Validated HPLC Method for the Determination of Nisoldipine

Nisoldipine, (±)3-isobutyl-5-methyl-1,4-dihydro-2,6-dimethyl-4(2-nitrophenyl) pyridine-3,5-dicarboxylate, is a second generation of dihydropyridine calcium antagonist which has a selective arteriolar vasodilatation but shows negligible effects on the other vessels and myocardium [1]. Nisoldipine is a yellow crystalline substance, practically insoluble in water but soluble in methanol. It has a molecular weight of 388.4 g/mol. It is used alone or together with other medicines to treat high blood pressure [2]. Nisoldipine is available as extended release tablets. Figure 1 shows the structural formula of Nisoldipine.


Introduction
Nisoldipine, (±)3-isobutyl-5-methyl-1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl) pyridine-3,5-dicarboxylate, is a second generation of dihydropyridine calcium antagonist which has a selective arteriolar vasodilatation but shows negligible effects on the other vessels and myocardium [1]. Nisoldipine is a yellow crystalline substance, practically insoluble in water but soluble in methanol. It has a molecular weight of 388.4 g/mol. It is used alone or together with other medicines to treat high blood pressure [2]. Nisoldipine is available as extended release tablets. Figure 1 shows the structural formula of Nisoldipine.
Many analytical chemical investigations have been published for the determination of Nisoldipine, including the determination in formulations by voltammetry [3][4][5][6], polarography [7] and HPLC [8][9][10][11][12]. Since Nisoldipine has light sensitivity, its stability, kinetics of degradation and determination of impurities are reported by various techniques including UV [13], polarography [5] and HPLC [14,15]. Therefore, the purpose of this investigation was to develop and validate a method using a simple, rapid, sensitive, precise, accurate and specific reversed phase HPLC-DAD assay. The method uses a simple mobile phase composition and the rapid run time of less than 10 min. Hence, this method can be used for the analysis of large number of samples in quality control laboratories of drugs.

Materials and reagents
Nisoldipine was supplied from NODCAR, Egypt, and its pharmaceutical form Sular tablets were manufactured by Rizhao Highrun Biotechnology Co., Ltd, China. All reagents and chemicals of the highest purity and analytical grade were available. Potassium dihydrogen phosphate and HPLC grade methanol were purchased from VWR. Hexane sulphonic acid sodium salt was obtained from Sigma-Aldrich. A standard stock solution 100 µg/ml of Nisoldipine was prepared in 100 ml methanol as solvent. Working solutions were prepared separately by making serial dilutions from the standard solution to obtain calibration graph in the range of 5-30 µg/ml. These solutions were stored at 20°C. Once prepared, analyzed daily for inter and intra-day variations of the method. 20 µl of these solutions were injected into LC system and chromatographic.

Chromatographic conditions
High performance liquid chromatography experiments were carried out using the model of LC system Agilent 1100 Series with degasser, quaternary pump, auto-sampler, column heater, UVdetector and Chemstation-software. The detector was set to scan from 200 to 500 nm and had a discrete channel set at 275 nm, which was the  wavelength used for quantification. Separation was performed on an Agilent ZORBAX Eclipse Plus C18 with particle size of 10 µm (4.6×250 mm). The mobile phase was consisted of methanol, 0.01 M potassium dihydrogen phosphate aqueous solution and 0.1 M Hexane sulphonic acid sodium salt (25:65:10, v/v) at pH 4.0 using orthophosphoric acid with a flow rate of 1.0 ml/min. Column temperature was set at 25°C and 20 µl of samples was injected to the HPLC system.

