Asian J. Research Chem. 2(2): April.-June, 2009 ,
ISSN 0974-4169
www.ajrconline.org
RESEARCH ARTICLE
RP-HPLC Method for Simultaneous Estimation of Omeprazole and Ondansetron in Combined Dosage Forms Zarna Dedania*, Ronak Dedania, Vaishali Karkhanis1, G Vidya Sagar, Meeta Baldania and NR Sheth2 Department of Pharmaceutical Analysis, Veerayatan Institute of Pharmacy, Kutch, Gujarat 1 A.R. College of pharmacy, Vallabh Vidhyanagar, Gujarat 2 Department of Pharmaeutical Sciences, Saurastra University, Rajkot, Gujarat *Corresponding Author E-mail:
[email protected]
ABSTRACT
A RP-HPLC method is developed for simultaneous estimation of omeprazole and ondansetron in combined tablet dosage form. Omeprazole is a proton pump inhibitor and in the treatment of gastro-oesophageal reflux disease (GERD), peptic ulcer and Zollinger-Ellison syndrome. Ondansetron is used as selective 5-HT3 receptor antagonist and used in the management of nausea and vomiting induced by cytotoxic chemotherapy and radio therapy and also post operative nausea and vomiting. The mobile phase used was a combination of Methanol: Acetonitrile (90:10). The detection of the combined dosage form was carried out at 218 nm and a flow rate employed was 0.5 ml/min. The retention time for omeprazole and ondansetron was found to be 5.39 and 11.08 min respectively. Linearity was obtained in the concentration range of 4 to 20 µg/ml of omeprazole and 4 to20 µg/ml of ondansetron with a correlation coefficient of 0.997 and 0.9967. Detector consists of photodiode array detector; the reversed phase column used was RP-C18 (2.27µm size, 250 mm´4.6 mm i.d.) at ambient temperature. The developed method was validated according to ICH guidelines and values of accuracy, precision and other statistical analysis were found to be in good accordance with the prescribed values. Thus the proposed method was successfully applied for simultaneous determination of ondansetron and omeprazole in routine analysis. KEY WORDS: Simultaneous Estimation; RP-HPLC; Omeprazole; Ondansetron;
INTRODUCTION
Omeprazole is chemically 5-methoxy-2-[[(4-methoxy-3, 5-dimethyl-2-pyridinyl) methyl] sulfinyl ]-1Hbenzimidazole and it is used as proton pump inhibitor and in the treatment of gastro-oesophageal reflux disease (GERD), peptic ulcer and Zollinger-Ellison syndrome1. Ondansetron is chemically (±) 1, 2, 3, 9-tetrahydro-9methyl-3-[(2-methyl-1H-imidazol-1-yl) methyl ]-4Hcarbazol-4-one, monohydrochloride, dehydrate and it is used as selective 5-HT3 receptor antagonist and used in the management of nausea and vomiting induced by cytotoxic chemotherapy and radio therapy and also post operative nausea and vomiting. Literature survey reveals that RP-HPLC in formulation2, 3 and in plasma and blood 4-6, HPTLC7, spectrophotometry8 are available for the determination of omeprazole and spectrometric9,10 in formulation, RPHPLC in formulation11 and blood12 for determination of ondansetron. Received on 29.01.2009 Accepted on 22.05.2009
Modified on 22.03.2009 © AJRC All right reserved
The review of the literature revealed that no RP-HPLC method has so far been reported for the combination of omeprazole and ondansetron. So an attempt has been made to develop a simple, precise, accurate reverse phase high performance liquid chromatographic method for the simultaneous estimation of omeprazole and ondansetron in combined tablet dosage forms.
EXPERIMENTAL:
Instrumentation: The present work was carried out on isocratic high pressure liquid chromatograph, LC system used consist of pump (Perkin Elmer, USA) with universal loop injector (Rheodyne) of injection capacity 20 µl. Detector consists of photodiode array detector; the reversed phase column used was RP-C18 (2.27µm size, 250 mm´4.6 mm i.d.) at ambient temperature. DORON-O containing 10 mg/tab of Omeprazole and 4 mg/tab of Ondansetron purchased from local market.
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Reagents and Chemicals: OME and OND were obtained as gift sample from BestoChem Formulation (I) Ltd., Delhi. All solvents were of HPLC grade obtained from Spectrochem, Mumbai, India.
