INTRODUCTION:

In the recent era of medication, combination therapy plays an important and distinct role with different mechanisms of action, which are used in patients who are not responding to the monothearpy¹. Telmisartan is a promising drug and is available in single and in combination with various antihypertensives. Telmisartan (TEL) and azelnidipine (AZE) most recently used combination in the management of hypertension which reduces the augmentation index in hypertensive patients ^2,3,4.

Telmisartan is an orally active angiotensin II blocker chemically (IUPAC) known as 4-[(1, 4- dimethyl-2-propyl [2, 6 –bi-1H-benzimidazol]-1-yl) methyl]-[1,1-biphenyl]-2-carboxylic acid (Figure1A) act as antihypertensive by acting on AT-1 receptor in adrenal gland along with vascular smooth muscles. Azelnidipine inhibits the influx of calcium through calcium dependent channels of smooth muscles and helps to reduce elevated blood pressure which is chemically known as 3-1(1-benzhydrylazetidine-3-yl) 5-isopropyl 2 amino-6- methyl-4-(3 –nitrophenyl)-1, 4 – dihydropyridine-3,5-dicaroxylate (Figure1B). As various analytical methods are available for single telmisartan^5,6,7 and for combinations of telmisartan^{8,9,10,11,12,13}, very few UV spectrophotometric¹⁴ and HPLC^15,16,17methods are reported for the simultaneous determination of Telmisartan and Azelnidipine. To achieve accuracy, precision, reproducibility and reliability of methods they need to be validated as per the International Conference on Harmonization (ICH) guidelines^18,19,20,21.

Figure 1A: Structural formula –Telmisartan

Figure 1B: Structural formula- Azelnidipine

Simultaneous equation method:

This method is significantly applicable for the simultaneous, concurrent determination of two drugs which are used in combinations. In this method absorbance of two different drugs at the λ max of one another is considered.

In this particular method λ max of telmisartan was 296± 1nm (λ₁) and 255±1nm (λ₂) for azelnidipine respectively. The concentration of the drugs calculated by equation

A2ay1 – A1ay2

Cx (Concentration of X drug) = --------------------

Ax2ay1 – ax1ay2

A1ax2 – A2ay1

Cy (Concentration of Y drug) = ----------------------

ax2ay1 – ax1ay2

In above equation, A1 and A2 is absorbance of the mixture at λ₁, and λ₂, ax₁and ax₂are absorptivity of the telmisartan at λ₁ and λ₂, ay₁and ay₂is absorptivity of the azelnidipine at λ₁ and λ₂ which can be calculated from linearity curve^21,22.

Absorbance ratio method/Q method:

It includes the measurement of absorbance of both the drug at isosbestic wavelength considered as λ₁ and wavelength of any one drug considered as λ_2.

Q_M - Q_YA₁

Cx (Concentration of X) = ----------- x -------

Q_X - Q_Y ax₁

Q_M A₂

Cy (Concentration of Y) = ----------- x -------

Q_X - Q_Y ay₁

Q_M= A₂/ A₁ , Q_X = ax₂/ ax₁ , Q_Y = ay₂/ay₁

Where, A₁ and A₂are absorbances of the mixture at the isosbestic point (λ₁) and λ max of one of the drugs (Azelnidipine) (λ₂), ax₁ and ax₂is absorptivity of the telmisartan at λ₁and λ₂, ay₁and ay₂ is absorptivity of the azelnidipine at λ₁ and λ₂^23,24,25.

In this particular method λ max of an isosbestic point was 275±1nm (λ₁) and 255±1nm (λ₂) for azelnidipine respectively.

MATERIAL AND METHODS:

Materials:

Instrumentation:

Shimadzu UV 1800 and Lab India UV 3200 double beam spectrophotometers, Shimadzu electronic balance, Labman LMUC6 sonicator were used.

Reagents and Chemicals:

Telmisartan and azelnidipine standard drug samples were kindly received from J.B. Chemicals and Pharmaceuticals Limited, Mumbai. All chemicals and reagents were used of AR grade procured from MERCK chemicals Ltd Mumbai. Distilled water was prepared in the laboratory using Barnstead pattern distillation unit, the marketed formulation containing telmisartan and azelnidipine (5:1 ratio) was purchased from local market.

