywords

Rabeprazole Sodium, Lafutidine, RP-UPLC, ICH guidelines.

INTRODUCTION

Lafutidine is chemically 2-(furan-2-ylmethylsulfinyl)- N-[4-[4-(piperidin-1-ylmethyl) pyridin-2-yl] oxybut-2- enyl] acetamide. Lafutidine is not official in any pharmacopoeias. Lafutidine is the new generation H2-receptor antagonist. It blocks the production of acid by inhibiting acid producing cells in the stomach and blocks histamine H2- receptors in the stomach and prevents histamine mediated gastric acid secretion. It is advised in hyperacidity, NSAID induced gastritis, gastric and duodenal ulcers and also used as preanesthetic medication. Apart from H2-receptor blockade activity, it has additional gastro protective action. Therefore it is used not only to inhibit acid secretion but also to provide gastric mucosal protection. [1, 2]

Rabeprazole Sodium is chemically 2-[[[4-(3- Methoxypropoxy)-3- Methyl-2-Pyridinyl]-Methyl] Sulfinyl]-1H-Benzimidazole Sodium salt. Rabeprazole sodium (RAB) is a Potent Proton Pump inhibitor that suppresses gastric acid secretion by specific inhibition of the gastric H+/K+-ATPase enzyme system at the secretory surface of the gastric parietal cell and is used in the treatment of Gastroesophageal reflux disease (GERD) and duodenal ulcers. It has a faster onset of action and lower potential for drug interaction compared to Omeprazole. [3, 4]

UPLC is a rising chromatographic separation technique whose packing materials have smaller particle size lesser than 2.5μm which improves the speed, resolution and sensitivity of analysis. When many scientists experienced separation barriers with conventional HPLC, UPLC extended and expanded the utility of chromatography. The main advantage is reduction of analysis time which also reduces solvent consumption. The analysis time, solvent consumption and analysis cost are very important factor in many analytical laboratories. The time consumption during optimization of new methods can also be greatly reduced. This results in many analysis in a day and quick results which is of very importance to the industries and research laboratories. [5, 6, 7]

Literature survey [8-15] revealed that a number of analytical methods have been reported for the estimation of Lafutidine (LAF) and Rabeprazole Sodium (RAB) in individual and combination with other drugs by spectrophotometry, HPLC, RPHPLC, HPTLC, but not even single method was reported for the simultaneous estimation of LAF and RAB in their combined dosage form by RPUPLC method.

MATERIALS AND METHODS

Instrument

UPLC Model: Mexera Shimadzu UPLC system Sample injector: S-5200 Pump: P-3000 Fixed Capacity Loop: 10 μl Detector: PDA detector Column: Phenomenex, C18 column, 150 mm. Other instruments: Double beam UV-visible spectrophotometer (SHIMADZU, Model 1800) Ultrasonicator: PEI, Ultra sonic bath pH meter: Chemiline, CL-180, Labline technology pvt ltd.

Electronic analytical balance: (AUX-220), Uni Bloc- SHIMADZU

Volumetric flask: 10, 25, 50, 100 ml (RASAYANBorosilicate glass)

Pipettes: 1, 2, 5, 10 ml

All instruments and glass wares were calibrated.

Reagents and chemicals

Pure drug samples of LAF and RAB were provided as a gift sample from Macleods & Ipca Laboratories Ltd, Mumbai, respectively. Acetonitrile and Water were of HPLC grade and procured from E. Merck, Darmstadt, Germany. Potassium di-hydrogen orthophosphate and analytical reagent grade supplied by Fischer Scientific Chemicals.

Marketed formulation

The commercial formulation LAFUMACPLUS (Macleods Pharmaceuticals Ltd., Mumbai) was purchased from Local pharmacy. Each Capsule contain 10mg Lafutidine and 20mg Rabeprazole Sodium.

Preparation and Selection of Mobile phase [4-10]

The preliminary isocratic studies on a reverse phase phenomenex C18 column with different mobile phase combination of Acetonitrile and Potassium di-hydrogen orthophosphate buffer were studied for simultaneous estimation of both drugs. The optimal composition of mobile phase determined to be Acetonitrile : 0.01M Potassium di-hydrogen orthophosphate pH 6.8 (60:40% v/v) and filtered through 0.22μ membrane filter.

Preparation of standard stock solution [11-12]

Accurately weighed quantity of LAF (100 mg) and RAB (100 mg) was transferred in to two separate 100 ml volumetric flasks, dissolved in diluents(mobile phase Acetonitrile:0.01M Potassium di-hydrogen orthophosphate pH 6.8 (50:50 v/v))and diluted to the mark with same solvent (concentration of stock solutions is 1000μg/ml of LAF and 1000μg/ml of RAB respectively). Appropriate volume of aliquots from standard lafutidine and rabeprazole sodium stock solutions were transferred to different volumetric flasks of 10 ml capacity. The volume was adjusted to the mark with mobile phase to give solution(s) containing 40, 60, 80,100,120μg/ml LAF and 80,120,160,200,240 μg/ml RAB.

