Stability Indicating HPLC Method for the Simultaneous Quantification of Aspirin and Pravastatin in bulk and Tablets : Method Development and Validation

Article history: Received on: 09/12/2016 Accepted on: 07/01/2017 Available online: 30/03/2017 The aim of the present study was to develop and validate a stability indicating HPLC method for the simultaneous determination of aspirin and pravastatin in bulk and pharmaceutical dosage forms. The chromatographic separation of aspirin, pravastatin and their degradation products was achieved on Phenomex C18 column with mixture of water, acetonitrile and acetic acid (40:59:01, v/v/v) as mobile phase in an isocratic elution mode at a flow rate of 1.5 mL/min. The method exhibited linearity in the concentration range of 20.561.5 μg/mL and 10-30 μg/mL for aspirin and pravastatin, respectively. The limits of detection and quantification were 0.204μg/mL and 0.680 μg/mL for aspirin and 0.077 μg/mL and 0.256 μg/mL for pravastatin, respectively. The developed method was validated with respect to accuracy, precision, selectivity, specificity and robustness. All the parameters examined were within the acceptance limits. The stability-indicating power of the proposed method was proved by subjecting the drugs to hydrolytic (acid and base), oxidative, photolytic and dry heat stress conditions. The developed method was found to be suitable for routine analysis of aspirin and pravastatin simultaneous in the presence of its stress degradation products.


Chemicals and solvents
HPLC grade acetonitrile was purchased from Merck Pvt Ltd., Mumbai, India.Analytical reagent grade glacial acetic acid, hydrochloric acid, sodium hydroxide and hydrogen peroxide were obtained from Sd Fine Chemicals Ltd., Mumbai, India.Purified water was obtained from Milli-Q system.Aspirin and pravastatin were kindly provided by Lara Drugs Private Limited (Telangana, India).Pravigard tablets labeled to contain 81 mg and 40 mg of aspirin and pravastatin, respectively are manufactured by Bristol-Myers squibb company, Princeton, New Jersey.

Instrumentation and chromatographic conditions
The Waters HPLC system, consisted of a binary HPLC pump model 2695, PDA detector model 2998 and a vacuum degasser, all controlled by a Waters Empower 2 software, was used.HPLC analysis was performed isocratically at 25 o C temperature using a Phenomex C18 (200 mm × 4.6 mm, 5 µm) column.The mobile phase consisted of a mixture of water, acetonitrile and acetic acid (40:59:01, v/v/v).The flow rate was 1.5 mL/min and injection volume was 20 µL.The eluent was monitored with a detector set at 260 nm.

General procedure
Stock standard solution of aspirin and pravastatin was prepared with mobile phase at a concentration of 410 μg/mL and 200 μg/mL, respectively.Stock solution was kept refrigerated at 4 °C until further use.For the construction of calibration curves, serial portions of the stock solution were separately transferred into 5 sets of 10 mL volumetric flasks to produce working standard solutions of concentrations 20.50 µg/mL, 30.75 µg/mL, 41.0 µg/mL, 51.25 µg/mL and 61.50 µg/mL for aspirin and 10 µg/mL, 15 µg/mL, 20 µg/mL, 25 µg/mL and 30 µg/mL for pravastatin.All the flasks were completed to volume with mobile phase.From these working standard solutions, 20 μL portions of each standard solution were injected into the HPLC column.The peak areas of aspirin and pravastatin were determined by following the operating chromatographic conditions.For each drug, the obtained peak areas were correlated to the corresponding concentrations to construct calibration curves.The regression equation was developed.

Analysis of tablets
For the analysis of aspirin and pravastatin in its pharmaceutical dosage form, twenty tablets were accurately weighed, finely powdered and thoroughly mixed.A quantity of the powdered tablets was precisely weighed so as to contain 41 mg of aspirin and 20 mg of pravastatin.The measured weight was extracted with 50 mL of mobile phase with the aid of sonication for 10 min.The obtained extract was filtered into a 100 mL volumetric flask and the volume was completed to mark with mobile phase.The produced tablet extract solution was labeled to contain 410 μg/mL aspirin and 200 μg/mL pravastatin.5 mL from the aforementioned extract solution was transferred into 50 mL volumetric flask to prepare working sample solution (aspirin -41 mg and pravastatin -20 mg) and the flasks were completed to volume with mobile phase.20 μL of working tablet sample solution was injected into the HPLC system.The peak areas of aspirin and pravastatin were determined by following the optimized chromatographic conditions.The concentration of aspirin and pravastatin in tablet was calculated either from the corresponding calibration curve or regression equation.

Degradation studies
This study was performed by using different ICH prescribed (acidic, basic, oxidative, thermal and photolytic) stress conditions (Q1A (R2) International Conference on Harmonisation, 2003).

