RP-HPLC METHOD FOR THE ESTIMATION OF PANTOPRAZOLE SODIUM
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2 Int. J. Pharm. Med. & Bio. Sc Kareti Srinivasa Rao et al., 2012 Research Paper ISSN Vol. 1, No. 1, July IJPMBS. All Rights Reserved RP-HPLC METHOD FOR THE ESTIMATION OF PANTOPRAZOLE SODIUM Kareti Srinivasa Rao 1*, Nargesh Kumar Keshar 1, Prasenjit Roy Choudhury 1 and Ajay Kumar Pattnaik 2 *Corresponding Author: Kareti Srinivasa Rao, ksrao108@gmail.com Aim: To develop and subsequently validate a simple reversed-phase HPLC method for estimation of Pantoprazole (PNZ) present in pharmaceutical dosage forms. Methods and Material: The proposed RP-HPLC method utilizes a Hibar 250-4, 6 LiChrospher 100 RP-18 end capped (5µ) column in an isocratic separation mode with mobile phase consisting of methanol: water of 80:20%v/v, at a flow rate 1ml/min and the effluent was monitored at 288nm. Results: The retention times of PNZ were and minute for pure drug and formulation respectively. The described method was linear over a range of µg/ml. The percentage recovery was 99.24±0.17. F-test and t-test at 95% confidence level was used to check the intermediate precision data obtained under different experimental setups; the calculated value was found to be less than critical value. Conclusion: The LC method described here is a very simple, sensitive, and accurate procedure for estimation of PNZ. The developed and validated LC method is specific, accurate, robust and precise analysis of PNZ in pure and its formulations. Keywords: Pantaprazole, HPLC, Validation, Assay INTRODUCTION Pantoprazole sodium (PNZ) is chemically Sodium 5-(difluoromethoxy)-2-[[(3,4-dimethoxy-2- pyridinyl) methyl] sulfinyl]-1h-benzimidazole sesquihydrate. It is a substituted benzimidazole proton pump inhibitors and used as anti ulcerative. It is official in The Merck Index (2001) and Martindale, The Extra Pharmacopoeia (Reynolds and Martindale, 1982). PNZ is very soluble in water and methanol. The proton-pump inhibitor PNZ inhibit gastric acid by blocking the H+/K+-adenosine triphosphatase enzyme system (the proton pump) of the gastric parietal cell. It is used for short-term treatment of erosion and ulceration of the esophagus. The PNZ oral dosage forms are supplied in enteric-coated tablets. Different analytical methods are reported in the literature for the assay of PNZ in dosage forms and in biological fluids including spectrophotometry (Ritter et al., 1999; Ewin, 1 Roland Institute of Pharmaceutical Sciences, Berhampur, Orissa , India. 2 Khallikote (Autonomous) College, Berhampur , Orissa. 48
3 2001; Ozaltin and Kocer, 1997; Sastry et al., 1997; Meyyanathan et al., 1997; and Moustafa, 2000), TLC (Wahbi et al., 2002), HPTLC (Salama et al., 2003; EI Sherif et al., 2005; and Renger, 1993). The proposed RP-HPLC method was validated by assessing its specificity, linearity, accuracy, precision, system suitability parameters, ruggedness and robustness. MATERIALS AND METHODS Chemicals and Reagents Gift sample of PNZ was received from Sun pharma limited (India). HPLC grade methanol was purchased from RANKEM, India. High pure distilled water was prepared by using Millipore Milli Q plus purification system. Commercial formulation Pantium (tablet) containing 40mg of PNZ were purchased from the local market. HPLC Instrumentation and Conditions Quantitative HPLC was performed on Shimadzu HPLC with LC 10AT VP series pumps besides SPD 10AVP UV-Visible detector. Shimadzu Class-VP version 6.12 SP1 software was used along with Hibar250-4, 6 LiChrospher 100 RP-18 endcapped(5µ) column for the separation, maintained at ambient temperature. Different mobile phases methanol: water, was used in different compositions of mobile phases (30:70, 40:60, 50:50, 70:30, 80:20) at different flow rates (0.5,0.75,1.0, 1.2, 1.5, ml/min). The mobile phase methanol: water of 80:20%v/v at a flow rate of 1.0ml/ min gave peak with good symmetric shape for PNZ was eluted at retention time around 3.558min. Optimized chromatographic conditions were shown in Table 1. Preparation of Mobile Phase Methanol and Water was properly mixed in the ratio of 80:20. Standard and Sample Preparation Stock solution of PNZ Sodium (1 mg/ml) was prepared by dissolving 25 mg of PNZ in 25 ml of volumetric flask containing 10 ml of methanol and 10ml of water. The solution was sonicated for about 10 min and then made up to volume with mobile phase. Working standard solutions of PNZ were prepared by taking suitable aliquots of drug solution from the standard stock solution 1000µg/ ml and the volume was made up to 10 ml with mobile phase. Table 1: Optimized Chromatographic Conditions Parameters Stationary phase (column) Mobile Phase Flow rate (ml/min) Conditions Hibar 250-4, 6 LiChrospher 100 RP-18 endcapped(5µm) Methanol: water (80:20%v/v) 1.0 ml Run time (minutes) 7 Column temperature ( C) Ambient Volume of injection loop (ml) 20 Detection wavelength (nm) 288 Drug RT (min)