Development of simultaneous equation method and absorbance ratio method using UV-Spectrophotometry:

Selection of wavelength maxima:

For the selection of wavelength for further analysis, stock solutions having a concentration of 100µg/ml telmisartan and azelnidipine were prepared separately in solvent methanol: distilled water (50:50). Each of them diluted separately to get 25µg/ml. This solution was scanned in the ultraviolet spectroscopic (UV) range of wavelength i.e. 200-400nm, the λmax of both the drugs were determined at the wavelength having maximum and acceptable absorption and those were selected as λmax of respective drugs.

Preparation of standard stock solutions:

Weighed accurately and dissolved 10mg of pure drug telmisartan and 10mg of pure drug azelnidipine in separate 100ml graduated flasks with a small amount of solvent methanol: distilled water (50: 50), sonicate it for 15 minutes at 30 to 35ºC and make up volume up to the mark using same solvent to get 100µg/ml stock solution. The stock solution was further used to make suitable dilutions like 2-12µg/ml for telmisartan and 6- 16µg/ml for azelnidipine separately.

Preparation of standard mixture (stock) solution:

Weighed accurately the 20mg of pure drug telmisartan and 4 mg of drug azelnidipine maintaining a ratio 5:1, mixed it properly and transferred to 100ml graduated flask dissolved in a small amount of solvent methanol: distilled water (50: 50), sonicate the solution for 15 minutes at 30 to 35ºC and made up the volume up to the 100ml using a same solvent to get 200µg/ml telmisartan and 40µg/ml azelnidipine mixture. Made the suitable dilution as required.

Preparation of sample (marketed) solution:

Weighed accurately 20 film-coated tablets containing telmisartan (40mg) and azelnidipine (8mg) procured from the local market and calculated the average weight per tablet. Crushed all the tablets and made the fine powered with the help of mortar and pestle, further accurately weighed the powdered mixture equivalent to 248.95 mg which contained 20mg of telmisartan and 4 mg of azelnidipine (5:1). Dissolved the powder using solvent methanol: distilled water (50:50) in 100ml graduated flask, stirred with sonicator for 15minutes at 30 to 35ºC and make-up volume up to the mark using same solvent to get 200µg/ml telmisartan and 40µg/ml azelnidipine. Filtered the solution with Whatman filter paper and used to prepare dilutions containing 20µg/ml telmisartan and 4µg/ml azelnidipine respectively and used for further analysis.

Linearity and Range:

From the 100µg/ml standard stock solution different aliquots having 2, 4, 6, 8, 10 and 12µg/ml of telmisartan and 6, 8, 10, 12, 14 and 16µg/ml of azelnidipine were prepared.

Determination of Limit of detection (LOD):

It helps to find out lowest amount of analyte that is able to be detected from the sample but is not often quantitated in the form of an exact value.

Limit of detection = 3.3x standred deviation/ Slope

Determination Limit of quantification (LOQ):

It helps to determine the lowest amount of analyte from the sample which able be quantified and determined with accuracy and having acceptable precision value.

Limit of quantification = 10 x standred deviation/ Slope

Accuracy:

Comparison to reference standard:

Accuracy with comparison to standard was carried out with a minimum six measurements using selected concentrations, this helps to reduce variability in sample preparation method particular sample and analysis for one sample at only one concentration.

Analyte recovery:

Analyte recovery study was performed at 80%, 100%, and 120% spiked levels. The recovery was determined with comparison to the known amount added and spiked level of analyte expected to be recovered in assay.

Precision:

Intraday and Interday precision were performed by selecting three different concentrations of analyte individually within the linearity range. For intraday six replicates for three different concentrations at three different times were determined. Interday precision was performed on three different days.

Ruggedness:

A method is considered rugged when results are reproducible as measured with % relative standard deviation (%RSD) between different laboratories, different analysts and equipment’s. Here two different analysts and two different UV spectrophotometers were used.

RESULT AND DISCUSSION:

Wavelength Selection: The prepared solutions of both the drug were scanned within 200- 400nm and it was 296±1nm for telmisartan [Figure 2] and 255±nm [Figure 3] for azelnidipine respectively. For the absorbance ratio method, the isosbestic point was determined by overlay of the spectra of both drugs. The 10µg/ml concentration of both samples were selected and it was observed at 275±1nm, Q value is 1.36±0.5 [Figure 4, 4A].

