Vol - 4, Issue - 2, Apr 2013 ISSN: Attar et al PHARMA SCIENCE MONITOR
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1 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES VALIDATED RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF RABEPRAZOLE SODIUM AND LEVOSULPIRIDE IN BULK DRUG AND FORMULATION M. S. Attar* 1, S. S. Pekamwar 2, T. M. Kalyankar 3 1 Assistant professor, B.S.P.M s B.Pharmacy College Ambajogai, Dist: Beed (MS) 2 Associate Professor, Department of Pharmaceutical Chemistry, School of Pharmacy, S.R.T.M.University, Nanded (MS) 3 Assistant Professor, Department of Pharmaceutical Chemistry, School of Pharmacy, S.R.T.M.University, Nanded (MS) ABSTRACT HPLC method has been described for simultaneous estimation of Rabeprazole sodium (RAB) and Levosulpiride (LEV) in formulation. This method is based on a HPLC separation of the two drugs on the Thermo Hypersil BDS.C18 (250 mm, 4.6 mm, 5.0 µm) with isocratic conditions and a mobile phase containing acetonitrile:phosphate buffer ph7.4 (60:40) at a flow rate of 0.7 ml/min using PDA detector. This method has been applied to a marketed formulation without interference of excipients. The linear regression analysis data for the calibration plots showed a good linear relationship over the concentration range of 4-24 µg/ml for RAB and 5-45µg/mL for LEV, respectively. The method was validated for precision, robustness and recovery. Statistical analysis showed that the method is repeatable and selective for the estimation of RAB and LEV. Keywords: Rabeprazole, Levosulpiride, HPLC, Validation. INTRODUCTION RAB is 2 - [[[4 (3 methoxy propoxy) 3 methyl 2 pyridinyl] methyl] sulfinyl] 1H benzimidazole sodium salt. RAB is proton pump inhibitor belongs to a class of antisecretory compounds. Suppress gastric acid secretion by inhibiting the gastric H + /K + ATPase at the secretory surface of the gastric parietal cell. 1,2 LEV, a purified levo isomer of sulpiride, chemically it is 5 (aminosulfonyl) N [(1 ethyl 2 pyrrolidinyl)methyl] 2 methoxy benzamide. LEV is a D2 dopamine receptor antagonist and commonly prescribed to patients with functional dyspepsia, psychosis, and depression. At low doses, LEV increases Dopaminergic neurotransmission primarily by IC Value
2 the blocking of the dopamine autoreceptors which inhibits the pre synaptic dopamine synthesis and release of dopamine. Compared with racemic and dextro forms, the levo form of sulpiride has greater central antidopaminergic activity4, antiemetic and antidyspeptic effects and lower acute toxicity. 3,4. H 3 C OCH 2 CH 2 CH 2 OCH 3 N H 2 C S N N Na O ( a ) O O N H N O S O NH 2 ( b ) Figure 1 (a) Structure of RAB (b) Structure of LEV. MATERIALS AND METHODS Reagents and Samples An analytically working standard of RAB was obtained from Enal Drugs Pvt. Ltd. (Hyderabad). All the Chemicals of HPLC grade were purchased from Rankem Pvt. Ltd. (Delhi). Sample used was Rabekind Plus tablets manufactured by Mankind Pharma Ltd. Delhi; Containing 20 mg of Rabeprazole sodium and 75 mg of Levosulpiride. Mobile Phase Component IC Value
3 The mobile phase components were acetonitrile: 0.01M phosphate buffer, ph 7.4(40:60 v/v). Before use these solutions were filtered through a 0.45-µm pore size Rankem filter and degassed in an ultrasonic bath for 15 min. Stock Solutions RAB: 1 mg of RAB pure standard reference drug was added to 100 ml volumetric flask. And then 0.01 M Phosphate Buffer was added up to the mark. Then it was mixed and degassed it by ultrasonication for 15 min. This will provide 100 ml solution of RAB with 100 μg/ml concentration. LEV: 1 mg of pure LEV was added to 100mL of volumetric flask M Phosphate Buffer was added up to the mark. It was mixed thoroughly and degassed for 15 min using ultrasonication bath. This gave 100 ml solution of 100 μg/ml of LEV. Sample Preparation Twenty tablets of Rabekind Plus were weighed; their average weight was calculated and crushed to fine powder. The tablet powder equivalent to 4 mg of RAB and 15 mg of LEV was weighed and transferred to 100 ml volumetric flask and dissolved in sufficient quantity of 0.01 M Phospahte Buffer and the content was kept in ultrasonication bath for 30 min. Finally the volume was made up to the mark with Phosphate Buffer. The solution was filtered through 0.45 μm membrane filter paper to give stock solution of 40 μg/ml of RAB & 150 μg/ml of LEV. From this Stock solution suitable aliquots of the solution were further diluted with 0.01 M Phosphate Buffer within concentration range for both drugs. Chromatographic System and Condition A HPLC method was performed in isocratic mode on HPLC system (Perkin Elmer series - 200) consisted of ODS HYPERSIL 5μm (250 mm 4.6 I.D.) column. A mobile phase consisted of Acetonitrile: 0.01 M Phosphate buffer (60:40 v/v) adjusted to ph 7.4 with Triethylamine, de-gassed and kept flow rate at 0.7 ml/min. The run time was set to 10 min, and PDA detector was set to nm. IC Value
