1Department of pharmaceutical quality assurance, Maharashtra College of Pharmacy, Nilanga.
2Faculty Department of Pharmacy, Maharashtra College of Pharmacy, Nilanga.
3Faculty Department of Pharmacy, Maharashtra College of Pharmacy, Nilanga.
4Department of pharmaceutical quality assurance, P. R. Pote Patil College of Pharmacy, Amravati.
The goal of the current study is to develop an RP-HPLC-based analytical method for determining the dosage of Azilsartan in bulk and tablet form that is fast, precise, sensitive, selective, and repeatable. Create a novel HPLC technique for Azilsartan estimation, and validate it in compliance with ICH guidelines. In order to make use of the accepted methodology for Azilsartan estimation in pharmaceutical formulations, an RP-HPLC method was utilized to create and validate a stability indicating method. Using an Inertsil-ODS C18 (250×4.6mm, 5µm) column and a 90:10 v/v methanol: acetonitrile mobile phase at a flow rate of 1 ml/min, the estimation was carried out using RP-HPLC. Azilsartan's HPLC linearity range was 20–70 µg/ml, and its R2 value was 0.999. Additionally, the method satisfies the robustness parameter requirement. The method's accuracy, precision, sensitivity, and economy are demonstrated by the findings. HPLC is a faster method. The medicinal dose form was successfully administered using the procedure.
In 2011, the USFDA authorized Azilsartan medoxomil (AZM), a chemically complex monopotassium salt of 5-methyl-2-oxo-1,3-dioxol-4-yl)methyl 2-ethoxy-1-{[2'-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)biphenyl 4yl]methyl}-1Hbenzimidazole-7 carboxylate-7 carboxylate.[1] Fig. 1 shows the structural formula for AZM. AZM is a prodrug that lowers blood pressure. When the medoxomil ester is hydrolyzed, the active component of AZM is exposed. This results in the conversion of azilsartan, an active blocker of the angiotensin II receptor that lowers blood pressure more quickly than valsartan and olmesartan in a 24-hour period.[2, 3]
There have been reports of several quantification methods for AZM from pure medication, various pharmaceutical formulations, and biological materials. Treatment for hypertension with azilsartan medoxomil and chlorthalidone works well together. Numerous analytical techniques, either by themselves or in conjunction with other medications, are demonstrated in the literature search. These techniques include UV spectrophotometry, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and high performance thin-layer chromatography (HPTLC). [4–13]
Fig. 1: Chemical structure of Azilsartan
Since RP-HPLC and HPTLC are the most widely used analytical techniques for estimating a drug's specificity and sensitivity, the current study aimed to develop an RP-HPLC method that is straightforward, accurate, sensitive, and precise for the analysis of azilsartan medoxomil in pharmaceutical formulations.
HPLC System:
Chromatographic separation was accomplished utilizing the RP-HPLC Waters system, which included the Waters Model No. 2690/5 with PDA detector and the Waters Empower-2 software from the Waters Corporation as the data processor. Column for analysis: Inertsil-ODS C18 (250 x 4.6 mm, 5?).
Sonicator:
Sonication of solvents and various preparation was done by using sonicator of IKON Industries Ultrasonic Bath.
UV System:
The wavelength of Azilsartan was determined using a Systronics-UV Model No. 119 with a pair of 10mm matched quartz cells. The wavelength was calculated by scanning a standard solution in a UV spectrophotometer from 200 nm to 400 nm in spectrum mode.
MERCK Limited, Mumbai, contributed HPLC grade methanol and acetonitrile, whereas Bharath Life Science Pvt. Ltd. provided a pure sample of working standard Azilsartan as a gift.
Take 100mg Azilsartan working standard in 100ml V.F add methanol sonicate it 30 minutes, (That is 1000ppm solution).
Take 100mg Azilsartan working standard in 100ml V.F add methanol sonicate it 30minutes, (That is 1000ppm solution).
Further Dilution (or) Optimized Method Solutions Preparation:
Take 4ml of above solution in 100ml V.F add Methanol up to mark sonicate it 10minutes (That 40ppm solution).
HPLC METHOD OPTIMIZATION:
For method optimization various mobile phase were tried in different ratio.
Optimized Method Stock Solution Preparation:
Mobile Phase: Methanol:
Acetonitrile (90:10)V/V. Sonicate it 30minets, Filter this mobile phase through 0.45micron filter paper.
