INTRODUCTION:

Telmisartan is a benzimidazole derivative and a non-peptide angiotensin II receptor antagonist with antihypertensive property^[1].. Telmisartan selectively antagonizes angiotensin II binding to the AT1 subtype receptor, located in vascular smooth muscle and adrenal gland. The antagonism results in vasodilation and inhibits the angiotensin II-mediated aldosterone production, which in turn leading to a decrease in sodium and water as well as an increase in potassium excretion leading to a subsequent reduction in blood pressure. Telmisartan is an angiotensin II receptor blocker used in the therapy of hypertension. Telmisartan is associated with a low rate of transient serum aminotransferase elevations, but has yet to be linked to instances of acute liver injury.

It is white to slightly yellowish solid and has melting point in the range of 261-263 °C. The practically insoluble in water and in the pH range of 3 to 9, sparingly soluble in strong acid (except insoluble in hydrochloric acid) and soluble in strong base. It is used alone or in combination with other classes of antihypertensives for the treatment of hypertension. Also used in the treatment of diabetic nephropathy in hypertensive patients with type 2 diabetes mellitus, as well as the treatment of congestive heart failure (only in patients who cannot tolerate ACE inhibitors)^[2].

Amlodipine Besylate is the besylate salt of amlodipine, a synthetic dihydropyridine with antihypertensive and antianginal effects. Amlodipine inhibits the influx of extracellular calcium ions into myocardial and peripheral vascular smooth muscle cells, thereby preventing vascular and myocardial contraction. These results in a dilatation of the main coronary and systemic arteries, decreased myocardial contractility, increased blood flow and oxygen delivery to the myocardial tissue, and decreased total peripheral resistance. This agent may also modulate multi-drug response (MDR) activity through inhibition of the p-glycoprotein efflux pump. benzenesulfonic acid;3-O-ethyl 5-O-methyl 2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate. This agent may also modulate multi-drug response (MDR) activity through inhibition of the p-glycoprotein efflux pump^[3].

Fig. 1: structure of Telmisartan

Fig. 2: Structure of Amlodipine besylate

The International Conference on Harmonization (ICH) guideline entitled “Stability testing of new drug substances and products” requires that stress testing be carried out to elucidate the inherent stability characteristics of the active substance^[4]. An ideal stability-indicating method is one that resolves the drug and its degradation products efficiently. Consequently, the implementation of an analytical methodology to determine Telmisartan and Amlodipine besylate simultaneously, in presence of its degradation products is rather a challenge for pharmaceutical analyst. Therefore, it was thought necessary to study the stability of Telmisartan and Amlodipine besylate under acidic, alkaline, oxidative, UV and photolytic conditions. This paper reports validated stability-indicating HPLC method for simultaneous determination of in presence of their degradation products. The proposed method is simple, accurate, reproducible, stability-indicating and suitable for routine determination of Telmisartan and Amlodipine besylate in combined dosage form. The method was validated in compliance with ICH guidelines^[5-9].

The literature review suggest that only very few analytical techniques like RP-HPLC, HPTLC, HPLC; spectroscopic methods have been reported for the determination of Telmisartan and its combination with different drugs in pharmaceutical dosage forms. Estimation of Amlodipine besylate with other drugs by spectrophotometric methods, LC-MS, HPLC and RP-HPLC^[10-13] has been reported. But there are no methods reported by simultaneous estimation methods for Telmisartan and Amlodipine besylate in combined dosage form 0.1N NaOH, as the combined dosage form is not official in any pharmacopoeia. A Combination of Amlodipine besylate and Telmisartan has been reported to show substantial and control of blood pressure in patients with hypertension to produce the maximum reduction in clinical cardiovascular end points, especially in patients with co morbidities like diabetes mellitus where more aggressive blood pressure lowering might be beneficial. Combination therapy is favored by the fact that multiple factors contribute to hypertension, and achieving control of blood pressure with single agent acting through one particular mechanism. Regimens can either be fixed dose combinations or drugs added sequentially one after other. Combining the drugs makes them available in a convenient dosing format, lower the dose of individual component, thus, reducing the side effects and improving compliance^[14].

