INTRODUCTION:

Azelnidipine¹, a new dihydropyridine calcium channel blockeris a third-generation, long-acting dihydropyridine calcium antagonist with chemical name ((±)-(3)-(1-diphenylmethylazetidin -3-yl)-5-isopropyl-2- amino-1, 4-dihydro-6-methyl -4-(3-nitrophenyl)-3, 5- pyridine dicarboxylate. Telmisartan²is a vasoconstrictor and blocks the aldosterone-secreting effects of angiotensin II and its chemical structure is 4'-[(1,4'-dimethyl-2'-propyl [2,6'-bi-1H-benzimidazol]-1'-yl)methyl]-[1,1'-biphenyl]-2-carboxylicacid^3,4.

Renin-angiotensin system with ACEinhibitors, which inhibit the biosynthesis of angiotensin II from angiotensin Literature survey of Azelnidipine and Telmisartan with chemical structure shown in figure 1 and 2 revealed HPLC, RP-HPLC, LC-ESI-MS/MS, HPTLC, LC-MS, and simultaneous UV spectrophotometric methods are reported^5-12 for quantitative estimation of Azelnidipine alone or in combination with other antihypertensive agents.

MATERIALS AND METHODS:

Materials:

Reagents and Chemicals:

All reagents and solvents were analytical and HPLC grade, including Acetonitrile, Triethylamine, and orthophosphoric acid fine-chem Ltd., Mumbai, India. High purity deionized water was obtained by double distillation and purification through a milli-Q water purification system. The 0.45µm nylon filter was purchased from advanced micro device Pvt Ltd.

Drug sample:

Azelnidipine and Telmisartan active pharma ingredient and marketed samples Azelnidipine and telmisartan UNIAZ T-40

Buffer preparation:

Triethylamine (2%) was dissolved in 100mL of HPLC grade water, and pH 3.0 was adjusted with orthophosphoric acid. and weigh accurately about adding 400mL of Acetonitrile. Mix well, filter, and degas. It was filtered through a 0.45µm nylon membrane filter and degassed. It was used as a diluent for the preparation of the sample and standard solution.

Selection of wavelength:

Accurately weighed quantity of the sample equivalent to 50mg of Azelnidipine and Telmisartan was taken in a 50 mL volumetric flask. 25mL of mobile phase was added, sonicated for 10min, and filtered through 0.45µm nylon filter. The above solution was scanned between 200 and 400nm by UV spectroscopy.

Methods:

Chromatographic conditions:

The experiments were performed with isocratic elution. The binary mobile phase consisted of a mixture of triethylamine buffer (2%) and Acetonitrile in the ratio of 20:80 V/V. The pH 3.0 was adjusted using 0.1% orthophosphoric acid, filtered through a membrane filter. The effluents were degassed before running at a flow rate of 1.0mL min. The column temperature was ambient at 27⁰C. The 20mL volume was injected per run, and effluents were detected using a UV detector at k = 226nm.

Preparation of standard solution and calibration graph:

The standard stock solution of Azelnidipine and Telmisartan of 1000mg mL was prepared by dissolving 25mg of standard drug in 20mL of diluent in a 25mL volumetric flask. The above solution was sonicated for 10min, and the volume was made with the same diluent. 0.5mL of the standard stock solution was diluted to 10 mL with the same diluent to get the final concentration of 500mg mL.

Sample solution:

Twenty tablets (Azelnidipine and Telmisartan) containing average weight 303mg of UNITAZ T-40 were accurately weighed, averaged, and finely powdered. A quantity of powder equivalent to the weight of one tablet was weighed and transferred to a 100mL volumetric flask, and 70mL of diluent was added. The volumetric flask was sonicated for 10min to dissolve the drug in the solution, filtered through a 0.45 lm nylon filter, and made up to the volume with diluent. 5 mL of the filtrate was taken in a 100mL volumetric flask and made up to the volume with the same diluent to the final concentration of 300mg mL. The chromatogram of the standards and combination are shown in Fig 1 and 2

Fig. 1: Standard chromatogram of Telmisartan and Azelnidipine

Fig. 2: Chromatogram of Standard solution of Azelnidipine and Telmisartan

Evaluation of system:

System suitability was studied under each validation parameter by injecting six replicate injections of the standard solution (10mg mL). System suitability parameters like column efficiency, plate count, and tailing factor were recorded. The results obtained were within the limit and are shown in the % RSD of six injections of standard solution is not more than 2%.