Acid degradation studies
For acid degradation study, 5 mL of tablet sample solution (410 μg/mL aspirin and 200 μg/mL pravastatin) was taken and placed in a 50 mL volumetric flask.10 mL of 0.1 N HCl was added to the flask and sonicated for 30 min.After completion of the stress, the solution was neutralized by using 0.1 N NaOH and completed up to the mark with mobile phase.

Base degradation studies
Base degradation study was performed by transferring 5 mL of tablet sample solution (410 μg/mL aspirin and 200 μg/mL pravastatin) to a 50 mL volumetric flask.10 mL of 0.1 N NaOH was added to the flask and sonicated for 30 min.After completion of the stress, the solution was neutralized by using 0.1 N HCl and completed up to the mark with mobile phase.

Oxidative degradation studies
For this study, 5 mL of tablet sample solution (410 μg/mL aspirin and 200 μg/mL pravastatin) was transferred to a 50 mL volumetric flask followed by addition of 10 mL of 30% H 2 O 2 .The contents of the flask were sonicated for 30 min.After completion of stress, the flask was completed up to the mark with mobile phase.

Thermal degradation studies
The thermal degradation study was performed at 105 o C for 30 min in oven.For this study, a quantity of the powdered tablets equivalent to 41 mg aspirin and 20 mg pravastatin was placed in petric disc and exposed to 105 o C for 30 min in oven.After the specified time, the tablet powder was cooled and transferred to a 100 mL volumetric flask containing 50 mL of mobile phase.After sonication for 10 min, the flask was made up to volume with mobile phase.This solution was diluted appropriately with the same solvent to get a final concentration of 41 μg/mL (aspirin) and 20 μg/mL (pravastatin).

Photolytic degradation studies
For photolytic degradation, a quantity of the powdered tablets equivalent to 41 mg aspirin and 20 mg pravastatin was placed in petric disc and exposed to sun light for 24 hrs.After the specified time, the solution was prepared as described in section 'Thermal degradation studies'.All the stress degraded samples were filtered before injection into the HPLC system.The peak areas of aspirin and pravastatin were recorded from the respective chromatogram.

Method development
The study was intended for the effective separation of aspirin, pravastatin and its stress induced degradation products.Various mobile phase combinations of acetonitrile with water, dipotsssium hydrogen phosphate, disodium hydrogen phosphate and glacial acetic acid were investigated to minimize peak tailing, improve peak symmetry, improve column efficiency, improve resolution and minimize total analysis time.Finally a mobile phase consisting of a mixture of water, acetonitrile and glacial acetic acid in the ratio of 40:59:01 (v/v/v) was selected as mobile phase to achieve maximum separation and sensitivity.Different flow rates of mobile phase were studied.A flow rate of 1.5 mL/min gave better results.Different temperatures (15°C -35°C) were evaluated.It was found that the temperature had a negligible influence on resolution and tailing factors.Therefore, room temperature was chosen.Using a reversed phase Phenomex C18 (200 × 4.6 mm, 5 µm) column and optimized HPLC conditions (mobile phase combination, flow rate and column temperature), the retention times for aspirin and pravastatin were observed to be 3.654 min and 4.664 min, respectively.Total time of analysis was <7 min.The chromatogram at 260 nm showed a complete resolution of aspirin and pravastatin peaks (Figure 3).The method was validated as per ICH guidelines (International Conference on Harmonization, 2005).

System suitability
Prior to analysis, the chromatographic system must satisfy system suitability test requirements.The system suitability test was performed by injecting working standard solution containing 41 μg/mL (aspirin) and 20 μg/mL (pravastatin) in five replicates.The percent relative standard deviation (RSD) of the peak area responses and retention times of analytes were determined.Also, USP tailing factor, USP plate count and USP resolution factor were calculated.The results of system suitability in comparison with the required limits are shown in Table 1.The system suitability parameters of the developed method are found to be within the recommended limits.

Linearity
The linearity of the method was evaluated by analyzing standard solutions of aspirin and pravastatin in the range of 20.5-61.5 μg/mL and 10-30 μg/mL, respectively.Calibration curve was constructed by plotting peak area versus concentration of analytes (Figures 4 and 5).The regression equation was calculated.The linear equations for the calibration curves were: Y = 39148 x+17786, R 2 = 0.9999 for aspirin Y = 50402 x + 944.2, R 2 = 0.9998 for pravastatin Where Y = peak area of drug; x = Concentration of drug in μg/mL; R 2 = regression coefficient.

Limits of detection (LOD) and quantitation (LOQ)
LOD and LOQ were calculated as signal-to-noise ratio of 3:1 and 10:1, respectively.LOD and LOQ, respectively, were found to be 0.204 μg/mL and 0.680 μg/mL for aspirin and 0.077 μg/mL and 0.256 μg/mL for pravastatin.The low values of LOD and LOQ indicate the adequate sensitivity of the proposed method.

