Manuscipt

THE RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF ASPIRIN AND
OMEPRAZOLE IN BINARY COMBINATION

Kashid A. M*., Sahoo S. G., Tathe S. V., Ghatge A. B., Wani R. M.
Correspondence address:
Department of Pharmaceutical Chemistry,
Sinhgad Institute of Pharmacy, Narhe, Pune, 411041, Maharashtra, India
Tel: 9011004372
E-mail: [email protected]
ABSTRACT
A simple, accurate, rapid and precise RP-HPLC method for the simultaneous estimation of
Aspirin and Omeprazole in binary mixture has been developed and validated. Separation of
drugs was achieved from HPLC Column (ACE 250 x 4.6 mm C 18 column) with a mobile phase
consisting of HPLC grade Methanol: Sodium acetate Buffer (70:30 v/v) at a flow rate of 1
ml/min with UV detection at 230 nm. The method was validated with respect to linearity,
sensitivity, accuracy, precision and robustness as per the International Conference on
Harmonization (ICH) guidelines. The method was specific and it was observed that no
interference with diluents.
The retention times of Aspirin and Omeprazole were 3.10 ± 0.3min and 5.01 ± 0.02min
respectively. The linearity was established over the concentration range of 40-140μg/ml and 20-
120μg/ml with correlation coefficients (r 2) 0.9998 and 0.9986 for Aspirin and Omeprazole
magnesium respectively. The mean recoveries were found to be in the range of 97.05%-99.75%
and 97.21% -98.76% for Aspirin and Omeprazole respectively. Conclusion: The % R.S.D. values
for intraday precision study and inter-day study were <1.0%, confirming that the method was
sufficiently precise. The method can be successfully employed for the simultaneous
determination of Aspirin and omeprazole in binary mixture.
Keywords: Aspirin, Omeprazole, HPLC, Simultaneous determination, 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 (OME) is chemically 6-methoxy-2-[(4-
methoxy3,5dimethylpyr-idin-2yl)methan-esulfinyl]-1H-1,3benz- odiazole (Figure 2).It is used in
treatment of peptic ulcer disease, NSAIDS associated ulceration and Zollinger-Ellison syndrome,
used as anti-ulcerative. ASP and OME in combined dosage form are is indicated for patients who
require aspirin for secondary prevention of cardiovascular and cerebrovascular events and who
[1, 2]
are at risk of developing aspirin associated gastric ulcers . The review of literature revealed
that various analytical method involving spectrophotometry [3-5], HPLC [5-7]
and HPTLC [8] have
been reported for ASP in single form and in combination with other drugs. Several analytical
methods have been reported for OME in single form and in combination with other drugs
including spectrophotometry, HPLC [10-14], and HPTLC [15].
The present work describes the development of a simple, precise, accurate, and reproducible
HPLC method for the simultaneous estimation of ASP and OME in binary mixture. The
[17, 18, 19]
developed method was validated in accordance with ICH Guidelines and successfully
employed for the assay of ASP and OME combine dosage form.
Materials and methods
Chemicals and Reagents
Pure drug samples of Aspirin and Omeprazole were provided by pelltech heath care, Mumbai,
Maharashtra and Methanol HPLC grade were manufacture by Merk Pvt. Ltd.
Chromatographic Conditions
The mobile phase consisted of methanol: sodium acetate buffer PH 5.5 in the ratio of 70:30 (v/v),
flowing through the column at a constant flow rate of 1 ml/min. An ACE C 18 column (250 x
4.6mm) was used as the stationary phase. By considering the chromatographic parameter,
sensitivity and selectivity of method for two drugs, 230 nm was selected as the detection
wavelength for UV-PDA detector. The HPLC system (Shimadzu) was operated at room
temperature 25 °C.
Preparation of Standard Stock Solutions
Accurately weighed aspirin (10 mg) was transferred to 10 mL volumetric flask, dissolved in and
diluted with methanol up to the mark to obtain (1000μg/mL). This solution was further diluted
with methanol to obtain final concentration of ASP 100μg/ml. For preparation of OME stock
solution, accurately weighed omeprazole (10 mg) was transferred to 10 mL volumetric flask,
dissolved in and diluted with methanol up to the mark to obtain (1000μg/mL). For preparation of
working standard solution, 2 ml of stock solution of OME (100μg/ml) and 4 ml of stock solution
of ASP (100μg/ml) were transferred to 10 ml volumetric flask and diluted with methanol up to
the mark to obtain final concentration containing 20μg/ml of OME and 40μg/ml of ASP.
Preparation of Sample Solution

Powder mixture equivalent to 80 mg of aspirin and 40 mg of omeprazole was transferred in
100ml volumetric flask containing 50 mL methanol, sonicated for 5 min and diluted to mark with
same solvent to obtain 0.8 mg/ml of ASP and 0.4mg/ml of OME. The resulting solution was
filtered using watmann filter paper. From the above solution 1mL was transferred into 10mL
volumetric flask and diluted to mark with same solvent. So, Resultant solution was found to
contain 40μg/ml of omeprazole and 80μg/ml of aspirin
RESULT AND DISCUSSION