Analytical Method Validation¹⁵:

Linearity and range:

To study the linearity of the drug, the serial dilution of standard stock solution was made in the range of Azelnidipine 1-5g mL and Telmisartan 5-25mL. A graph was plotted as the concentration of drug versus peak area response. It was found to be linear for the given concentration of the drug as shown in fig:3

Fig. 3: Linearity Curve of Azelnidipine and Telmisartan

To study the linearity of the drug, the serial dilution of standard stock solution was made in the range of Azelnidipine 1-5gmL and Telmisartan 5-25mL. A graph was plotted as the concentration of drug versus peak area response. It was found to be linear for the given concentration of the drug as shown in figure 6 and 7

System suitability and system precision:

ICH guidelines followed system suitability checking out is an integral part of many analytical procedures.

According to ICH specification:

The theoretical plate should not be less than 2000Tailing factor should not be more than 2.0 of each analyte, Theoretical plate, tailing factor and resolution was within the acceptance limit, RSD (Relative standard deviation) of six Replications of area and Retention time for Azelnidipine and Telmisartan was Less than 2.0%. System suitability and system precision result were shown in table:1.

Table 1: System suitability studies

S. No	Telmisartan					Azelnidipine
	Rt in min		Plate count		Tailing factor	Rt in min	Plate count		Tailing factor
1	3.889		2516		1.10	6.058	2804		0.97
2	3.885		2560		1.13	6.051	2865		0.94
3	3.882		2531		1.09	6.061	2854		1.05
4	3.885		2554		1.08	6.055	2894		1.11
5	3.876		2547		1.15	6.061	2857		1.02
6	3.890		2532		1.05	6.056	2841		1.08
7	3.870		2521		1.11	6.054	2876		0.94
S. No		Telmisartan				Azelnidipine
		Rt in min		Area		Rt in min		Area
1		3.889		2283790		6.058		125830
2		3.885		2266448		6.051		126624
3		3.882		2299019		6.061		127098
4		3.885		2275011		6.055		124420
5		3.876		2282190		6.061		124124
6		3.890		2272101		6.056		123210
Avg		3.884		227975		6.06		125211
%RSD		0.45		0.69		0.52		0.9

Specificity:

Specificity is the ability to assess the analyte in the presence of components that may be present. Typically these might include impurities, degradants, matrix,etc.. blank and placebo solutions were injected into the HPLC system. The blank and placebo chromatograms were shown in Figure 4

Fig. 4: Chromatogram of Blank without Standard drug and Placebo

Acceptance criteria:

Blank solution and placebo solution interfered at the retention time of the prominent analyte peaks. No Blank and placebo interference was observed at two prominent analyte peaks.

Method precision:

Sample solutions were six prepared individually and each injected into the HPLC system, calculated % Assay using the average area of six standards. The %RSD for %Assay of all peak areas and retention time of Azelnidipine and Telmisartan were calculated and shown in table:2. The Average and individual %Assay was obtained between 95.0 to 105.0% individual preparation and %RSD for %Assay of six replicate preparations was obtained below 2.0.