4 From the Tablet equivalent to 25 mg of PNZ was taken and extracted with 10ml methanol and 10 ml water in a 25ml volumetric flask using ultra sonicator for 15 minutes. The solution obtained was diluted with the mobile phase so as to obtain a concentration in the range of linearity previously determined. This solution was filtered through 0.45µm filter paper. All determinations were carried out in triplicate. Procedure for Calibration Curve The contents of the mobile phase were filtered before use through 0.45 µm filter paper, and pumped from the respective solvent reservoirs to the column at a specified flow rate. Prior to injection of the drug solutions, the column was equilibrated for at least 30 min with the mobile phase flowing through the systems. Then, 20ml of each of standard and sample solutions were injected into the HPLC system for to get the chromatograms. The retention time, peak areas of drug were recorded. Taking conc. plotted a graph on X-axis and areas under curve of drug on Y-axis. The linearity range was found to be in between mg/ml for PNZ. RESULTS AND DISCUSSION Optimization of the Method The proposed RP-HPLC method utilizes a Hibar 250-4, 6 LiChrospher 100 RP-18 end capped (5 µ) column in an isocratic separation mode with mobile phase consisting of methanol: water of 80:20%v/v, at a flow rate 1ml/min and the effluent was monitored at 288nm ( max for PNZ). The retention time for PNZ was The linearity range was found to be in between mg/ml for PNZ. Method Validation The described method has been validated for linearity, precision, accuracy, specificity, system suitability parameters, ruggedness and robustness. Linearity Least square regression analysis was carried out for the slope, intercept and correlation coefficient (Table 2). The linear fit of the system was illustrated graphically. The linearity range was found to be µg/ml. Regression equation for PNZ was, y = x (R 2 = ). Linearity Experiment (n=5) PNZ Range (µg/ml) Mean R value Slope Intercept Accuracy Table 2: Regression Characteristics of the Proposed HPLC Method This experiment was performed at three levels, in which sample stock solutions were spiked with standard drug solution containing 80, 100 and 120% of labeled amount of the drugs (40mg PNZ) in tablet. Three replicate samples of each concentration level were prepared and the % recovery at each level (n = 3), and mean % recovery (n=9) were determined (Table 3). The mean recovery was ±1.350 %. Precision Method Precision The precision of each method was ascertained separately from the peak areas obtained by actual determination of six replicates of a fixed amount of drug. The percent relative standard deviations were calculated for Pantoprazole Sodium and presented in the Table 4. 50
5 Table 3: Results of Accuracy Experiment Using Proposed Method PNZ Taken(µg) Recovered (µg) % Recovery LEVEL 1(80) LEVEL 2(100) LEVEL 3(120) Mean % recovery (n=9) ±1.350 %. %RSD Table 4: Results of Precision study Method Precision (PNZ) System Precision (PNZ) S. No. Concentrations Peak Statistical Concentrations Peak Statistical (10mg/ml) Area Analysis (10mg/ml) Area Analysis 1 Sample Injection Sample Mean= Injection Mean= Sample SD= % Injection SD= % 4 Sample RSD=1.61 Injection RSD= Sample Injection Sample Injection System Precision: The precision of each system was ascertained separately from the peak areas obtained by actual determination of six injections of the same sample solution. The percent relative standard deviations were calculated for Pantoprazole Sodium and presented in the Table 4. Specificity Specificity is the ability to measure accurately and specifically the analyte of interest in the presence of other components that may be expected to present in the sample matrix. It was found that the proposed method was specific as there is no interference of other active ingredients and excipients ensuring that the peak response is due only to a