Fig: 1 Chromatogram containing 16µg/ml standard Omeprazole (OME) and Ondansetron Hydrochloride (OND) in Methanol: Acetonitrile (90:10), Flow rate – 0.5ml/min
Experimental Condition: The HPLC system was operated isocratically at flow rate of 0.5ml/min. at 25°C ± 0.5°C for 15 min. The mobile phase found to be most suitable for analysis was Methonal: Acetonitrile 90:10%v/v, detection was carried out at 218 nm. Preparation of Standard Solution: Standard stock solution of 1000 g/ml of each OME and OND were prepared by dissolving 10 mg of each drug in mobile phase. Table 1: System Suitability Test Parameters System Suitability Proposed Method Parameters OME OND Retention times (RT) min 5.39 11.08 Theoretical plates (N) 5164.81 7857.04 Tailing factor (AS) 1.36 1.67 Resolution (RS) 14.225
Fig 2: Chromatogram containing Tablet formulation of Omeprazole (OME) and Ondansetron Hydrochloride (OND) in Methanol: Acetonitrile (90:10), Flow rate – 0.5ml/min, retention time 5.39 min. for OME and 11.08 min. for OND
Sub stock solution was prepared from stock solution by diluting each standard stock solution (1 ml) up to 10 ml to get 100 g/ml of each drug. The nominal concentrations in range of 4-20 g/ml were prepared for calibration. All solutions were stored at room temperature. Each standard solution (20µl) was injected into the column after filtration using 0.2 micron membrane filter. Sample Preparation: Twenty tablets were weighed and crushed to fine powder. Powder equivalent to 10 mg of OME and 4mg OND was accurately weighed and dissolved in mobile phase, sonicated for 10 min and filtered through whatmann filter paper No.42, finally different concentrations of tablet sample were prepared by serial dilution technique. PROCEDURE: Chromatographic Condition: Chromatographic separation was achieved by using mobile phase consisted of Methanol: Acetonitrile (90:10), flowing through RP-C18 column at a constant flow rate of 0.5 ml/min for 15 min. A RP-C18 column was used as the separation phase. Detection was carried out using a photo diode array detector at 218 nm. Linearity: To establish the linearity a series of dilutions ranging from 4-20 g/ml for OME and 4-20 g/ml for OND were prepared separately and calibration graph was plotted between the mean peak area Vs respective concentration and regression equation was derived.
Method Validation: The accuracy, precision and robustness were determined by analyzing a set of laboratory sample (n=5) with each of the five concentrations ranging from 4-20 g/ml for both drugs.
RESULTS AND DISCUSSION:
Chromatographic Method: Initially Methanol: Water was tried as mobile phase, in which satisfactory peak was not obtained. Then different ratio of Methanol: Acetonitrile were tried but resolution was not satisfactory. Finally the system containing Methanol: Acetonitrile (90:10) was found to be satisfactory and gave two well resolved peaks for OME and OND with retention time for OME and OND 5.39 min and 11.08 min respectively. A representative graph of this is shown in Fig.1.
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Table 2: Determination of Accuracy Amt of sample
Amt. of drug added
Amt. recovered
OME ( g/ml)
OND ( g/ml)
OME ( g/ml)
OND ( g/ml)
OME ( g/ml)
12 12 12 12
12 12 12 12
0 6 12 18
0 6 12 18
12.07 18.02 23.96 30.11
Table 3: Summary of Validation Parameters of RP-HPLC Parameters OME OND Recovery% 99.83 – 100.4 99.54 – 100.2 Repeatability (RSD, 0.45 – 0.79 0.67 - 0.89 n=5) Precision(CV) Intra-day (n=3) 0.58 – 0.78 0.58 – 0.73 Inter-day (n=3) 0.68 – 0.81 0.69 – 0.85 Specificity Specific Specific Solvent suitability 99.53 – 100.5 99.23 – 100.7 Limit of Detection 0.24 0.28 ( g/ml) Limit of Quantitation 0.72 0.86 ( g/ml)
System Suitability: The system suitability test was applied to a representative chromatogram to check the various parameters such as column efficiency, resolution, precision and peak tailing. The result obtained is shown in Table 1. The resolution between OME and OND was 14.22. The number of theoretical plate for OME and OND were 5164 and 7857 respectively. All these parameters were evaluated with the background of regulatory requirements, which also suggests good chromatographic condition. Linearity: OME and OND showed a linearity of response between 4-20 g/ml. This linearity was represented by a linear regression equation as follows. YOME = 209551 x - 166332 (r2 = 0.997) YOND = 92120 x - 50850 (r2 = 0.9967) Accuracy and Precision: The recovery experiment was carried out by spiking the already analyzed sample of the tablets with their different known concentration of standard OME and OND. The result is summarized in Table 2.The percent recovery for OME ranges from 98.83 to 100.4% and OND ranges from 99.54 to 100.2%. The summary of other validation parameter shown in Table 3. Assay: The content of OME and OND found in the tablets by the proposed method are shown in Table 4. and chromatograph shown in Fig 2. The low R.S.D indicates that the method is precise and accurate.
OND ( g/ml) 11.88 17.95 23.89 30.07
% Recovery OME %
OND %
100.1 99.83 100.4
99.72 99.54 100.2
CONCLUSIONS:
The proposed RP-HPLC method allows for accurate, precise and reliable measurement of OME and OND simultaneously in combined dosage form. The developed RP-HPLC method was found to be simple, rapid, selective, accurate and precise for the concurrent estimation of drugs in respective two-component tablet dosage form of OME and OND. The method was evaluated in a mass of facets, such as best condition, linear relation including coefficient of correlation, robustness, accuracy, reproducibility and precision. The RSD for all parameters was found to be less than one, which indicates the validity of method and assay results obtained by this method are in fair agreement. The developed method can be used for routine quantitative simultaneous estimation of OME and OND in pharmaceutical preparation.
ACKNOWLEDGMENTS:
The authors are grateful to Institute of Science and Technology for Advanced Studies and Research (ISTAR) and A. R. college of Pharmacy, Vallabh Vidhyanagar, India, for providing the facilities to carry the experiment and BestoChem Formulation (I) Ltd., Delhi for providing gift samples of Omeprazole and Ondansetron. Table 4: Assay Results of Marketed Formulation Formulation Actual % OME % OND concentration µg/ml OME OND Tablet 20 8 100.2 % 99.78 %
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