Table 2: Method precision

Name of the Sample	% Assay of Telmisartan	%Assay of Azelnidipine
Method precsion -01	99.51	99.01
Method precsion -02	99.41	99.41
Method precsion -03	99.31	98.83
Method precsion -04	99.61	98.56
Method precsion -05	99.32	99.21
Method precsion -06	99.46	98.61
AVG	99.35	99.05
Std dev	0.35	0.8
%RSD	0.35	0.8

Accuracy and recovery studies:

The closeness of agreement between the value accepted either as a true conventional value or the value found and shown in table: 3. Each level of Accuracy and %Recovery mean was obtained 99.4% for Telmisartan and 99.5%for Azelnidipine

Table 3: Details of accuracy

Name of the level	Telmisartan	Azelnidipine
50% Accuracy	98.91	100.21
100%Accuracy	99.31	99.54
150%Accuracy	100.21	98.76
Mean	99.47	99.50
Std dev	0.3	0.4
%RSD	0.3	0.4

Limit of detection:

The limit of detection can be calculated using the following equation according to ICH guidelines: LOD = 3.3x N/S where N is the standard deviation of peak areas of the drug and S is the slope of the corresponding calibration curve. The results obtained in LOD were for Azelnidipine 5.9876 and Telmisartan 0.8356.

Limit of quantification:

The limit of quantification can be calculated using the following equation according to ICH guidelines:

LOQ = 10 x N/S where N is the standard deviation of peak areas of the drug and S is the slope of the corresponding calibration curve. The results obtained in LOQ were for Azelnidipine 18.1276 and Telmisartan 2.453.

Robustness studies:

The robustness was determined by deliberately changing some operating conditions such as flow rate and wavelength. It was observed that there were no remarkable changes in the chromatogram, the tailing factor, and platecounts. Hence the robustness of the method is established as shown in table: 4 to the extent of variations applied to analytical conditions.

Table 4: Robustness studies

Chromatographic Condition	Retention time		% Assay
Chromatographic Condition	Azelnidipine	Telmisartan	Azelnidipine	Telmisartan
Actual condition	3.9	6.1	99.78%	99.58%
Change in ratio (80:20)	3.6	5.1	98.57%	99.04%
Change in ratio (76:24)	3.9	5.8	98.31%	98.76%
Flow rate 1.ml	3.85	5.78	99.51%	98.01%
Flow rate 1.2ml	4.04	5.87	97.46%	99.79%

RESULTS AND DISCUSSION:

Chromatographic parameters were optimized to develop an HPLC method to determine Azelnidipine6.0min and Telmisartan 3.9min with a short analysis time. Various compositions of mobile phase like water: methanol (65:35 V/V), buffer: methanol (30:70 V/V), and phosphate buffer: Acetonitrile (70:30 V/V) in different ratios were tried at a flow rate of 1.0 mL min1. Triethylamine (2%) buffer pH 3.0 and Acetonitrile(20:80) showed symmetrical peaks with good resolution. The optimum wavelength for detection was 226 nm, and the retention time was found to Azelnidipine 6.0 min. The linear curve was obtained in the concentration range of Azelnidipine and Telmisartan 1–5 mg/ mL and 5-25mg /mL. LOD and LOQ were determined from the slope and standard deviation of the Y-intercept of the regression line of the calibration curve., respectively. The method's precision and instrument precision were evaluated, and the % RSD values were less than 2.0%. Recovery studies determined the accuracy of the process. The recovery studies were close to 100%, which complies with FDA approval of the drug. The developed method was robust while changing the flow rate, wavelength detection, temperature, and composition of mobile. There was no considerable change in the peak areas and retention time. The parameters like tailing factor and retention time showed adherence to the limit. The proposed method is superior to the reported method with less retention time, high theoretical plate count, and composition of the mobile phase with good separation.

CONCLUSION:

A simple precise RP-HPLC method for Azelnidipine and Telmisartan was developed and validated in a tablet dosage form as per ICH Guidelines., using Shimazu LC-20 AT HPLC series with PDA Detector and Phenomenex C18 (250x4.6mm, 5µ) column, injection of 20µl is injected and eluted with the mobile phase of

0.2% Triethylamine in the buffer with pH 3 and Acetonitrile in the ratio 20:80, pumped at a flow rate of 1.0ml at 226nm. The peak of Azelnidipine and Telmisartan was found well separated at 6.0 min and 3.8min.