MATERIALS AND METHODS:

Telmisartan and Amlodipine besylate of pharmaceutical grade were supplied by Yarrow Chem Pvt Ltd. Methanol and water used was of HPLC grade and were purchased from Spectrochem Pvt. Ltd. Mumbai, India. The tablet formulation containing 5mg of Telmisartan and 40mg of Amlodipine besylate was procured from local market and used for analysis of marketed formulation. Digital weighing machine from Shimadzu, Lab India Analytical UV 3092. Double beam UV –Visible Spectrophotometer, pH meter (Systronics model EQMK VI), a sonicator (Spectra Lab, model UCB 40), a hot air oven (Labhosp), UV chamber (Labhosp) were used in this study.

Preparation of Standard Stock Solutions:

The standard stock solution of Amlodipine besylate and Telmisartan were prepared by dissolving 10mg of each in a separate standard flask 50ml of 0.1N Sodium Hydroxide were added and sonicated for 20 min and then diluted to 100ml to produce a concentration of 100µg/ml respectively.

Calibration curves for Telmisartan and Amlodipine besylate:

Standard stock solution containing 100µg/ml was prepared of Amlodipine and Telmisaartan in 0.1N sodium hydroxide. The aliquots were prepared from the stock and diluted the volume to 10ml to obtain different concentration in the UV range 200-400nm. Amlodipine and Telmisartan show absorbance at 240nm and 230nm respectively. it is tabulated in table 1.

TABLE 1: PARAMETERS FOR CALIBRATION CURVES

Parameters (Units)	Telmisartan	Amlodipine besylate
Linearity range (µg/ml)	0-6	0-25
r²	0.999	0.997
Slope	0.109	0.0387
Intercept	0.013	0.017

Analysis of Marketed Formulations:

About 5 mg of Amlodipine besylate and 40mg of Telmisartan was accurately weighed and transferred into a 100 ml volumetric flask containing 50 ml of 0.1N Sodium hydroxide were added and sonicated until the tablet formulation get dissolved and diluted up to the mark with same solvent to get final concentrations of 50µg/ml of

Amlodipine besylate and 400µg/ml of Telmisartan respectively. The above solution was filtered using Whatman filter paper No 41.

Fig. 3: Chromatogram of Telmisartan

Fig. 4: Chromatogram of Amlodipine besylate

Method Validation:

The method of analysis for stability studies was validated as per the recommendations of ICH^[21] and USP^[22] for Telmisartan and Amlodipine besylate the parameters like accuracy, linearity, precision, detection limit, quantitation limit and robustness. The accuracy of the method was determined by calculating percentage recovery of Telmisartan and Amlodipine besylate. For both the drugs, recovery studies were carried out by applying the method to drug sample to which known amount of Telmisartan and Amlodipine besylate to 80, 100 and 120% of label claim had been added (standard addition method). At each level of the amount six determinations were performed and the results obtained were compared. Intraday and interday precision study of Telmisartan and Amlodipine was carried out by estimating the corresponding responses of every two hours for 5 times on the same day and on 6 different days for the concentration of 10 µg/ml of Telmisartan and Amlodipine and the results were tabulated in table 2.

TABLE 2: LINEAR REGRESSION DATA FOR CALIBRATION CURVES

Linearity
Concentration µg/ml	Telmisartan	Concentration µg/ml	Amlodipine besylate
0	0.008	0	0.001
2	0.234	5	0.211
3	0.345	10	0.401
4	0.456	15	0.599
5	0.5567	20	0.784
6	0.663	25	0.936

Fig. 5: Linearity chart for Telmisartan

Fig. 6: Linearity chart for Amlodipine besylate

The limit of detection (LOD) and limit of quantitation (LOQ) were calculated using following formulae: LOD= 3.3(SD)/S and LOQ= 10 (SD)/S, where SD=standard deviation of response (peak area) and S= average of the slope of the calibration curve. The summary of the results were tabulated in table 4.