Method Development
The HPLC procedure was optimized for simultaneous determination of ASP and OME. The
developed analytical method consist mobile phase of Methanol: Sodium acetate buffer PH 5
(70:30 v/v) resulted in good resolution and sharp and symmetrical peaks. Using a reversed-phase
C18 column, the retention times for ASP and OME were observed to be 3.09 ± 0.3 for ASP and
5.1 ± 0.05 for OME respectively. Total time of analysis was 10 min (Fig.4) The maximum
absorption of ASP and OME together as detected at 230 nm and this wavelength was chosen for
the analysis (Fig. 3)
Method validation:
The method was validated according to the ICH guidelines. The following validation
characteristics were addressed: linearity, accuracy, precision, specificity, limits of detection and
quantitation.
System suitability
The suitability of the chromatographic system was tested. Five replicate injections of standard
preparation were injected and resolution, Retention time, No. of theoretical plate and Tailing
factor were determined as shown in Table I.
Linearity:
From the sample solution various aliquots were pipetted out to prepare six standard solutions
covering the concentration range of 40-140 μg/ml for ASP and 20-120 μg/ml for OME
Calibration curve showing concentration versus peak area was plotted and the data obtained was
subjected to regression analysis using the least square method. The result of linearity and
calibration curve is shown in Table II, III and Fig. 6, 7.
Precision:
Repeatability
Repeatability of sample application was assessed by injecting 100μg/ml and 80μg/ml of drug
solution of Aspirin and omeprazole respectively six times and measurement of peak areas and
retention times of solutions for ASP and OME without changing the parameter of the proposed
chromatographic method. The result of Repeatability is shown in Table IVA.
Intermediate precision
The method was also validated for both Intraday and Interday precision, by injecting replicate
injections six times with similar concentrations on the same day and on six different days. The
result of reproducibility was reported in terms of % RSD shown in Table IVB and C.
Accuracy /recovery study:

The accuracy of the method was determined by calculating the recoveries of ASP and OME by
the standard addition method. Known amounts of standard solutions of ASP and OME were
added at 80%, 100% and 120% concentration to pre quantified sample solutions of ASP and
OME and the amount of drug recovered was estimated. The Result of Accuracy is shown in
Table VA and B.
Robustness:
In this method, wavelength (± 2 nm) and flow rate (± 0.1 mL/min) were slightly changed to
lower and higher sides of the actual values to find if the change the peak area and retention time
were within limits. Evaluate the system suitability values as required by the test method for both
the altered parameters as shown in Table VIA and B.
Sensitivity
The limit of detection (LOD) of the method refers to that minimum concentration of the active
component that can be effectively estimated based on visual evaluation and the limit of
quantitation (LOQ) is the lowest amount of analyte in a sample, which can be quantitatively
determined with suitable precision and accuracy.
LOD= 3.3 σ/S, LOQ = 10 σ/S
Where, σ = the standard deviation of the response, S = the slope of the calibration curve
LOD and LOQ were determined from the standard deviations of the responses for six replicate
determinations as shown in Table VII.
Specificity & selectivity

Commonly used excipients (starch, microcrystalline cellulose and magnesium stearate, lactose,)
were spiked in to a pre weighed quantity of drugs .The chromatogram was taken by appropriate
dilution and the quantities of drug were determined. The specificity of the HPLC method is
illustrated in Fig. 5. Where complete separation of ASP and OME in presence of excipients.
Analysis of ASP and ESO in synthetic mixture

Content of ASP and OME found in the synthetic mixture from the proposed method are shown in
Table VIII. The % purity was 99.23% for ASP and 99.88% for OME.
CONCLUSION
A simple, precise, reliable, sensitive and accurate reverse phase HPLC method has been
development for simultaneous determination of ASP and OME. The method was validated as per
International Conference on Harmonization (ICH) guidelines . The developed method is suitable
for identification and quantification of binary combination of ASP and OME. High percentage of
recovery and run time less than 6 minutes allow its application for routine determination of ASP
and OME in Tablet dosage form
ACKNOWLEDGEMENT
Authors are thankful to Pelltech Healthcare PVT. LTD, Mumbai, India for providing gratis
sample of Aspirin & Omeprazole to complete this work successfully.
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THE RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF ASPIRIN AND

OMEPRAZOLE IN BINARY COMBINATION

Department of Pharmaceutical Chemistry,

Sinhgad Institute of Pharmacy, Narhe, Pune, 411041, Maharashtra, India

Keywords: Aspirin, Omeprazole, HPLC, Simultaneous determination, Validation

Preparation of Sample Solution

RESULT AND DISCUSSION

Accuracy /recovery study:

Specificity & selectivity

Analysis of ASP and ESO in synthetic mixture

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