Figure 2: Spectrum of telmisartan to determine λ max (296±1nm)

Figure 3: Spectrum of azelnidipine to determine λ max (255±1 nm)

Figure 5: Linearity of Telmisartan at different wavelength

Table 2. Calibration curve data for Azelnidipine at 296±1nm (λ max for telmisartan), 255±1nm (λ max for azelnidipine), 275nm±1nm (isosbestic point).

Concentration (µg/ml)	λ max (nm)
Concentration (µg/ml)	296 ± 1 Mean *	255 ±1 Mean *	275 ± 1 Mean *
6	0.089	0.4019	0.2440
8	0.1157	0.5121	0.3209
10	0.1446	0.6437	0.3866
12	0.1778	0.7590	0.4741
14	0.2025	0.8997	0.5671
16	0.2293	0.9932	0.69

*Mean of three replicates considered

Figure 6: Linearity of Azelnidipine at different wavelength

Table 3: Optical and regression characteristics

Parameters	Telmisartan			Azelnidipine
λ max (nm)	296 ± 1	255 ±1	275 ± 1	296 ± 1	255 ±1	275 ± 1
Beer’s law limit (µg/ml)	2-14	2-14	2-14	6-16	6-16	6-16
Coefficient of correlation (R²)	0.9975	0.9985	0.9966	0.9986	0.9981	0.9959
Regression equation Y= mx+c	0.0546x + 0.0039	0.0544x + 0.0179	0.0392x + 0.0133	0.0142x + 0.0035	0.0605x + 0.0362	0.0415x+0.0146
Slop (m)	0.0546	0.0544	0.0392	0.0142	0.0605	0.0415
Intercept (c)	0.0039	0.0179	0.0133	0.0035	0.0362	0.0146

Table 4: Determination of Limit of detection (LOD) and Limit of quantification (LOQ)

Parameters	Telmisartan (µg/ml)			Azelnidipine (µg/ml)
λ max (nm)	296 ± 1	255 ±1	275 ± 1	296 ± 1	255 ±1	275 ± 1
LOD	0.2095	2.5510	2.8442	0.47653	0.5197	0.0369
LOQ	3.8375	7.7303	0.1119	1.4440	1.5748	09961

Table 5: Analysis of the standard mixture of telmisartan and azelnidipine.

Sr. No	Name of the Drug	Concentration Taken (µg/ml)	By Simultaneous method				Absorbance ratio method
Sr. No	Name of the Drug	Concentration Taken (µg/ml)	Concentration Obtained (µg/ml)*	% Obtained	S. D	% RSD	Concentration Obtained (µg/ml)	% Obtained	S.D	% RSD
1	Telmisartan	20	19.98	99.41	0.1755	0.1766	19.89	99.69	0.2154	0.2166
2	Azelnidipine	4	3.91	97.51	0.5919	0.6051	3.95	98.58	0.4206	0.4249

* Average of six determinations

Table 6: Statistical validation of recovery study

For the simultaneous equation method
Level of recovery	Concentration Taken (µg/ml)		Amount added (µg/ml)		% Recovery		S. D		% R S D
	TEL	AZL	TEL	AZL	TEL	AZL	TEL	AZL	TEL	AZL
80	20	4	16	3.2	99.59	98.27	0.2154	0.02025	0.2163	0.2061
100	20	4	20	4	99.76	98.57	0.1159	0.1184	0.1161	0.1201
120	20	4	24	4.8	100.43	99.98	0.6653	0.4046	0.6624	0.4046
For absorbance ratio method
80	20	4	16	3.2	99.77	98.27	0.1341	0.2470	0.1344		0.2513
100	20	4	20	4	99.50	98.69	0.0014	0.0023	0.1417		0.2337
120	20	4	24	4.8	99.70	99.10	0.0064	0.0052	0.6448		0.5289

