Dayanand College of Pharmacy Latur, Swami Ramanand Teerth Marathwada University, Nanded, Latur- 413512, Maharashtra, India
The simultaneous quantification of irbesartan and amlodipine besylate in bulk and pharmaceutical formulation has been achieved by the development of a reverse phase high performance liquid chromatographic technique employing an RP-C18 column. At a flow rate of 1.0 mL/min, the mobile phase (0.2% Triethylamine: Acetonitrile) PH 5.0 adjusted with orthophosphoric acid was pumped in a 50:50% v/v ratio, and its wavelength was measured at 236 nm. The suggested HPLC method's high degree of accuracy and precision was demonstrated by the statistical validation of the procedure, which revealed an RSD of less than 2%. Irbesartan and amlodipine besylate have retention times of 4.4 and 4.1, respectively. Additionally, there is no diluent interference with the standard and sample chromatograms. In accordance with ICH requirements, its accuracy, precision, specificity, and robustness were statistically validated. We are able to state that this method can be used for additional analysis after analyzing the validation data. Because routine analysis can be conducted using this suggested strategy.
Based on anhydrous calculation, irbesartan has a C25H28N6O content of at least 99.0 percent and up to 101.0 percent. The drug irbesartan is used to treat high blood pressure and shield the kidneys against diabetic kidney disease. It belongs to the group of drugs known as angiotensin II receptor blockers (ARBs). Irbesartan relaxes blood arteries to facilitate easier blood flow, which lowers blood pressure1,2. Irbesartan prevents the body's natural production of angiotensin II, which narrows blood vessels and lowers blood flow. Irbesartan causes blood arteries to dilate and reduce blood pressure by blocking the actions of angiotensin I3,4.
The IUPAC name of this compound is :
2-butyl-3-[[4-[2-(2H-tetrazol-5-yl)phenyl]methyl][4.4] -1,3-diazaspironon-1-en-4-one
Formula for a molecule:
C25H28N6O
Weight in molecules:
428.5 g/mol
Type:
Medicine for hypertension
Solubility:
Almost insoluble in water, soluble in methanol, ethanol, and isopropyl alcohol.
Melting point:
180–181 degrees Celsius;
pKa:
4.12–7.40?
Fig. No:1 Irbesartan
Fig. No: 2 Amlodipine Besylate
Based on anhydrous calculation, Amlodipine Besylate has a minimum of 97.0 percent and a maximum of 102.0 percent C26H31Cl N2O8S. Amlodipine Besylate is a dihydropyridine calcium antagonist, also known as a slow-channel blocker or calcium ion antagonist, which prevents calcium ions from entering cardiac and vascular smooth muscle5,6. Additionally, some forms of coronary artery disease and angina (chest pain) are treated with it (narrowing of the blood vessels that provide blood to the heart)7,8.
IUPAC Name:
3-O-ethyl benzenesulfonic acid 5-O-methyl 2-(2-chlorophenyl)-4-(2-aminoethoxymethyl)-6-methylDihydropyridine-1,4-d-3,5-dicarboxylate
The Molecular Formula:
C26H31ClN2O8S
Molecular Weight:
567.1 g/mol;
Category:
Antihypertensive, Antianginal;
Solubility:
slight solubility in water and sparingly soluble in ethanol and soluble in Iso propyl alcohol
Melting point:
200 °C
pKa:
8.6?
MATERIALS AND METHOD
Instrumentation
HPLC equipment was an Agilent 1260 Infinity. For chromatographic separation and quantification, an Agilent 5TC C18 (250 mm × 4.6 mm) analytical column was utilized, and it was maintained at room temperature the entire time. Tablet samples, solution samples, and mobile phases were filtered using Whatman filter paper before being injected into the HPLC apparatus.
Chemicals, solvents, and drugs
We bought free gift samples of Amlodipine Besylate and Irbesartan from Aurochem Pharmaceuticals in Palghar, India. Acetonitrile of HPLC quality was provided by Research Laboratories Ltd. (Mumbai, India). tablets called AMIX R that contain 10 mg of amlodipine besylate and 100 mg of irbesartan. Water of HPLC quality was utilized throughout the entire analysis..
Conditions for chromatography
Mobile phase :
TEA(PH-5.0) : Acetonitrile (50:50 v/v)
Flow rate :
1mL/min
Wavelength of detection:
236 nm
Column temperature :
Room temperature
Injection volume :
20 ?L
Run time :
7 min
Detector:
PDA detector
Standard drug solution
Weigh out 100 milligrams of irbesartan and 13.9 milligrams of amlodipine besylate precisely, and then transfer each amount into a separate 100 milliliter volumetric flask. Next, fill the volumetric flask with irbesartan and amlodipine besylate. Add 55 ml of isopropyl alcohol and 45 ml of water (acting as a diluent). Filter the mixture using filter paper No. 41. Using the distill water, adjust the final volume to the mark on the volumetric flask. 5 ml of each of the previously prepared solutions should be pipetted out and transferred to a second 100 ml volumetric flask. Distill water should be added to the volumetric flask until the desired volume is reached.(Figure 1)
Tablet sample solution
Calculate the average weight of the ten tablets by weighing each one. Make a fine powder out of the crushed tablets. Weighed out of the powdered tablet, 100 mg of irbesartan and 13.9 mg of amlodipine besylate were added to a 100 ml volumetric flask. To get rid of any contaminants or solid particles, strain the mixture containing the tablet powder through filter paper No. 41. 10 milliliters of the filtered solution were pipetted into a second 100 milliliter volumetric flask. Distill water should be added to the volumetric flask until the desired volume is reached. After the solution is ready, add it to the HPLC apparatus for examination.(Figure 2)
Preparation of mobile phase
After dissolving 1 milliliter of triethylamine in 500 milliliters of water, adjust the pH to 5.0 with the aid of orthophosphoric acid.
