Research Journal of Pharmacy and Life Sciences: Volume 1, Issue 1; January-April 2020: Page 42-50 Research Article NEW RP-HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF OMEPRAZOLE AND ASPIRIN IN BULK AND TABLET DOSAGE FORM V. Anil Kumar1, Soraj Kumar Raul1, Gopal Krishna Padhy2* 1 Department of Pharmaceutical Analysis and Quality Assurance, Maharajah’s College of Pharmacy, Phool Baugh, Vizianagaram-535002, Andhra Pradesh. 2 Department of Pharmaceutical Chemistry, School of Pharmacy, Centurion University of Technology and Management, Odisha. ARTICLEINFO ABSTRACT Article history: Received 29 December 2019 Revised 09 January 2020 Accepted 15 January 2020 A reverse phase HPLC method was successfully developed and validated for concurrent assessment of omeprazole and aspirin for bulk and pharmaceutical formulation. The method was developed employing a reversed phase C18 column. A optimized mobile phase having a composition of acetonitrile-methanol (20:80 v/v) was used. The experiment was carried out at flow rate of 0.6 ml/min and wavelength of 233 nm. Retention times for omeprazole and aspirin were found at 2.667 and 1.720 min respectively. Across a concentration span of 10-50 µg/ml and 5-25 µg/ml the method was linear for aspirin and omeprazole, respectively. The proposed method have potential for the regular quality scrutiny of this combination in bulk and pharmaceutical dosage form. ã2020 Published by HOPE on behalf of RJPLS This is an open access article under the CC-BY-NC-ND License. Keywords: RP-HPLC, Aspirin, Omeprazole, ICH, Simultaneous estimation. ______________________ * Corresponding author: Gopal Krishna Padhy, Department of Pharmaceutical Chemistry, School of Pharmacy, Centurion University of Technology and Management, Pitamahal, Rayagada, Odisha-765002, India. E mail ID: gopalmedchem@gmail.com Introduction Aspirin (ASP), inflammatory incidence of heart failure and used in long a non-steroidal (NSAID) anti- term basis to prevent strokes and heart medication attacks.1 Omeprazole (OME, Figure 2), a indicated to cure pain, inflammation and selective and irreversible proton pump fever ( Figure 1). Due to it’s antithrombotic inhibitor indicated in the treatment of peptic action, aspirin is also given to decreases the ulcer and Zollinger–Ellison syndrome.2 Yosprala is a pharmaceutical formulation Methanol, approved by USFDA in 2016 for treatment Fischer Scientific, India). Methanol was of of sick person who need aspirin for an analytical grade, (SD-Fine chemicals, avoidance India). of cerebrovascular and Acetonitrile, (HPLC grade, cardiovascular measures and those are at Instruments: danger of getting aspirin linked gastric RP- HPLC- Agilent 1220 Infinity LC system ulcers.3 UV spectroscopy and HPTLC provided with a G4 288c series 2-Channel, methods for estimation of Aspirin in Dual plunger pump, Variable wavelength combination with omeprazole have been Detector (VWD) with Deuterium lamp and reported in the literature.4-6 Recently few Holmium oxide filter. Eclipse XDB plus HPLC simultaneous C18 Column (4.6 × 150 mm, 5µm particle identification of omeprazole and aspirin in size) , Agilent Cary 60 UV-Visible double bulk and pharmaceutical formulation have beam Spectrophotometer, manual injector. methods been developed. employed 7-12 for Majority of the methods complex mobile phases The analysis was done using data analysis program (Ez Chrome-version A.04.05). containing buffer. Therefore an endeavour Preparation of standard working solutions was made to develop a new, simple, Precisely measured OME and ASP standard sensitive, accurate and cost effective method drugs (10 mg) were taken in a 100 ml for concurrent estimation of aspirin and volumetric flask. Afterward the volume was omeprazole. adjusted with the mobile phase and mixed thoroughly to get standard working solutions of ASP and OPZ with concentration of (100 µg/mL) Figure 1: Structure of Aspirin Chromatographic conditions Mobile phase composition of acetonitrilemethanol (20:80 v/v) was employed at a flow rate of 0.6 mL/min. injection volume Figure 2: Structure of Omeprazole was 20 µL is used to develop the method. Material and Methods An Agilent C18 column (150 mm x 4.6 Pure ASP (99 %) and OME (99 %) kindly mm), having particle size of 5 µm was used. supplied by Lee pharma Ltd as gift sample. The analytes were measured at 233 nm. 43 Determination of calibration curves for 20 µL of aliquots were injected into the OME and ASP column and then run with the mobile phase Precise amount of standard OME and ASP employing solutions were taken in separate sets of conditions. Amount of both drugs present in volumetric flasks (10 ml) and diluted with the tablets were calculated by means of mobile regression equation. phase to achieve ultimate the most advantageous concentrations of 5-25 and 10-50 µg/mL for Results and Discussion OME and ASP respectively. 20 µL of the Determination of λmax solutions were eluted with the mobile phase The standard solution was scanned between using the most favorable chromatographic 200 to 400 to nm to calculate the λmax. The conditions. Calibration graphs were obtained λmax were observed at 299.4nm and 229.2nm by plotting the peak areas of OME and ASP for omeprazole and Aspirin respectively. versus The spectrum of both drugs were overlaid. different drug concentrations regression From the overlain spectrum, the isobestic equations were calculated from the standard point was found to be at 233.8nm (Figure 3). plot. This wavelength was utilized for HPLC Analysis of tablet dosage form method development. (µg/mL). Subsequently the Twenty tablets (containing 40 and 81 mg of OME and ASP per tablet) were measured and made into fine powder. Suitable quantity of powder equivalent to one tablet was accurately measured and transferred to a volumetric flask (100 mL) and 50 mL of mobile phase was added. Solution obtained was vigorously shaken, sonicated for 30 min Figure 3: Isobestic point for Aspirin and and filtered. Then volume was adjusted