Determination of Nifedipine by Validated RP-HPLC Method in Bulk and Pharmaceutical Dosage Form

The present paper deals with the development and validation of reverse phase HPLC method for the determination of Nifedipine on Nucleosil 100, 5 μm, C 8 , 250 x 4.0 mm column. A mobile phase consisting of 40 ml 2-propanol: 60 ml phosphoric acid 0.85% was employed in this study. The flow rate was kept at 0.8 ml/min and the injection volume was 10 µl. The separation was performed at 40°C. Eluents were monitored by UV detector set at 237 nm. The developed method was statistically validated for the linearity, precision, robustness, specificity and solution stability. The specificity of the method was ascertained by force degradation studies by acid and alkali hydrolysis, oxidation, heat and photo degradation. The degraded products were well resolved from the analyte peak with significant differences in their retention time values.


Reagents and chemicals used
HPLC grades Methanol, AR grade ortho phosphoric acid, Hydrochloric acid, Sodium hydroxide, Hydrogen peroxide was procured from E. Merck.
Note: Nifedipine is extremely light sensitive, especially in solution.Hence, all labor must be done protected from light and as fast as possible.

Preparation of standard stock solution
Accurately, about 20 mg of standard Nifedipine was weighed and transferred to a 100 ml volumetric flask with 65 ml 2-Propanol and sonicated for 10 minutes cooling with ice.Equilibrated to room temperature and made up to graduation with ortho phosphoric acid 85%.The final concentration for the standard solution is 200 µg/ml.

Selection and preparation of mobile phase
Pure drug of Nifedipine was injected into the HPLC system and run in different solvent systems.It was found that 2-propanol and phosphoric acid 0.85% gives satisfactory results as compared to other mobile phases.Finally, the optimal composition of the mobile phase employed was 40 ml 2-propanol: 60 ml phosphoric acid 0.85%.The prepared mobile phase was ultrasonicated for 20 mins.

Selection of analytical wavelength
By appropriate dilution of standard stock solution with mobile phase, various concentrations of Nifedipine were prepared separately.The solutions were scanned using the double beam UV visible spectrophotometer in the spectrum mode between the wavelength ranges of 400 nm to 200 nm.The λmax of Nifedipine was found to be 237 nm which was selected as the analytical wavelength for further analysis.

System suitability
System suitability parameters were calculated at the start of study of each validation parameter.The values of system suitability results obtained during the entire study are recorded in Table 1.Linearity was determined at five levels over the range of 70% to 130% of test concentration.A standard Linearity solution was prepared to attain concentration of 70%, 80%, 100%, 120%, and 130% of the test concentration.Each linearity solution was injected in triplicate.The mean area at each level is calculated and a graph of mean area versus concentration is plotted.The correlation co-efficient (r), Yintercept, slope of regression line, residual sum of squares is calculated and recorded in Table 2.The plot of peak area response against concentration is presented in Fig 2 .The Beer Lambert's law was obeyed in the concentration range of 70% to 130% for Nifedipine.The linearity of calibration graphs and adherence of the system to Beer's law was validated by high value of correlation coefficient.(r 2 = 0.9989).

Specificity
The specificity of the HPLC method was determined by complete separation of Nifedipine in the presence of its degradation products.There was no interference from sample and its degraded products the peak purity of Nifedipine is 0.9991.It shows that developed analytical method is specific for the analysis of Nifedipine.

Precision
The precision of the method was established by carrying out the analysis of the analyte (n=6) using the proposed method.The chromatogram for standard was given in Fig. 3.The value of standard deviation shows that the method is precise.The results obtained are presented in Table 3.

Recovery studies
To check the accuracy of the proposed method, recovery studies were carried out at 70, 100 and 130 % of the test concentration as per ICH guidelines.The recovery study was performed three times at each level.The results of the recovery studies are given in Table 4.