Preparation of Sample solution [11-12]

Twenty Capsules were weighed and content crushed to obtain a fine powder. An accurately weighed powder equivalent to about 100 mg of lafutidine and 200 mg of rabeprazole sodium was transferred to 100 ml volumetric flask and the volume was made up to the mark using mobile phase. The solution was sonicated for 20 minutes. The solution was filtered through Whatman filter paper No.42. First few ml of filtrate were discarded. 8.0 ml of the solution from above filtrate was diluted to 100 ml with mobile phase to make the final concentration of working sample equivalent to 100% of target concentration.

Optimized Chromatographic Conditions

The mobile phase, Acetonitrile : 0.01M Potassium di-hydrogen orthophosphate pH 6.8 (60:40% v/v) pumped at a flow rate of 1.2 ml/min through the column Phenomanex, C18 column, 150 mm. The mobile phase was degassed prior to use under vacuum by filtration through a 0.22μ membrane filter. Both drugs showed good absorbance at 215 nm (isobestic point), which was selected as wavelength for further analysis.

Development and Validation of RP-UPLC Method

System Suitability

System suitability study of the method was carried out by six replicate analysis of solution containing 100% target concentration of Lafutidine and Rabeprazole Sodium. Various chromatographic parameters such as retention time, peak area, tailing factor, theoretical plates of the column and resolution between the peaks were determined and the method was evaluated by analyzing these parameters.

Specificity

Specificity test determines the effect of excipients on the assay result. To determine the specificity of the method, standard sample of Lafutidine and Rabeprazole Sodium were injected first. Then commercial product, blank and excipients solution were run in the instrument one after another.

Linearity

Linearity of the method was determined by constructing calibration curves. Standard solutions of Lafutidine and Rabeprazole Sodium of different concentrations level (50%, 75%, 100%, 125% and 150%) were used for this purpose. Each measurement was carried out in 6 replicates and the peak areas of the chromatograms were plotted against the concentrations to obtain the calibration curves and correlation coefficients.

Accuracy (Recovery Studies)

To check the degree of accuracy of the method, recovery studies were performed in triplicate by standard addition method at 50%, 100% and 150%. Known amounts of standard Lafutidine and Rabeprazole Sodium were added to preanalyzed samples and were subjected to the proposed UPLC method.

Precision

Precision of the method was determined by performing intraday variation, interday variation and method repeatability studies. Three replicates of three different concentrations were injected on the same day and the percent relative standard deviations (%RSD) were calculated to determine intra-day precision. These studies were also repeated on three consecutive days to determine inter-day precision. Repeatability study was performed by injecting the six replicates of the same concentration and the percent relative standard deviations (%RSD) were calculated.

Robustness

To evaluate the robustness of the developed RPUPLC method, small deliberate variations in the optimized method parameters were done. The effect of change in flow rate and temperature on the Area of Chromatograms were studied. The method was found to be unaffected by small changes ± 0.2 change in flow rate and temperature.

Analysis of marketed formulation

Twenty Capsules were weighed and content crushed to obtain a fine powder. An accurately weighed powder equivalent to about 100 mg of Lafutidine and 200 mg of Rabeprazole Sodium was transferred to 100 ml volumetric flask and the volume was made up to the mark using mobile phase. The solution was sonicated for 20 minutes. The solution was filtered through Whatman filter paper No.42. First few ml of filtrate were discarded. 8.0 ml of the solution from above filtrate was diluted to 100 ml with mobile phase. The prepared sample solution was chromatographed for 10 minutes run time using same mobile phase at 215 nm at a flow rate of 1.2 ml/min. From the peak area obtained in the chromatogram, the amounts of both the drugs were calculated by fitting peak area responses into the equation of the straight line representing the calibration curves for Lafutidine and Rabeprazole sodium.

RESULT AND DISCUSSION

The proposed method was validated as per ICH guideline Q2R1. Results obtained for various validation parameters are as follow:

CONCLUSION

The proposed UPLC method is found to be specific, accurate, precise and rapid for determination of LAF and RAB in combination. The proposed method was also applied for the estimation of these drugs in commercial dosage form and was successfully estimated. This method is reliable and can be useful for the rapid estimation in industries during inprocess quality control testing.

ACKNOWLEDGEMENT

Authors are very much thankful to Principal for providing the facilities. Authors are also thankful to Prof. S. S. Jalapure, Deputy director and Mr. PVSN. Malleswara rao of Dr. Prabhakar Kore Basic Science Research Center, Belagavi forproviding the facilities to carry out this project work.

Tables at a glance

Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9

Figures at a glance

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9

References

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