single component. System Suitability Parameters System suitability parameters can be defined as tests to ensure that the method can generate results of acceptable accuracy and precision. The system suitability parameters like Theoretical plates (N), Resolution (R), Tailing factor (T) were calculated and compared with the standard values to ascertain whether the proposed RP-HPLC method for the estimation of PNZ in pharmaceutical formulations were validated or not. System suitability is usually developed after method development and validation has been completed. The obtained value of Theoretical plates (N) in this method was and the tailing factor was found to be
6 Ruggedness Ruggedness is the degree of reproducibility of the results obtained under a variety of conditions. It is checked that the results were reproducible under differences in reagents, analysts and experimental periods. And hence the proposed method was found to be rugged. Robustness The percentage recovery of PNZ was good under most conditions and didn t show any significant change when the critical parameters were modified. The tailing factor was always less than 2.0 and the components were well separated under all the changes carried out. Thus the method conditions were robust. Assay The validated method was applied to the determination of PNZ in commercially available Pantium (tablet). Figures 1 and 2 illustrates typical HPLC chromatograms obtained from PNZ standard solution and from the assay of Pantium. The observed concentration of PNZ was found to be 39.69±0.17mg (mean±sd) for Pantium. The results of the assay (n = 9) undertaken yielded 99.24±0.17 % (%RSD = 0.629) of label claim for PNZ in Pantium. The retention times for PNZ were found to be and minutes respectively for standard drug and formulation respectively. The results of the assay indicates that the method was selective for the estimation of PNZ without interference from the excipients used to formulate and produce these tablets. Figure 1: A Typical Chromatogram of PNZ (10 g/ml) in Pure Form 50 Detector A - 1 (288nm) Pantoprazole pan065 Retention Time 50 Volts Volts Minutes Figure 2: A Typical Chromatogram of PNZ (10 g/ml) in Formulation 40 Detector A - 1 (288nm) Pantoprazole pan066 Retention Time 40 Volts Volts Minutes 52
7 CONCLUSION The LC method described here is a very simple, sensitive, and accurate procedure for estimation of PNZ. The developed and validated LC method is specific, accurate, robust and precise analysis of PNZ in pure and its formulations. The method was sensitive enough for quantitative detection of the analyte in pharmaceutical preparations. The proposed method can thus be used for routine analysis, quality control and for studies of the stability of pharmaceutical tablets containing these drugs. The sample recoveries in all formulations were in good agreement with their respective label claims and they suggested noninterference of formulation excipients in the estimation. REFERENCES 1. Argekar A P and Kunjir S S (1996), J. Planar- Chromator. Mod., Vol. 9, pp EI Sherif Z A, Mohamed A O, EI-Bardeicy M G and EI-Tarras M F (2005), Spectroscopy Lett., Vol. 38, pp Ewin K J (2001), Goodman & Gilman s,the Pharmacological Basis of Therapeutics, 10 th Edition, McGraw-Hill Inc., p. 1007, London. 4. Meyyanathan S N, Raj J R A and Suresh B (1997), Indian Drugs, Vol. 34, pp Moustafa A A M (2000), J. Pharm. Biomed Anal., Vol. 22, pp Ozaltin N and Kocer A (1997), J. Pharm. Biomed. Anal., Vol. 16, pp Pandya K K, Mody V D, Satia M C, Modi I A, Modi R I, Chakravarthy B K and Gandhi T P (1990), J. Chromatog. B. Biomed. App., Vol. 693, pp Renger B J (1993), AOAC. Int., Vol. 76, pp Reynolds J F and Martindale (1982), Extra Pharmacopoeia, 35 th Edition, Pharmaceutical Press, p. 1217, London. 10. Ritter J M, Lewis L D and Mant T G K (1999), A Textbook of Clinical Pharmacology, 4th Edition, Arnold LTD., p. 365, London. 11. Salama F, Abasawy N E I, Abdel Razeq S A, Ismail M F and Fouad M M (2003), J. Pharm. Biomed Anal., Vol. 32, pp Sastry C S P, Naidu P Y, Murty S S N, Talanta (1997), Vol. 44, pp The Merck Index (2001), An Encyclopedia of Chemicals, Drugs, and Biologicals, 13 th Edition, Monograph No. 7084, O Neal, M J (Ed.), Merck Research Lab, Whitehouse Station, New Jersey. 14. Wahbi A A M, Abdel-Razak O, Mahgoub Gazy A A H and Moneeb M S (2002), J. Pharm. Biomed. Anal., Vol. 30, pp
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