Vani et al.

Universal Journal of Pharmaceutical Research 2017; 2(4):25-28

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Universal Journal of Pharmaceutical Research
An International Peer Reviewed Journal
ISSN: 2831-5235 (Print); 2456-8058 (Electronic)
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RESEARCH ARTICLE

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.

Article Info: Abstract

_______________________________________________ ____________________________________________________________________________________________________

Article History: Objectives: A new simple, accurate, precise and reproducible RP-HPLC method
Received: 22 May 2017 has been developed for the simultaneous estimation of Aspirin and Omeprazole in
Reviewed: 2 July 2017 bulk and pharmaceutical dosage form using C18 column (Agilent, 250 x 4.6 mm, 5
Accepted: 28 August 2017 μm) in isocratic mode.
Published: 15 September 2017 Methods: 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
Cite this article: 256 nm. The method was linear over the concentration range for Omeprazole 50-
Vani R, Sunitha M. Analytical method 250 μg/ml and for Aspirin 10-50 μg/ml.
development and validation for the Results: The recoveries of Omeprazole and Aspirin were found to be 100.07 and
determination of Omeprazole and Aspirin using 100.06% respectively. The validation of method was carried out utilizing ICH-
reverse phase HPLC method in bulk and dosage guidelines. The described HPLC method was successfully employed for the
form. Universal Journal of Pharmaceutical analysis of pharmaceutical formulations containing combined dosage form.
Research 2017; 2(4): 25-28. Conclusion: Study concludes that the proposed method is accurate, precise, rapid
http://doi.org/10.22270/ujpr.v2i4.R6
and selective 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
*Address for Correspondence:
R Vani, Shadan Women’s College of Pharmacy, of their dosage form.
Hyderabad, India, Tel: +91-9885685042 E-mail: Keywords: Aspirin, Omeprazole, reverse phase HPLC, validation.
vrathipelli@gmail.com

INTRODUCTION HPTLC have been reported for ASP in single form and
in combination with other drugs12. Several analytical
Aspirin (ASP) is chemically 2-(acetyloxy)-benzoic acid methods have been reported for ESO in single form
(Figure 1). It is nonselective cyclooxygenase inhibitor and in combination with other drugs including
used as an antipyretic, analgesic, anti-inflammatory, spectrophotometry13,14, HPLC15,16, and HPTLC17.
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
disorders1-3
Figure 2: Structure of Esomeprazole.

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
Figure 1: Structure of Aspirin. was validated in accordance with ICH Guidelines and
The review of literature revealed that various analytical successfully employed for the assay of ASP and OMP
methods involving spectrophotometry5-7, HPLC8-11 and combine dosage form1,18.

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Vani et al. Universal Journal of Pharmaceutical Research 2017; 2(4):25-28

MATERIALS AND METHODS Number of mobile phase and their different proportions
were tried and finally was selected as 0.1 M Di-
ASP and OMP were received gratis from Hetero drugs, potassium Phosphate buffer (pH 3) and Methanol in the
Hyderabad and were used as received. HPLC grade ratio of 40:60 v/v appropriate mobile phase which gave
Methanol was purchased from SD Fine Chem Pvt. Ltd. good resolution and acceptable system suitability
(Mumbai, Maharashtra). Ultra-pure water was obtained parameters. The results of system suitability parameters
from ELGA (Bucks, UK) water purification unit. were shown in Table 2. The chromatogram of working
Waters total recovery vials (Waters, Milford, MA, standard solution is shown in Figure 3. The summary
USA) were of glass type 1, class A with 950 μL of Chromatographic conditions was given in Table 1.
maximal injectable volumes. All other chemicals were
of analytical reagent grade. Table 1: Summary of chromatographic conditions.
Chromatographic conditions Parameter Description/ Value
The HPLC system (LC Waters, Milford, MA, USA) Stationary Phase Water’s C18 (250X4.6X5)
consisted of quaternary gradient system (600 0.1 M Dipotassium Phosphate
Controller), in-line degasser (Waters, model AF), Mobile Phase buffer (pH 3) and Methanol in
photodiode array detector (Water, 2998 model) and the ratio of 40:60v/v
auto sampler (Waters, model 717 plus). Data was Flow rate 1 ml/min
processed using Empower Pro software (Waters, Detection
256 nm
Wavelength
Milford, MA, USA). Isocratic elution of the mobile Detector Photo diode array
phase 0.1 M Di-potassium Phosphate buffer (pH 3) and auto sampler-Waters, model
Methanol in the ratio of 40:60 v/v with the flow rate of Injection
717 plus
1ml/min. Separation was performed on a Waters C18 Omeprazole– 2.323Min
(250x4.6 mm i.d, 5 μ particle size) analytical column Rt’s
Aspirin– 4.342 Min
and a pre-column to protect the analytical column from Injection
10μl
strongly bonded material. Integration of the detector volume
output was performed using the Waters Empower Column
35°C
software to determine the peak area. The contents of Temperature
the mobile phase were filtered through a 0.45 µm Run time 6 min
Diluent Mobile Phase
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 110–112 kg/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. Figure 3: Typical Chromatogram of Omeprazole
Preparation of standard solutions and Aspirin.
ASP and OMP were weighed (10 mg each) and
transferred to two separate 10 ml volumetric flasks and Table 2: System suitability parameters.
dissolved in 5 ml of water and make up the volume up Parameter Result
to the mark with mobile phase. Working standards of Omeprazole Aspirin
the drugs were prepared from this solution. Retention Time 2.323 min 4.325 min
Preparation of sample solution Tailing 1.079 1.189
Theoretical Plates(n) 5076 7837
Twenty tablets (Yosprala, Make: Aralez
Pharmaceuticals) were weighed. An accurately Resolution factor(R) 3.08
weighed amount of the finely powdered tablets Similarity Factor 1.0124 (Limit: 0.98–1.2)
equivalent to 10mg was made up to 10 ml with mobile
phase. The solution was filtered followed by serial Method Validation
dilution to the required concentrations for each Accuracy
experiment. Recovery assessment was obtained by using standard
addition technique which was by adding known
RESULTS AND DISCUSSION quantities of pure standards at three different levels in
50%, 100% and 150% to the pre analyzed sample
Method Development formulation.