The developed method was validated for various parameters as per ICH guidelines. System suitability, accuracy, precision, linearity, specificity, ruggedness, robustness, and solution stability.The analytical method validation of Azelnidipine and Telmisartan by RP HPLC was satisfactory and could be used for the routine pharmaceutical analysis of Azelnidipine and Telmisartan.

REFERENCES:

1. Dibya Das, Dhiman Halder, Himangshu Sekhar Maji, Pintu Kumar De, Tapan Kumar Pal. Special Emphasis on Bioanalytical Method Development and Validation of an Anti-Hypertensive Drug Azelnidipine by LC-ESI-MS/MS in Healthy Human Volunteer’s Blood Plasma. Research Journal of Pharmacy and Technology. 2021; 14(7):3571-7. doi: 10.52711/0974-360X.2021.00618

2. Mrs. Aishwarya P. Joglekar. Method Development and Validation for the Estimation of Telmisartan and Chlorthalidone in Bulk and Pharmaceutical Dosage form by HPTLC Method. Research J. Pharm. and Tech. 8(4): April, 2015; Page 376-381. doi: 10.5958/0974-360X.2015.00063.3

3. https://www.go.drugbank.com/drugs/ db09230

4. https://www.go.drugbank.com/drugs/ db00966

5. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4245158/

6. A.R. Chabukswar, S.C. Jagdale, S.V. Kumbhar, D.J. Desai, B.S. Kuchekar, P.D. Lokhande. HPLC Method Development for Telmisartan and Amlodipine. Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1227-1230. doi:

7. Krishanu Pal, M Bhagavan Raju, K Pavan Kumar, Rahul Chakravorthy. Simultaneous Determination of Telmisartan and Ramipril in Tablet Dosage Form by Spectrophotometry. Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 861-864.

8. Sheetal, Sonia. K, K. S. Lakshmi. Validation of Telmisartan by UV Spectrophotometry Method. Research J. Pharm. and Tech. 2019; 12(5):2413-2415. doi: 10.5958/0974-360X.2019.00404.9

9. Chandrakant S. Satpute, Prashant K. Pagare, Varsha M. Jadhav, Vilasrao J. Kadam. Simultaneous Estimation of Telmisartan and Indapamide in Bulk Drug and Finished products by RP-HPLC method. Research J. Pharm. and Tech. 6(11): November 2013; Page 1217-1224.

10. Chandrakant S. Satpute, Prashant K. Pagare, Varsha M. Jadhav, Vilasrao J. Kadam. Simultaneous Estimation of Telmisartan and Indapamide in Bulk Drug and Finished products by RP-HPLC method. Research J. Pharm. and Tech. 6(11): November 2013; Page 1217-1224.

11. MP Yeole, A J Asnani. Simultaneous Determination of Telmisartan and Amlodipine in Tablets by Reverse Phase High Performance Liquid Chromatography. Research J. Pharm. and Tech. 4 (1): January 2011; Page 75-77.

12. Jawed Akhtar, B. Srivastava, Parth Bhatt. Simultaneous Estimation of Telmisartan and Indapamide in Bulk and Capsule Dosage Form by Reverse Phase High Performance Liquid Chromatography. Research J. Pharm. and Tech. 5(1): Jan. 2012; Page 60-62

13. NirmaChavda, Suresh Kumar. A Review article on Analytical Method Development for the combination of Azelnidipine and Telmisartan. Asian Journal of Pharmaceutical Analysis. 2021; 11(3):227-4.

14. Rele R.V., Patil S.P.. Ultra-Violet Spectrophotometric Method for Estimation of Azelnidipine from Bulk Drug and Pharmaceutical Formulation. Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 1077-1079.

15. International Conference of Harmonization Quality Guidelines Q2R1 validation of Analytical Procedure :Text and Methodology.

Received on 26.11.2021 Modified on 09.06.2022

Research J. Pharm. and Tech 2023; 16(6):2638-2642.

DOI: 10.52711/0974-360X.2023.00433