Selectivity
The chromatograms of mobile phase blank, placebo blank and tablet sample solution were checked with the standard solution for the appearance of any extra peaks.Placebo blank solution was prepared in the same way of the tablet sample solution by common excipients (starch-40 mg, acacia-30 mg, sodium citrate-35 mg, hydroxyl cellulose-40 mg, magnesium stearate-40 mg, talc-30 mg and sodium alginate-30 mg) of the tablet dosage form but without aspirin and pravastatin.In the chromatogram of tablet sample, single peak for aspirin (retention time-3.653min) and pravastatin (retention time-4.624min) was obtained under optimized conditions (Figure 6d).The retention times of aspirin and pravastatin in the chromatograms of standard and tablet sample solutions are same (Figures 6c and 6d).No peaks were observed in the chromatograms of mobile phase blank and placebo blank solutions (Figures 6a and 6b), showing no interference from common tablet excipients and components of mobile phase.

Precision
Precision of the proposed method was studied by making six repeated injections of the standard solution (41 μg/mL-aspirin and 20 μg/mL-pravastatin).Relative standard deviation of aspirin and pravastatin peaks was then calculated to represent precision.The relative standard deviation after six determinations was 0.385% at 41 μg/mL concentration of aspirin and 0.347% at 20 μg/mL concentration of pravastatin (Table 2).The low relative standard deviation (<0.5%) values indicate that the developed method is precise.

Accuracy
Accuracy of the proposed method was established by making six repeated injections of the standard solution (41 μg/mLaspirin and 20 μg/mL-pravastatin).The mean percent recovery of aspirin and pravastatin was calculated to represent the accuracy of the method.The mean percent recovery was 99.58% for aspirin and 99.16% for pravastatin (Table 2).The high percent recovery values indicate good accuracy of the developed method.The accuracy of the proposed method was further assessed by recovery studies at three different concentration levels by standard addition method.For recovery study, fixed amount of standard aspirin and pravastatin was spiked into preanalyzed sample at three different concentration levels (50%, 100% and 150%).The samples were injected into the HPLC system in triplicate and their percent recovery was determined.
The mean percent recoveries obtained for aspirin and pravastatin were in the range of 98.43%-100.16%and 100.06%-100.81%,respectively (Table 3).The results indicated that there was no interference from tablet excipients.

Robustness
Influence of small changes in chromatographic conditions, such as change in flow rate of the mobile phase (±0.1 mL/min) and column temperature (± 5 o C), on the method performance was studied to verify the robustness of the developed HPLC method.The method robustness was determined at a concentration of 41 μg/mL and 20 μg/mL of aspirin and pravastatin, respectively.In all deliberately varied chromatographic conditions, the system suitability parameters were found to be well within the acceptable limit (Table 4) indicating the method robustness.

Forced degradation studies/specificity
The forced degradation study was intended to make sure the effective separation of aspirin, pravastatin and their degradation peaks.Forced degradation studies were performed to assess the stability indicating properties and specificity of the proposed method.Tablet sample solutions for use in stress studies were prepared at a concentration of 41 μg/mL (aspirin) and 20 μg/mL (pravastatin).Forced degradation study was performed by subjecting the analytes to acid hydrolysis, base hydrolysis, hydrogen peroxide mediated hydrolysis, photolytic and dry heat degradation conditions.The results (percent assay, percent degradation, peak purity angle and purity threshold) of degradation studies are summarized in Table 5.The chromatograms of the degraded samples are shown in Figures 7a-7e.The percentage degradation of aspirin is more in hydrogen peroxide mediated oxidative degradation condition and less in alkali hydrolysis.The pravastatin is degraded more in acid hydrolysis and less in hydrogen peroxide mediated oxidative degradation condition.The determination of the peak purity angle and peak purity threshold values demonstrated that the aspirin and pravastatin peaks were pure in all situations.The stability indicating nature and specificity of the method was demonstrated as no degradation products from different stress conditions affected the detection and quantification of aspirin and pravastatin.

CONCLUSION
A simple stability indicating HPLC method with PDA detection for the simultaneous determination of aspirin and pravastatin in bulk and in its pharmaceutical dosage forms has been described for the first time.The proposed method was successfully validated in terms of system suitability, linearity, precision, accuracy, selectivity, specificity, LOD, LOQ and robustness as per the guidelines of ICH.The results of validation parameters were found to be within the acceptable limits.The short run time (≤ 6 min) make the method suitable for quick and routine analysis.The results of forced degradation studies showed that the proposed method is stability indicating and capable of simultaneous quantification of aspirin and pravastatin in the presence of their degradation products.

Table 2 :
Precision and accuracy of the method.

Table 3 :
Recovery of aspirin and pravastatin.

Table 4 :
Robustness of the method.