Method Validation
Fig.1: chromatogram of standard
Fig.2:chromatogram of tablet sample
System suitability
The production of irbesartan and amlodipine besylate followed protocol, and the HPLC system received five injections of the working standard solution. Standard chromatograms were used to assess the appropriateness of the system by computing the percentage RSD of the retention period, tailing factor, theoretical plates, and peak regions from five replicate injections.
Table1:System suitability of Amlodipine Besylate and Irbesartan
Fig.3: Specificity blank chromatogram
Fig.4:Specifity sample chromatogram
Fig.5:specificity standard chromatogram
Specificity
Specificity is the ability to isolate the target analyte from other sample constituents. The relationship between the chromatograms for the standard, sample, blank, and placebo ensured the procedure's specificity.
Table 2:Specificity data for sample
Table 3:Specificity data for standard
Linearity and Range
To demonstrate the assay's linearity, inject six reference solutions containing irbesartan and amlodipine besylate. A 0.99 correlation coefficient was discovered. There was a linearity plot created.
Table 4:Data sheet for linearity of Amlodipine Besylate
Fig.6: linearity graph of Amlodipine Besylate
Table 5:Data sheet for linearity of Irbesartan
Fig.7:linearity graph of Irbesartan
Accuracy
The classic addition recovery approach was used to gauge the procedure's accuracy. The previously analyzed sample solutions were mixed with a standard stock solution at three distinct concentrations: 80%, 100%, and 120%. The standard stock solution contained known levels of irbesartan and amlodipine besylate. While going over these options again, three analyses of the answers at every level were carried out. The accuracy was caSlculated and reported as a percentage of the recovery.
Table 6:Data for Accuracy of Amlodipine Besylate
Table 7:Data for Accuracy of Irbesartan
Precision
The degree of agreement between several measurements made from repeated samplings of the same homogenous material under specified conditions is known as the analytical process' precision. System and method implementation both exhibit precision
Table 8:Method Precision of Amlodipine Besylate
Table 9:Method Precision of Irbesartan
Robustness
An intended change to the process is the addition of mobile phases minus and plus. Additionally, the proportion of RSD is calculated.
Table 10:Robustness data for Amlodipine Besylate and Irbesartan (for flow rate 0.8ml/min)
Table 11:Robustness data for Amlodipine Besylate and Irbesartan (for flow rate 1.1ml/min)
Solution stability
For a whole day at room temperature, standard and sample solutions were tested to demonstrate the stability of the solutions during analysis.
Table 12:Standard Solution Stability data for Amlodipine Besylate and Irbesartan
Table 13:Sample Solution Stability data for Amlodipine Besylate and Irbesartan
RESULT AND DISCUSSION
The measures for system appropriateness satisfy the requirements for approval. Verify the apparatus is appropriate before beginning any analytical tests, including HPLC analysis. (Reference Table 1). Irbesartan and amlodipine besylate-containing sample solution and reference solution's chromatograms are similar.(Figures 4, 5, and 3, 4) The gap has no effect on the retention time of the primary peak.(Tables 2 and 3). It is possible to establish a positive correlation with a correlation coefficient of 0.99 by modifying the area between the drug and its concentration.(Tables 4 and 5) (Figures 6 and 7). The results of multiple tests showed that the cure rate ranged from 95% to 103%. This approach was selected, as evidenced by the fact that excipients do not interfere with the analyte measurement (Table 6&7). The precision percentage RSD values of the method are less than 2%, indicating that it has good precision for the simultaneous analysis of irbesartan and amlodipine besylate. (Tables 8 and 9). The results demonstrate the applicability of the method by demonstrating that minute changes in the mobile phase's composition and temperature have no bearing whatsoever on the drug's peak area.(Tables 10 and 11). At room temperature, samples and reference solutions remain stable for a whole day.(Tables 12 and 13). In conclusion, we can state that the established, validated RP HPLC method provides accurate, precise, cost-effective, and user-friendly quantitative determination of irbesartan and amlodipine besylate in formulation. It was determined that the procedure complies with ICH criteria. Consequently, the method can be effectively used to the routine assessment of irbesartan and amlodipine besylate in tablet form.
ACKNOWLEDGMENT
The authors would like to thank Aurochem Laboratories, Palghar, for providing gift samples of Amlodipine Besylate and Irbesartan.
CONFLICT OF INTEREST
The authors claim to have no competing interests.
REFERENCES:
Khadakumarge Chavan Kashish Arvind, Jadhav Mahima Murlidhar, Satpute K. L, Development And Validation Of RP-HPLC Method For Simultaneous Estimation Of Irbesartan And Amlodipine Besylate In Pharmaceutical Dosage Form, Int. J. of Pharm. Sci., 2024, Vol 2, Issue 6, 1335-1334. https://doi.org/10.5281/zenodo.12588711
10.5281/zenodo.12588711