up Omeprazole to 100 mL to produce stock solution Method development containing 810 and 400 µg/mL of ASP and Different OME respectively. Stock solution was suitable separation of the drugs were refined diluted with the mobile phase to get diverse considering resolution among the drugs. concentrations of ASP and OME of choice. Numerous mobile phases were tested one at circumstances affecting the 44 a time to get better separation. Solvents such up by plotting the drug concentrations as (µg/mL) versus peak areas for both ASP and acetonitrile and methanol in different ratios were used. Method development was OME. carried out on Agilent C18 column (150 mm corresponding peak areas are presented in x 4.6 mm, 5 µm particle size). Optimum table 1. Regression line equations, slopes resolution was observed intercepts, in mobile phase The linearity ranges and and correlation coefficient for consisting of acetonitrile-methanol (20:80) the calibration curve were furnished in v/v at flow rate 0.6 ml/ min and wave length Figure 5 and 6. 233.8nm. The chromatogram (Figure 4) Table 1: Linearity results for Aspirin and exposed that ASP and OME were evidently Omeprazole separated from one another with retention times of 1.717 and Figure 4: HPLC chromatogram combination of 2.667 Aspirin min. (ASP) for and Omeprazole (OME) Figure 5: Calibration curve of Aspirin Method validation (Linearity) The method developed was validated according guidelines prescribed by ICH. Validation parameters viz: accuracy, linearity, specificity, precision, limit of quantification (LOQ), limit of detection (LOD) and robustness were checked.13,14 Linearity Underneath Figure 6: Calibration curve of Omeprazole (Linearity) the better chromatographic conditions, the calibration graphs were put Recovery study (Accuracy) Accuracy of the method was resolved by 45 using the method outlined in ICH. Accuracy repeatability. Mean area, standard deviation were and % RSD were calculated for the two determined using 40 µg/mL concentration of ASP and 20 µg/mL drugs. The data were given in Table 3. concentration of OMP. Three levels of Table 3: Precision results of Aspirin and samples were prepared to calculate accuracy Omeprazole by standard addition method. Injections were given in triplicate to determine each level of accuracy and the % recovery was determined using the consequent regression equation. The mean %Recovery was found to be 99.93% and 99.84% for Aspirin and Omeprazole respectively. The results were represented in Table 2 Table 2: Accuracy results for Aspirin and Omeprazole Specificity Specificity is described as accurate computation of the response of analyzed drugs without any interferences from additives. The specificity test was performed for Omeprazole and Aspirin. It was found that there was no interference of sample matrix in retention time of analytical peak. Precision The chromatograms are represented in Precision is expressed as level of accord Figure 7, 8 and 9. among the individual test result when the method is employed repetitively to manifold homogenous samples. Intra-day precision was determined by applying six injections on the same day following the procedure of Figure 7: Chromatogram of blank (mobile 46 phase) Limit of detection (LOD) is mentioned as the minimum quantity of analyte that could be ascertained under the experimental condition and Limit of quantification (LOQ) is the minimun quantity of analyte in the sample that could be resolved with accepted Figure 8: Chromatogram of standard injection (ASP and OME) precision and accuracy. The slope (M) and standard deviation (SD) was used to determine the quantification and detection limits. LOD and LOQ was determined using the formula LOQ = 10(SD/M) and LOD = 3.3 (SD/M). The results were given in Table 5. Table: 5 LOD & LOQ table of Aspirin and Figure 9: Chromatogram of sample injection Omeprazole (ASP and OME) System suitability Different HPLC parameters were assessed by injecting five replicate of each drug concentration (40 µg/mL) to HPLC column. A range of parameters such as resolution factor (R), theoretical plates (N) and tailing factor (T) were calculated. The data were given in Table 4. Table 4: System suitability parameters for Aspirin and Omeprazole Robustness The robustness study was conducted by making small changes in special parameters like composition of mobile phase and flow rate. Negligible parameters could changes not in HPLC influenced the separation, which affirmed sensitivity of the developed method. The results obtained were represented in Table 6. Table 6: Robustness results of Aspirin and Omeprazole: Determination of LOQ and LOD 47 Acknowledgments The authors are thankful to Lee pharma Ltd for providing the gift sample of aspirin and omeprazole. They are also thankful to Principal of Maharajah’s College of pharmacy, Dr. P. Udaya Shankar, , , for Utilization of the validated method to pharmaceutical tablet dosage form giving required infrastructure and facility. Conflict of Interest Simultaneous estimation of OME and ASP in tablet dosage form was determined by the projected method. Acceptable outcomes were obtained, which are in good accord with the label claim representing no hindrance from additives. The results are shown in Table 7. The authors have no conflict of interest References 1. leland JG, Bulpitt CJ, Falk RH, Findlay IN, akley CM, Murray G, Poole-Wilson PA, Prentice CR, Sutton GC. Is aspirin safe for patients with heart failure. Br Heart J. 1995 Sep; 74(3):215-219. Table 7: Determination of aspirin and omeprazole combination in tablet dosage forms by the proposed method. 2. Aton PN. Omeprazole. N Engl J Med. 1991 Apr 4; 324(14):965-975. 3. Eltri KT. Yosprala: a fixed dose combination of aspirin and omeprazole. Cardiol Rev. 2018 Jan 1; 26(1):50-53. 4. Atta S, Afreen S, Tappa S, Nagarajan C, GnanaPrakash K. Simultaneous stimation of aspirin and omeprazole (y0sprala) in bulk by Conclusions uv-spectroscopy. 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