Stability Indicating Reverse Phase High-Performance Liquid Chromatographic Method for the Analysis of Azelnidipine in Bulk Drug and Pharmaceutical Dosage Forms

Introduction

The chemical formula for azelnidipine 3-(1-diphenylmethylazethidin 3-yl) 5- isopropyl 12-amino-1,4 dihydro-6-methyl-4-(3nitrophenyl) 3,5-pyri-dinedicarboxylate.¹ It is used to treat hypertension in clinical circumstances.² A literature review indicates that several analytical methods have been documented for the estimation of azelnidipine. However, their number is limited. These include a novel analytical technique for measuring the calcium channel blocker azelnidipine and method validation using RP-HPLC.^3,4 Other documented approaches include an innovative liquid chromatography-electrospray technique for simultaneous estimation of Olmesartan and azelnidipine in human plasma, the application of ionization tandem mass spectrometry for pharmacokinetic studies, and the development of a UV spectrophotometric method for pharmaceutical dosage forms.^5,6 Additionally, RP-HPLC methods have been used to validate azelnidipine straightforwardly, including simultaneous measurement of aelmisartan and azelnidipine in tablet dosage form.^7-9 Other techniques involve UV-visible spectroscopy, a mathematically processed UV spectroscopic method for azelnidipine and Chlorthalidone quantification, and a stability-indicating RP-HPLC method for simultaneous estimation of telmisartan and azelnidipine in fixed-dose combinations.^10-12 Overall, the literature review suggests that limited analytical techniques are available for estimating azelnidipine using RP-HPLC in combination dosage forms. The primary goal of this research is to create an RP-HPLC technique that is very simple, sensitive, quick, accurate, and precise for determining the amount of azelnidipine in pharmaceutical dosage forms.

Materials and Methods

Equipment: Compact HPLC (WATERS Model No. 486 series system) with Inertsil-C18 ODS column, electronic balance (Sartorius), digital pH meter (POLOMAN), sonicator (FAST CLEAN), and PDA detector.

Reagents and chemicals: All of the chemicals utilized were of an analytical variety. As a gift sample, azelnidipine in bulk was acquired from Moleculochem Pvt. Ltd (Ahmedabad, India). Acetonitrile used was 100% pure and degassed methanol of HPLC quality. The pills, which go by the trade names Azovas16 and Zeblong 16, were bought from the market.

Mobile phase preparation: A 30:70 v/v combination of acetonitrile and methanol was filtered and degassed to create the mobile phase's content. A 0.45 μ membrane filter was used to filter the mobile phase. A 20 μl injection volume was used. At a 1.0 ml/min flow rate, the mobile phase was pumped from the solvent reservoir to the column. The temperature in the column was room temperature. At 280 nm, the eluents were seen.

Preparation of standard solution: To obtain a 1000 ppm solution, precisely weigh about 100 mg of azelnidipine, place it into a 100 ml volumetric flask, add 100 ml mobile phase, and sonicate for 20 minutes. To create a working standard solution containing 100 ppm of azelnidipine, 10 ml of the previously mentioned solution was placed in a 100 ml volumetric flask. The volume was then filled up with the mobile phase. To produce a concentration range of 20-80 ppm, 2 to 8 ml of the aforementioned solution was diluted with mobile phase to a volume of 10 ml.

Preparation of sample solution: A precisely weighed 100 mg tablet powder was transferred to a 100 ml volumetric flask, filled to the brim with mobile phase, and sonicated for 10 minutes to get 1000 ppm. An additional 100 ppm was obtained by diluting 10 ml of the solution to 100 ml in a volumetric flask. To receive 20-80 ppm, 2-8 ml of the stock solution above was pipetted into a 10-ml volumetric flask and diluted with mobile phase to make 10 ml.

Assay procedure: Twenty commercially available pill brands were weighed and powdered. The exact weight of one hundred milligrams of tablet powder was measured and then transferred to a 100 ml volumetric flask with 100 milliliters of mobile phase. The flask was then sonicated for approximately ten minutes, and the solution was filtered through a 0.45-micrometer filter. A working standard solution of one thousand parts per million was created by diluting the extract further with the mobile phase. The aforementioned solution was used to make further dilutions of azelnidipine in the 20-80 ppm range. Azelnidipine’s retention duration in bulk medication was determined to be 3.237 minutes, whereas its retention duration in pharmaceutical formulation was discovered to be 3.236 minutes.