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Vani et al. Universal Journal of Pharmaceutical Research 2017; 2(4):25-28

Table 3: Results of accuracy.

% Concentration Omeprazole Aspirin
(at specific level) Amount Amount Mean % Amount Amount Mean %
added (µg) found (µg) Recovery added (µg) found (µg) Recovery
50 75 75 100* 15 15 100*
100 150 149.25 99.13** 30 30 100**
150 225 224.89 99.69* 45 44.55 99*
*Mean % Recovery of 6 replicates; **Mean % Recovery of 3 replicates

From the amount of drug found, amount of drug 10:1, respectively according to International
recovered and percentage recovery were calculated Conference on Harmonization guidelines.LOD values
which sense to conformation that the proposed method for ASP and OMP were found to be 3.08and
was accurate. The results were tabulated in Table 3. 3.041µg/ml respectively. LOQ values for ASP and
Precision OMP were found to be 9.24 µg/ml and 10.37 µg/ml
The intraday and inter day precision of the proposed respectively.
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.

Table 4: Results of precision (% Assay).

Sample Omeprazole Aspirin
No. Sample % Sample %
Area - 1 Assay-1 Area-2 Assay-2
1 2194758 100.06 1456296 100 Figure 5: Linearity of Aspirin.
2 2195700 99.49 1457422 100
3 2196191 99.14 1456513 98
4 2195326 100.27 1454579 99 Assay of the tablet dosage form
5 2200951 100.27 1451483 99 The proposed validated method was successfully
6 2196585 100.39 1455259 99 applied to determine ASP and OMP in tablet dosage
Average 100 Average 99 form. The result obtained for ASP and OMP were
Assay: Assay: comparable with corresponding labeled amounts. The
STD 0.51 STD 0.82
results were tabulated in Table 4.
% RSD 0.51 % RSD 0.83
CONCLUSIONS

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
Figure 4: Linearity of Omeprazole. adopted for routine analysis.
Linearity ACKNOWLEDGEMENTS
Calibration graphs were constructed by plotting peak
area vs concentration of ASP and OMP and the The authors are grateful to Principal, Management of
regression equations were calculated. The calibration Shadan Women’s College of Pharmacy, Hyderabad,
graphs were plotted over 5 different linear India for providing necessary facilities to carry out this
concentrations in the range of 10-50 µg/ml for ASP research project. Authors are thankful for Hetero drugs,
and 50-250 µg/ml for OMP. Aliquots (10 µl) of each Hyderabad, AP for kindly providing the gift sample of
solution were injected under the operating OMP and ASP.
chromatographic condition described above [Number
of replicates (n=6)]. The linearity graphs were shown AUTHOR’S CONTRIBUTION
in Figure 4 and Figure 5.
Limit of detection (LOD) and limit of quantitation Vani R: writing original draft, methodology,
(LOQ): investigation, formal analysis, data curation,
The limit of detection (LOD) and limit of quantitation conceptualization. Sunitha M: writing, review and
(LOQ) of ASP and OMP were determined by editing, methodology, formal analysis, data curation,
calculating the signal-to-noise (S/N) ratio of 3:1 and

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Vani et al. Universal Journal of Pharmaceutical Research 2017; 2(4):25-28

conceptualization. All the authors approved the method for determination of atorvastatin calcium and
finished version of the manuscript. aspirin in a capsule dosage form. Indian J Pharm Sci
2007; 69 (4): 546–549.
https://doi.org/10.4103/0250-474X.41473
DATA AVAILABILITY 9. Montgomery ER, Taylor S, Segretario J, Engler E,
Sebastian D. Development and validation of a reversed-
The data and material are available from the phase liquid chromatographic method for analysis of
corresponding author on reasonable request. aspirin and warfarin in a combination tablet formulation,
J Pharm Biomed Anal 1996; 15 (1): 73–82.
CONFLICTS OF INTEREST https://doi.org/10.1016/0731-7085(96)01813-4
10. Deconinck E, Sacré PY, Baudewyns S, Courselle P, De
Beer J. A fast ultra high pressure liquid chromatographic
The authors declared no conflicts of interest. method for qualification and quantification of
pharmaceutical combination preparations containing
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