Robustness of method
The robustness of the developed method was studied by making small deliberate variations in the method parameters such as the small components in the mobile phase, flow rate, wave length and the

Ruggedness
Ruggedness test was determined between two different analysts, instruments and columns.The value of percentage RSD was below 2.0%, exhibits the ruggedness of developed analytical method and results are presented in Table 6.

Solution stability studies
The sample solution was prepared at test concentration and initial assay was determined.Solution was stored up to 24 hours at room temperature and about 4°C and assay was determined at 4 hours, 8 hours, 12 hours and 24 hours against freshly prepared standard and also analyzed about 4 °C at 24 hours.The assay obtained at different time intervals was compared with the initial assay value and recorded.The relative standard deviation was found below 2.0%.It proves that both standard and sample solutions are stable up to 24 hours at room temperature and at 4 °C.

Analysis of the marketed formulation
To determine the content of Nifedipine in conventional tablets [CALCIGARD RTD-tab (Torrent Pharmaceuticals Ltd.) label claim: 20 mg/tab], twenty tablets of Nifedipine were weighed; their average weight was determined and crushed to fine powder.The tablet powder equivalent to 20 mg of Nifedipine was weighed and transferred to a 100 ml volumetric flask with 65 ml 2-Propanol and sonicated for 10 minutes cooling with ice.Equilibrated to room temperature and made up to graduation with ortho phosphoric acid 85%.The solution was filtered through a 0.22 μ membrane filter.The final nline at: le o b ila Ava -99 -concentration for the standard solution is 200 µg/ml.A 10 μl volume of sample solution was injected into the sample injector of HPLC under the optimized chromatographic conditions.Area of peak was measured at 237 nm.The amount of drug present in the sample was determined using the prepared calibration curve of standard Nifedipine.

Results and Discussion
Nifedipine is an important calcium channel blocker with peripheral and coronary vasodilator activity.Literature survey reveals that there is no stability indicating HPLC method reported so far for the determination of Nifedipine.Keeping this point into consideration an attempt was made develop a simple and accurate RP-HPLC method to determine Nifedipine in presence of its degradation products.
The mobile phase consisting of 40 ml 2-propanol: 60 ml phosphoric acid 0.85% was employed in this study.The flow rate was kept at 0.8 ml/min and the injection volume was 10 µl.The separation was performed at 40°C.Eluents were monitored by UV detector set at 237 nm.The separation was carried on on Nucleosil 100, 5 μm, C8, 250 x 4.0 mm column.The run time was set at 20 min.The retention times of Nifedipine was found to 9.35 mins (Fig No .2).The peak areas of the drug were reproducible as indicated by the low coefficient of variation.The amount of drug found was between 98 to 102%.The sample recoveries in formulation were in good agreement with their respective label claim which suggested non-interference of formulation excipients in the estimation.Also, the % RSD for both the tablet analysis and recovery studies was less than 2 % indicating high degree of precision and accuracy of the proposed method.The mean % assay of intra-day and inter-day precision was found to be 100.11and98.92 respectively.The results of the robustness study also indicated that the method is robust and is unaffected by small variations in the chromatographic conditions.

Conclusion
Also, the developed method was capable of determining Nifedipine in presence of its degradation products.The solutions of both standard and sample solutions are stable up to 24 hours at room temperature and at 4 °C.Hence, it can be concluded that the developed RPHPLC method is a stability indicating simple, accurate, precise and robust method and can be employed successfully for the estimation of Nifedipine in bulk and formulation.

Table 1 .
System Suitability and System Precision

Table 2 .
Characteristics Of The Analytical Method Derived From The Standard Calibration Curve Fig. 2: Linear calibration curve for Nifedipine.

Table 5 .
temperature.The solution containing 200 µg/mL of Nifedipine was injected into sample injector of HPLC under the different conditions.The results of the robustness study are given in Table5.Method Robustness Determination of Nifedipine by Validated RP-HPLC Method in Bulk and Pharmaceutical Dosage Form online at: https://jazindia.com-98 -column