4 RESULTS AND DISCUSSION Validation of the Method The method was validated for accuracy, precision, sensitivity, recovery, linearity and robustness. The method validation was performed as per ICH guidelines.[1] Linearity For each drug appropriate aliquots were pipette out from each standard stock solution into a series of 10ml volumetric flasks. The volume was made up to the mark with Phosphate Buffer to get a set of solutions for RAB having concentration range 4, 8, 12, 16, 20 and 24µg/mL for LEV 15, 30, 45, 60, 75, and 90 µg/ml. Triplicate dilutions of each concentration of each drug were prepared separately. From these triplicate solutions, 10 μl injections of each concentration of each drug were injected into the HPLC system two times separately and run under the conditions as described above. Evaluation of both drugs was performed with UV detector at 210 nm. Peak areas were recorded for all the peaks as shown in Table 1 and 2. Working calibration curves for RAB and LEV were plotted separately with peak area Vs the respective concentration of RAB and LEV as shown in Figure 2 and 3. TABLE 1: CALIBRATION TABLE FOR LEV Sr. No. Concentration AUC (μg/ml) (mv) IC Value
5 Figure 2 Calibration curve for LEV TABLE 2: CALIBRATION TABLE FOR RAB Sr. No. Concentration AUC (μg/ml) (mv) Figure 3 Calibration curve for RAB IC Value
6 Analysis of marketed formulation For the analysis of tablet formulation sample solution was prepared as per the above mentioned procedure and results were shown in Table 3. TABLE 3: RESULTS OF ANALYSIS OF MARKETED FORMULATION. Label Claim Amt of drug found % Drug found RAB LEV RAB LEV RAB LEV Mean SD RSD Sensitivity The sensitivity of measurement of RAB and LEV was estimated in terms of the limit of quantitation (LOQ). The smallest amounts detected under the chromatographic conditions used were estimated in terms of the limit of detection (LOD). LOQ and LOD were calculated by use of the equations LOD = 3 N/B and LOQ = 10 N/B, where N is the standard deviation of the peak areas of the drugs, taken as a measure of noise, and B is the slope of the corresponding calibration plot. LOD and LOQ for RAB were found to be μg/ml and μg/ml, for LEV, μg/ml and μg/ml. Precision: The precision of the method was investigated with respect to repeatability. For intraday precision, six concentrations of each compound were analyzed on the same day each concentration of sample was injected two times. For intraday samples are injected two IC Value
7 times in a day, and for interday, samples are injected for one time once in a day & it is for two days. Tables 4 summarize the SD and relative standard deviation (RSD) of precision studies. TABLE 4: RESULTS OF PRECISION STUDY Repeatability Intraday Interday RAB LEV RAB LEV RAB LEV % Mean S.D % R. S. D *Mean of six determinations Accuracy (Recovery studies) To check the accuracy of proposed method, recovery studies were carried out at 80%, 100% and 120% of the test concentration as per ICH Guidelines 1. As per label claim, the tablet consisted of 20 mg of RAB & 75 mg of LEV. For recovery studies different levels of the standard concentration according to 80%, 100% and 120% are made and % mean recoveries are calculated. (Table 5) Level of % recovery TABLE 5: RESULT OF RECOVERY STUDY Label Claim Amt. of Total amount mg/tab standard added recovered % Recovery RAB LEV RAB LEV RAB LEV RAB LEV Mean SD %RSD IC Value
8 Robustness studies The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. The evaluation of robustness should be considered during the development phase and depends upon the type of procedure under study. It should show the reliability of an analysis with respect to deliberate variations in Method parameters. The parameters included flow rate, ph, composition of mobile phase ratio, and column temperature. The solution containing rationale amount of RAB and LEV was injected into sample injector of HPLC three times using variation in the parameters like flow rate, percentage of Acetonitrile in the mobile phase, column temperature. The results obtained are given in the form of data in Table 6 along with their statistical validation parameters. TABLE 6: RESULTS OF ROBUSTNESS STUDY Factor Retention Time Tailing Factor Flow Rate (ml/min) (ml/min) Level LEV RAB LEV RAB Mean ± SD 5.63 ± ± ± ± 0.20 % of ACN in mobile phase (v/v) % Level LEV RAB LEV RAB Mean ± SD 5.98 ± ± ± ±0.12 Column Temperature o C o C Level LEV RAB LEV RAB IC Value
