ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF OMEPRAZOLE AND ASPIRIN USING REVERSE PHASE HPLC METHOD IN BULK AND DOSAGE FORM

R Vani, M Sunitha

Shadan Women’s College of Pharmacy, Hyderabad, India

*Corresponding Author’s Email: vrathipelli@gmail.com

DOI: http://doi.org/10.22270/ujpr.v2i4.R6

ABSTRACT

A new simple, accurate, precise and reproducible RP-HPLC method has been developed for the simultaneous estimation of Aspirin and Omeprazole in bulk and pharmaceutical dosage form using C18 column (Agilent, 250 x 4.6 mm, 5μm) in isocratic mode. The mobile phase consisted of Methanol and 0.1 M Di-potassium Phosphate buffer (pH 3) in the ratio of 60:40 v/v. The detection was carried out at 256 nm. The method was linear over the concentration range for Omeprazole 50-250 μg/ml and for Aspirin 10-50μg/ml. The recoveries of Omeprazole and Aspirin were found to be 100.07 and 100.06% respectively. The validation of method was carried out utilizing ICH-guidelines. The described HPLC method was successfully employed for the analysis of pharmaceutical formulations containing combined dosage form.

Keywords: Aspirin, Omeprazole, reverse phase HPLC, validation.

INTRODUCTION

Aspirin (ASP) is chemically 2-(acetyloxy)-benzoic acid (Figure 1). It is nonselective cyclooxygenase inhibitor used as an antipyretic, analgesic, anti-inflammatory, and antithrombotic agent. omeprazole magnesium (ESO) is S-isomer of omeprazole and proton pump inhibitor. It is magnesium, bis [5-methoxy-2-[[(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulfinyl]1H-benzimidazolato] (Figure 2). It is used in treatment of peptic ulcer disease, NSAIDS-associated ulceration and Zollinger-Ellison syndrome, used as antiulcerative. ASP and ESO in combined dosage form are used in cardiovascular disorder and cerebrovascular

disorders^1–3

Figure 1: Structure of aspirin.

The review of literature revealed that various analytical methods involving spectrophotometry^5-7, HPLC^8-11 and HPTLC have been reported for ASP in single form and in combination with other drugs¹². Several analytical methods have been reported for ESO in single form and in combination with other drugs including spectrophotometry^13,14, HPLC^15,16, and HPTLC¹⁷.

The present work describes the development of a simple, precise, accurate, and reproducible HPLC method for the simultaneous estimation of ASP and ESO in combined dosage form. The developed method was validated in accordance with ICH Guidelines and successfully employed for the assay of ASP and OMP combine dosage form^1,18.

Figure 2: Structure of Esomeprazole 

MATERIALS

ASP and OMP were received gratis from Hetero drugs, Hyderabad and were used as received. HPLC grade Methanol was purchased from SD Fine Chem Pvt. Ltd. (Mumbai, Maharashtra). Ultra-pure water was obtained from ELGA (Bucks, UK) water purification unit. Waters total recovery vials (Waters, Milford, MA, USA) were of glass type 1, class A with 950μL maximal injectable volumes. All other chemicals were of analytical reagent grade.

EXPERIMENTAL WORK

Chromatographic conditions

The HPLC system (LC Waters, Milford, MA, USA) consisted of quaternary gradient system (600 Controller), in-line degasser (Waters, model AF), photodiode array detector (Water, 2998 model) and auto sampler (Waters, model 717 plus). Data was processed using Empower Pro software (Waters, Milford, MA, USA).

Isocratic elution of the mobile phase 0.1 M Di-potassium Phosphate buffer (pH 3) and Methanol in the ratio of 40:60 v/v with the flow rate of 1ml/min. Separation was performed on a Waters C₁₈ (250x4.6mm i.d, 5μ particle size) analytical column and a pre-column to protect the analytical column from strongly bonded material. Integration of the detector output was performed using the Waters Empower software to determine the peak area. The contents of the mobile phase were filtered through a 0.45µm membrane filter and degassed by sonication before use. Mobile phase was used as diluents.