* Average of six determinations

Table 7: Intraday and Interday precision

For simultaneous equation method
Drug	Concentration Taken (µg/ml)	Interday precision			Intraday precision*
Drug	Concentration Taken (µg/ml)	Mean* (µg /ml)	S.D.	% RSD	Mean* (µg /ml)	S.D.	% RSD
Telmisartan	4	3.5350	0.0437	1.2363	3.535	0.0124	1.2363
	6	5.9000	0.0510	0.8642	5.9866	0.0066	0.6569
	8	7.9233	0.0635	0.8009	7.9233	0.0080	0.8009
Azelnidipine	8	8.0100	0.0932	1.1631	8.1	0.0058	0.5791
	10	9.8380	0.0928	0.9437	9.8380	0.0094	0.9437
	12	12.001	0.0920	0.7662	11.9833	0.0065	0.6542
For absorbance ratio method
Telmisartan	4	3.8917	0.0467	1.1988	3.8917	0.0120	1.1988
	6	5.9518	0.0693	1.1637	5.9518	0.0116	1.1637
	8	7.9483	0.0631	0.7934	7.9483	0.0079	0.7934
Azelnidipine	8	7.9417	0.0475	0.5982	7.9417	0.0060	0.5982
	10	9.7980	0.1262	1.2878	9.7980	0.0129	1.2878
	12	11.981	0.1021	0.8519	11.9818	0.0085	0.8519

*Average of six determinations

Determination of precision (Repeatability and intermediate precision):

Precision is expressed in terms of the percentage of relative standard deviation (% RSD) and for both methods it was observed less than 2 %.

Tablet formulation Analysis:

Prepared an aliquot of the sample (marketed) solution having 20 µg/ml telmisartan corresponding to 4 µg/ml azelnidipine (5:1), recorded the absorbance of the solution and calculated the amount present in the sample. The percentage relative standard deviation for both methods was below 2%.

Table 8: Statistical validation of tablet formulation

Drug	Concentration Taken (µg/ml)	*Mean (µg/ ml)**	S.D.	% RSD
For simultaneous equation method
Telmisartan	20	20.81	0.2453	1.1790
Azelnidipine	4	3.96	0.3741	0.9448
For absorbance ratio method
Telmisartan	20	19.71	0.160	0.8165
Azelnidipine	4	3.77	0.0321	0.8519

*Average of six determinations

Ruggedness:

The results obtained from two different analysts by using two different made of instruments were found to be within the limits and overall RSD was below 2%.

Table 9: Statistical validation of ruggedness.

Drug	Concentration Taken (µg/ml)	*Mean (µg/ ml)**	S.D.	% RSD
For simultaneous equation method
Telmisartan	20	20.01	0.1724	0.8615
Azelnidipine	4	3.95	0.06	1.5189
For absorbance ratio method
Telmisartan	20	19.88	0.1616	0.8120
Azelnidipine	4	3.93	0.03785	0.9625

*Mean of six determinations

CONCLUSION:

Developed and validated simultaneous equation and absorbance ratio (Q) method, applicable for the simultaneous determination of telmisartan and azelnidipine in combined dosage forms and formulation. Both techniques, which make use of UV spectrophotometry, have been proven to be successful. Beer's law was followed by the linearity of both approaches in the concentration ranges of 2–12 µg /ml for telmisartan and 6–16 µg /ml for azelnidipine, respectively. Recovery studies of both techniques were conducted to guarantee the methods' reproducibility and dependability. The results demonstrate that the techniques are accurate, reliable, reproducible, and cost-effective, making both methods appropriate for routine quality control analysis of telmisartan and azelnidipine in bulk as well as pharmaceutical dosage forms and formulations.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank to J.B. Chemicals and Pharmaceutical Limited, Mumbai for providing a gift sample for the analysis, and also thankful to Progressive Education Society’s, PES’s Modern College of Pharmacy for Ladies, Moshi, Pune for providing all necessary facilities.

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Received on 26.10.2024 Revised on 17.02.2025

Accepted on 22.04.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):6035-6041.

DOI: 10.52711/0974-360X.2025.00872

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Concentration (µg/ml)	λ max (nm)
Concentration (µg/ml)	296 ± 1 Mean *	255 ±1 Mean *	275 ± 1Mean *
2	0.112	0.1247	0.0885
4	0.2366	0.2555	0.1843
6	0.3221	0.3426	0.2415
8	0.4278	0.4398	0.3182
10	0.553	0.5623	0.4056
12	0.6657	0.6765	0.4896