9 Mean ± SD 6.08 ± ± ± ±0.31 ph of Mobile Phase ph Level LEV RAB LEV RAB Mean ± SD 5.55 ± ± ± ±0.26 * Each observation is mean of three observation (n = 3) System suitability parameters: To ascertain resolution and reproducibility of the chromatographic system, system suitability parameters are studied and results are summarized in Table 7. TABLE 7: SYSTEM SUITABILITY PARAMETERS Parameter Observations LEV RAB Retention time (min) Theoretical Plates Resolution Tailing Factor CONCLUSION A combination of RAB with LEV is used for the treatment of ulcer. Literature survey revealed that no analytical method is yet reported, for the simultaneous estimation of these drugs till date to the best of my knowledge. Hence an attempt has been made to develop simple, accurate, precise, economic and validated analytical method on RP-HPLC for determination of both the candidates in formulation. Statistical data indicated that the proposed method requires short duration of analysis and appear to be equally suitable for routine determination of RAB and LEV in formulation in quality control laboratories, where economy and time saving are essential The chromatographic method was validated as per ICH guidelines. IC Value
10 The results of recovery studies, LOD and LOQ for the method were found to be satisfactory. Intra and inter-day variation of HPLC method was well within limit with % RSD < 2%. The method can be used to determine the purity of the drug available from various sources by detecting the related impurities. It could be implemented in the industries for the analysis of drugs. ACKNOWLEDGEMENT Authors are thankful to Enal Drugs Pvt. Ltd. Hyderabad, for providing us the gift sample of the pure drug and also to the Director School of Pharmacy, S R T M University, Nanded, for providing research facilities. REFERENCES: 1. Indian Pharmacopoeia Volume-3, The Indian Pharmacopoeia Commission, Sweetman SC: Martindale: The Complete Drug Reference. The Pharmaceutical Press 36 th Edition, Prasanna Reddy Battu, M S Reddy: Development and Validation of RP-HPLC for the Rabeprazole Sodium in Pharmaceutical formulations and Human Plasma. Asian Journal Of Research In Chemistry 2009; 1: A. Lakhmana Rao, B. N. V. Ravikumar and G. G. Shanker: Development of RP- HPLC of Rabeprazole in Pure and Tablet Dosage Form. E-Journal of Chemistry 2008; S2: IC Value
11 7. M. Kalyan Obula Reddy, Chiranjeevi Bodepudi, P. Shanmugasundaram: Method Development of rabeprazole in Bulk and Tablet Dosage form by RP-HPLC Method, International Journal of ChemTech Research 2011; 3: Tushar G. Barot, Vipul Prajapati, Dr. P. K. Pate, D. K. Bhoi, Dr. L. D. Patel: A Method Development and Validation of Rabeprazole Sodium (API) by High Performance Liquid Chromatography. International Journal of ChemTech Research 2009; 4: V. L. Kulkarni and P. P. Mahulikar: Estimation of Rabeprazole Sodium in tablet dosage form by rapid Isocratic Reversed Phase High Performance Liquid Chromatography. Journal of Scientific and Industrial Research 2006; 65: S.P. Silambarasan, K. Anandakumar, R. Venkatalakshmi and C. Sasikala: Development of UV Spectrophotometry and RP-HPLC Methods for the Estimation of Levosulpiride in Bulk and in Tablet Formulation. Asian journal of Research In Chemistry 2010; 1: Satish Middi, Shobha Manjunath, Venkatesh Chouhan, RP-HPLC Quantification of Levosulpiride in bulk drug and Pharmaceutical formulation, IPC Hea Young Cho, Yong Bok Lee: Improvement and Validation of a liquid chromatographic method for the determination of Levosulpiride in serum and urine. Journal of Chromatography B 2003; 2: Su-Eon Jin, Eunmi Ban, Yang Bae Kim, Chong-Kook Kim: Development of HPLC Method for the Determination Levosulpiride in Human Plasma. Journal of Pharmaceutical and Biomedical Analysis 2004; 4: ICH, Q2A, Harmonised Tripartie Guideline, Text on Validation of Analytical Procedures, IFPMA, in: Proceedings of the International Conference on harmonization, Geneva, March IC Value
12 15. ICH, Q2B, Harmonised Tripartie Guideline, Validation of Analytical Procedure: Methodology, IFPMA, in: Proceedings of the International Conference on harmonization, Geneva, March1996. For Correspondence: M. S. Attar IC Value
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