Method validation
Validation of an analytical method is the process by which it is established by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical application of International Conference on Harmonization (ICH) guidelines. Validation is required for any new or amended method to ensure that it is capable of giving reproducible and reliable results, when used by different operators employing the same equipment in the same or different laboratories. The type of validation program required depends entirely on the particular method and its proposed applications. Typical analytical parameters used in assay validation include: linearity range, precision, accuracy, limit of detection, limit of quantification, robustness, selectivity and specificity.
Linearity range: Calibration curve was determined for six different concentrations of standard solution of the drug analyzed in the proposed range of 5-30 µg/ml. Each calibration was injected three times. The calibration curve was performed in triplicate. Calibration curves were calculated applying the least-squares method to concentration versus peak area. Each plot as a linear relationship between concentration and peak area was observed in the range of study.
Precision: Precision, expressed in terms of %RSD, was determined in terms of intraday and interday precisions, analyzing the drug at three different concentrations, determining each concentration thrice, and reflects a high degree of precision. In other words, Intra-day precision was determined by analyzing Nisoldipine (5-30 µg/ml) at three different time points on the same day whereas interday precision on different days and %RSD was calculated.
Accuracy: Accuracy was determined by performing recovery studies by the standard addition method by spiking different concentrations of pure drug in the solution containing Nisoldipine powder for tablet samples within the analytical concentration range of the proposed method at three different set at level of 50%, 100% and 150%. The amount of Nisoldipine was calculated at each level and % recoveries were computed. The results were within the specified limits of ICH guidelines.

Limit of detection (LOD) and limit of quantification (LOQ):
The LOD and LOQ were calculated by using the following equations: LOD=3 S.D./m and LOQ=10 S.D./m, where "S.D." is the standard deviation of the intercept of the calibration curve and "m" is the slope of the calibration curve [16].
The LOD and LOQ of Nisoldipine were 0.4 and 1.0 µg/ml, respectively.

Robustness:
The robustness of an analytical procedure refers to its ability to remain unaffected by small and deliberate variations in method parameters and provides an indication of its reliability for the routine analysis. Robustness was carried out by varying three parameters (deliberate change) from the optimized chromatographic conditions like mobile phase composition (± 2 ml methanol), flow rate (± 0.1 ml/ min.) and pH (± 0.20). No significant change was observed. Also, to assess the stability of sample solutions of Nisoldipine, the samples tested were maintained at 2-8ºC for 24 h and also placed into the auto sampler, the stability of these solutions was studied by performing the experiment and observing any change in the chromatographic pattern, compared with freshly prepared solutions.
Selectivity and Specificity of the method were determined by screening five batches of drug. The extraction recovery of Nisoldipine was calculated by comparing the peak area ratio measured for the standard solution with that obtained for drug in dosage form. Results from this testing studies indicated the method was enabled highly selective analysis of the drugs. Because the specificity or selectivity of a developed method is often difficult to ensure, several simple and easy techniques are used in method validation experiments to increase confidence in selectivity (peak purity) [17]. Results from these procedures confirmed the method was selective and specific.
System suitability test: The system suitability test was also carried out to evaluate the resolution and reproducibility of the system for the analysis, using five replicate injections of a reference solution containing 10 µg/ml of Nisoldipine. The parameters measured were peak area, retention time, theoretical plates and tailing factor (peak symmetry).

Procedure for tablets
Twenty tablets of formulation (Sular tablet) were powdered finely and an amount equivalent to one tablet of Nisoldipine was weighed and then dissolved in the methanol. Solutions were then filtered through ordinary filter paper. The desired concentrations in the range of 5-30 µg/ml for Nisoldipine were obtained by accurate dilution and these solutions were then sonicated. Finally, all the solutions were filtered through 45 μm Millipore™ filter, in order to separate out the insoluble excepients before chromatographed.