Method of Validation

Linearity: The range of 20 to 80 ppm yielded the standard curve. The least squares approach in regression analysis was used to assess the method's linearity.

Figure 2 displays the linearity graph. Six duplicates of a set medication dosage were determined, and the peak area ratios were used to determine the accuracy of the methods. For azlnidipine, the percent range of errors and percent relative standard deviation (at 0.05 and 0.01 confidence levels) were computed and are shown in Table 1. The assay’s repeatability and intra- and inter-day fluctuations in the peak regions for a series of drug solutions were also measured, and the results were expressed in terms of percent RSD (Table 2).

 Study of Forced Degradation

Radiation: Studies on forced degradation were carried out to assess the method's stability and specificity.

a. Thermal degradation: Studies on heat deterioration were conducted for 0, 2, 4, 8, 16, and 24 hours. The peak did not alter significantly. The medication remained stable even after being heated to 80°C for 24 hours.

b. Acidic conditions: Azelnidipine was dissolved in 0.1N HCl for the acidic breakdown investigation, and the solution was kept at 80ºC in the dark for 30 and 60 minutes. When azelnidipine was exposed to acidic conditions, there was no discernible medication deterioration.

c. Basic conditions: Azelnidipine was dissolved in 0.1N NaOH for a basic breakdown investigation, and this mixture was heated to 80ºC for 60 minutes in a hot air oven. The medication azelnidipine did not significantly degrade when exposed to simple circumstances.

d. Oxidation conditions: Azelnidipine was dissolved in 3% hydrogen peroxide, and for 30-60 minutes, this mixture was heated to 80ºC respectively, in a hot air oven for the oxidation breakdown investigation. Applying 3% hydrogen peroxide to azelnidipine caused a notable deterioration of the medication.

Results

The comprehensive validation of the recommended method is shown in Table 1. With a %RSD of less than 1%, azelnidipine's recovery was constant across tablet formulations, demonstrating excellent accuracy and precision. Azelnidipine was found among its breakdown products, demonstrating the method's exceptional specificity.

The stability order determined by forced degradation investigations was NaOH (0.1N) < HCl (0.1N) < H2O2 (3%) < thermal. Using acetonitrile: methanol as the mobile phase, the developed RP-HPLC method is a novel stability-indicating technique that hasn't been documented in the literature before regulatory compliance. The effect of different flow rates, temperatures, and pH levels on the chromatogram was analyzed according to the ICH Q2 (R1) guidelines, as shown in Table 4.

Discussion

The newly developed RP-HPLC technique for azelnidipine analysis demonstrates a high degree of accuracy and precision in the validation of the method. The technique's high recovery percentage demonstrated that tablet formulations had no detrimental effects on measurement. The approach exhibits high accuracy and precision with a %RSD of less than 1%, essential for regulatory compliance, as shown in Table 2.

Crucially, the technique is extremely specific, successfully identifying azelnidipine even when its breakdown products are present. Azelnidipine is most stable in NaOH (0.1N), followed by HCl (0.1N), H2O2 (3%), and heat environments, according to the forced degradation experiments. This data supports the method's application in stability testing and improves our comprehension of the stability of the drug.

The best separation is provided by the creative combination of methanol and acetonitrile as the mobile phase, indicating this approach is a new way to analyze azelnidipines. The results confirm that the RP-HPLC method is a trustworthy instrument for stability and quality control research, with possible uses in standard pharmaceutical analysis.

Conclusion

The established RP-HPLC technique is stable, suggesting that it is quick, easy, precise, accurate, and specific. This may be used to accurately determine the dose form of azelnidipine in pharmaceutical and bulk medication forms.

Conflict of Interest

Nil

Acknowledgement

We are thankful to Moleculochem Pvt, Ahmedabad, India. For providing the gift sample of azelnidipine, we are also grateful to the principal, HKES’s MTRIPS Kalaburagi, for providing the facilities and continuous support needed for this study.