Optimized Method Stock Solution Preparation:
Take 100mg Azilsartan working standard in 100ml V.F add methanol sonicate it 30minets,(That is 1000ppm solution).
Further Dilution (or) Optimized Method Solutions Preparation:
Take 4ml of above solution in 100ml V.F add Methanol up to mark sonicate it 10minets(That 40ppm solution).
Chromatographic Conditions:
Table no.1: Chromatographic conditions for optimized trial.
Fig 2. Optimized Method Development Trial Chromatogram of Azilsartan
RESULTS AND DISSCUSSION:
Validation:
A Standard solution was prepared by using Azilsartan working standard as per test method and was injected Five times into the HPLC system. The system suitability parameters were evaluated from standard chromatograms by calculating the % RSD from five replicate injections for Azilsartan, retenton times and peak areas.
Table no.2: System suitability data
Repeatability:
System precision: Standard solution prepared as per test method and injected five times.
Method precision: Prepared six sample preparations individually using single as per test method and injected each solution.
System Precision:
Table no.3: System precision data
Method Precision:
Table no.4: Method precision data
Intermediate precision:
Table no.5: Intermediate precision data
A study of Accuracy was conducted. Drug Assay was performed in triplicate as per test method with equivalent amount of Azilsartan into each volumetric flask for each spike level to get the concentration of Azilsartan equivalent to 50%, 100%, and 150% of the labeled amount as per the test method. The average % recovery of Azilsartan was calculated.
Table no.6: Accuracy data
A Series of solutions are prepared using Azilsartan working standard at concentration levels from 20ppm to 70 ppm of target concentration.
Table no.7: Linearity data (concentration vs peak area)
Statistical Analysis of Linearity
Table no.8: Statistical analysis of Linearity
Fig 3: Plot of Linearity (concentration vs peak area)
System to system variability:
System to system variability study was conducted on different HPLC systems, under similar conditions at different times. Six samples were prepared and each was analyzed as per test metho
d. Comparison of both the results obtained on two different HPLC systems, shows that the assay test method were rugged for System-to-system variability. For system 1, refer to system suitability data; Table no.2
Table no.9: Ruggedness data for system 2
Effect of variation of flow rate:
A study was conducted to determine the effect of variation in flow rate. Standard solution prepared as per the test method was injected into the HPLC system using flow rates, 1.0ml/min and 1.2ml/min. The system suitability parameters were evaluated and found to be within the limits for 1.0ml/min and 1.2ml/min flow.
Table no.10: Data for robustness (Flow rate 0.8ml)
Table no.11: Data for robustness (Flow rate 1.0ml)
Table no.12: Data for robustness (Flow rate 1.2ml)
Table no.13: Values of LOD AND LOQ
Marketed Sample Analysis:
Table no.14: Information of marketed sample of Azilsartan
Table no.15: Marketed sample analysis data
CONCLUSION
Various parameters were examined in order to develop the analytical methodology. To begin with, it was found that Azilsartan had a maximum absorbance of 236 nm. A great peak region was obtained by setting the injection volume at 20µl. In this job, the Inertsil C18 column was used, and ODS selected a good peak form. It was found that the ambient temperature was suitable for the kind of pharmaceutical solution. The flow rate was set at 1.0 ml/min due to the good peak area, sufficient retention length, and good resolution. Several mobile phase ratios were examined; but, due to its symmetrical peaks and good resolution, the mobile phase with a Methanol: Acetonitrile (90:10) ratio was selected. Consequently, this mobile phase was utilized in the intended investigation.
It was found that the system and technique both had precise and well-within-range accuracy. The linearity investigation yielded the correlation coefficient and curve fitting. The analytical method was demonstrated to be linear for both medications throughout a range of 20–70 ppm of the target concentration. The analysis passed the ruggedness and robustness testing. In both cases, the relative standard deviation was very good.
REFERENCES
Sagar N. Katke, Santosh P. Kumbhar, Vinod D. Usnale, Siddhant M. Sawant, The Method Development And Validation Of A High-Performance Liquid Chromatographic Method For Azilsartan Analysis , Int. J. of Pharm. Sci., 2024, Vol 2, Issue 5, 1152-1160. https://doi.org/10.5281/zenodo.11240815
10.5281/zenodo.11240815