Results and Discussion
The present work describes development and validation of HPLC method for the determination of Nisoldipine in the bulk powder and in the pharmaceutical tablet formulations.
The mobile phase for the assay of Nisoldipine was optimized and selected by taking different proportions of aqueous and organic phases which gave acceptable asymmetry and theoretical plates with appropriate run time. From the different mobile phases tried mobile phase consisting of methanol, 0.01 M potassium dihydrogen phosphate aqueous solution (25:65, v/v) was found to be satisfactory. However due to amino group present in the structure and the polymeric nature, the chromatogram displayed a tailing peak of Nisoldipine, which was avoided by adding 10% v/v of 0.1 M Hexane sulphonic acid sodium salt. The pH of the mobile phase was also optimized since it is a basic drug, so ionization of the drug was found at pH 4.0 using orthophosphoric acid, where the drug gave symmetric and sharp peak for Nisoldipine at 1.0 ml/min. as flow rate with good theoretical plates and acceptable tailing factor on Agilent ZORBAX Eclipse Plus C18 (4.6×250 mm) with particle size of 10 µm. For quantitative analytical purpose wavelength was set at 275 nm, which provided better reproducibility with minimum interference. Under the chosen experimental conditions, the liquid chromatogram of Nisoldipine showed a single peak of the drug at retention time (RT) 7.43 min with asymmetry of 0.91 (Figure 2).

System suitability
The system suitability was evaluated by calculating the %RSD values of peak area, retention time, asymmetry and theoretical plates of  (Table 1) showed that the values were within the acceptable range indicating that the system was suitable for the intended analysis.
Linearity range: The calibration range was established by considering the practical range necessary for assay and to give accurate and precise results with good linearity. Detector response (area of peak) was plotted against concentration to obtain calibration curves. Calibration range of drug concentration of Nisoldipine was as (5,10,15,15,20,25 and 30 μg/ml) in methanol as solvent. Regression analysis was carried out on calibration curve and results are summarized in Table 2. Linearity of the calibration curve shown in Figure 3 and the adherence to Beer's law were validated by the high value of the correlation coefficient (Figure 3).

Limit of detection and limit of quantification:
The parameters LOD and LOQ were determined on the basis of signal to noise ratio, LOD and LOQ were calculated by the method which was based on the standard deviation (S.D.) of the response and the slope (S) of the calibration curve at levels approximating the LOD and LOQ were found to be 0.4 and 1.0 µg/ml, respectively ( Table 2).
Precision: Intra-day and inter-day precision of the assay samples containing Nisoldipine (5, 10, 15, 20 and 25 μg/ml) were analyzed five times in the same day (intraday) and for three consecutive days by different analysts. Precision was calculated as intra and inter-day coefficient of variation (CV) as shown in Table 3.
Accuracy: Accuracy of the method was studied by applying the developed method to prepared synthetic mixtures of tablet excipients to which known amount of Nisoldipine corresponding to 50-150% of the labeled claim had been added. The mean recovery values for Nislodipine were shown in Table 4, indicating that the developed method was accurate for the determination of Nisoldipine in pharmaceutical formulation (Table 4).

Robustness:
The robustness of the method was evaluated by assaying the same sample under different analytical conditions deliberately changed from the original analytical condition. The results obtained were not affected by varying the conditions and were in accordance with the results for original conditions ( Table 5). The % RSD value (0.51) of assay determined for the same sample under original conditions and with all the conditions of robustness indicates that the developed method was robust and not affected by deliberate changes in the method parameters.

Conclusion
The results of the validation studies show that the LC method is specific, accurate and possesses significant linearity and precision      characteristics without any interference from the excipients, demonstrating the advantages of the LC technique, very well established for the quality control of most of the pharmaceuticals due to its simplicity, high resolution and satisfactory precision and accuracy. Therefore, the proposed method was successfully applied and suggested for the quantitative analysis of Nisoldipine in the bulk powder and in the pharmaceutical dosage forms, contributing to improve the quality control and to assure the therapeutic efficacy.

Future scope for work on Nisoldipine
Simply the current method, the probable degradation pathways of the drug in different stressed conditions can be predicted by analyzing the stressed samples by LC-method, whereby molecular weights and the fragmentation results of the degradation products of Nisoldipine will confirm the most probable degradation pathways of the drug.