The flow rate of the mobile phase was optimized to 1 ml/min which yields a column back pressure of 110–112kg/cm. The run time was set at 6 min and a column temperature was maintained at 35°C. The volume of injection was 10 µl, prior to injection of the analyte, the column was equilibrated for 30–40 min with the mobile phase. The eluents were detected at 256 nm. The developed method was validated in terms of specificity, linearity, accuracy, limit of detection (LOD), limit of quantification (LOQ), intra-day and inter-day precision and robustness for the assay of ASP and OMP as per ICH guidelines.

Preparation of standard solutions

ASP and OMP were weighed (10mg each) and transferred to two separate 10ml volumetric flasks and dissolved in 5ml of water and make up the volume up to the mark with mobile phase. Working standards of the drugs were prepared from this solution.

Preparation of sample solution

Twenty tablets (Yosprala, Make: Aralez Pharmaceuticals) were weighed. An accurately weighed amount of the finely powdered tablets equivalent to 10mg was made up to 10 mL with mobile phase. The solution was filtered followed by serial dilution to the required concentrations for each experiment.

RESULTS AND DISCUSSION

Method Development

Number of mobile phase and their different proportions were tried and finally was selected as 0.1 M Di-potassium Phosphate buffer (pH 3) and Methanol in the ratio of 40:60 v/v appropriate mobile phase which gave good resolution and acceptable system suitability parameters. The results of system suitability parameters were shown in Table 2. The chromatogram of working standard solution is shown in Figure 3. The summary of Chromatographic conditions was given in Table 1.

Method Validation

Accuracy

Recovery assessment was obtained by using standard addition technique which was by adding known quantities of pure standards at three different levels in 50%, 100% and 150% to the pre analyzed sample formulation. From the amount of drug found, amount of drug recovered and percentage recovery were calculated which sense to conformation that the proposed method was accurate. The results were tabulated in Table 3.

Precision

The intraday and inter day precision of the proposed method was determined by analyzing mixed standard solution of OMP and ASP at concentration 150 µg/mL and 30 µg/mL, 3 times on the same day and on 3 different days. The results shown in Table 4 were reported in terms of relative standard deviation.

Linearity

Calibration graphs were constructed by plotting peak area vs concentration of ASP and OMP and the regression equations were calculated. The calibration graphs were plotted over 5different linear concentrations in the range of 10-50µg/ml for ASP and 50-250 µg/ml for OMP. Aliquots (10µl) of each solution were injected under the operating chromatographic condition described above [Number of replicates (n=6)]. The linearity graphs were shown in Figure 4 and 5.

Limit of detection (LOD) and limit of quantitation (LOQ):

The limit of detection (LOD) and limit of quantitation (LOQ) of ASP and OMP were determined by calculating the signal-to-noise (S/N) ratio of 3:1 and 10:1, respectively according to International Conference on Harmonization guidelines.LOD values for ASP and OMP were found to be 3.08and 3.041µg/mL respectively. LOQ values for ASP and OMP were found to be 9.24µg/mL and 10.37µg/mL respectively.

Assay of the tablet dosage form

The proposed validated method was successfully applied to determine ASP and OMP in tablet dosage form. The result obtained for ASP and OMP were comparable with corresponding labeled amounts. The results were tabulated in Table 4.

CONCLUSION

The proposed method has advantage of simplicity and convenience for the separation and quantitation of ASP and OMP in the combination which can be used for the assay of their dosage form. Also, the low solvent consumption and short analytical run time lead to environmentally friendly chromatographic procedure. The method is accurate, precise, rapid and selective for simultaneous estimation of Aspirin and Omeprazole in tablet dosage form. Hence it can be conveniently adopted for routine analysis.

ACKNOWLEDGMENTS

The authors are grateful to Principal, Management of Shadan Women’s College of Pharmacy, Hyderabad, India for providing necessary facilities to carry out this research project. Authors are thankful for Hetero drugs, Hyderabad, AP for kindly providing the gift sample of OMP and ASP.

CONFLICTS OF INTEREST

The authors declared no conflicts of interest.

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Figure 3: Typical Chromatogram of Omeprazole and Aspirin

Table 1: Summary of chromatographic conditions

Table 2: System suitability parameters

Table 3: Results of accuracy

*Mean % Recovery of 6 replicates; **Mean % Recovery of 3 replicates

Table 4: Results of precision (%Assay)

Figure 4: Linearity of Omeprazole

Figure 5: Linearity of Aspirin