TABLE 4: SUMMARY OF VALIDATION AND STRESS PARAMETERS

Parameter (Units)	Telmisartan	Amlodipine besylate
Linearity range (µg/ml)	50-300	10-60
Correlation coefficient	0.999	0.997
LOD (mg/ml)	1.41	1.96
LOQ (mg/ml)	4.24	5.72
Recovery (%)	100.57	100.35
Precision (%RSD)
Interday (n=3)	1.5	0.44
Intraday (n=3)	1.9	0.49

Forced degradation studies:

Forced degradation studies of both the drugs were carried out under various conditions like hydrolysis, dry heat, oxidation, UV light and photolysis. To 1ml of stock solution of Telmisartan and Amlodipine (tablet) which is used for forced degradation studies.

Forced degradation in acidic media was performed by keeping the 1ml of stock solution of drug in contact with 1N HCl and made up the volume to 10ml volumetric flask kept for 24hours and reading is taken. Forced degradation in basic media was performed by taking 1 ml of stock solution of Telmisartan and Amlodipine. To 1ml of solution of Telmisartan and Amlodipine (tablet) , 5ml of 1N Sodium hydroxide was added in 10ml volumetric flask flask kept for 24hours and reading is taken. Degradation with hydrogen peroxide was performed by taking 1 ml of stock solution of Telmisartan and Amlodipine and adding 5 ml of 3% (w/v) hydrogen peroxide and volume was made upto 10ml and reading is taken after 24 hours. For dry heat degradation, the mixture was heated at 60⁰ C for 24 hours and the readings were taken. The photo stability was also studied by exposing above stock solutions of both the drugs to direct sunlight. For UV degradation study, the stock solutions of both drugs (1000 µg/ml) were exposed to UV radiation of a wavelength of180-340nm for 24 hours. The results are tabulated in table 3.

TABLE 3: SUMMARY OF DEGRADATION STUDIES FOR TELMISARTAN AND AMLODIPINE

Degradation	Time	% Assay of active substance present in the drug Degradation
Condition		Telmisartan	Amlodipine besylate
Acidic degradation	24h	88.23%	98%
Basic degradation
	24h	96%	97.7%
Oxidative degradation		99%	94%
	24h
Dry Heat induced	24h	92%	96%
Photolytic degradation		94%	98%
	24h
UV Radiation at 256 nm	24h	95%	97%

RESULTS AND DISCUSSION:

The method of analysis for Telmisartan and Amlodipine in combination is validated as per the ICH guidelines parameters are included and stress studies were done. The drug obeys Beer’s law in the concentration range of 0-25µg/ml for Amlodipine and 0-6µg/ml for Telmisartan with the correlation coefficient of 0.997 and 0.999 respectively. The Intraday and Interday variation and %RSD were calculated and tabulated table 2. The Stress studies were included which contains parameters like acid, base, peroxidase, photolytic and the summary is tabulated in table 3.

A simple, precise, accurate and rapid UV Visible spectrophotometric method was done for the simultaneous estimation of Amlodipine and Telmisartan in pharmaceutical dosage form. The present study concluded the stress studies for the Amlodipine and Telmisartan according to ICH guidelines. The drug shows degradation at different stages. This method is accurate and be used routinely for analysis in normal lab condition.

REFERENCES:

1. https://www.healthline.com/health/telmisartan-oral-tablet#modal-close

2. https://en.wikipedia.org/wiki/Telmisartan

3. https://www.drugs.com/amlodipine.html

4. Dr.M.Nappinnai, Sonia.K,Stability studies of Nitroglycerine in different dosage forms (as per ICH guidelines). World Journal of Pharmacy and Pharmaceutical Sciences.2015 4(2):237-24.

5. ICH Q2B: Guidance for Industry: Validation of Analytical Procedures: Methodology U.S. Department of Health and Human Services Food and Drug Administration, (CDER), (CBER), 1996, 1-10.

6. ICH, Q1A, Stability Testing of New Drug Substances and Products, in: Proceedings of the International Conference on Harmonisation, Geneva, October, 1993.

7. ICH, Q2A, Hamonised Tripartite Guideline, Test On Validation of Analytical Procedures, IFPMA, in: Proceedings of the International Conference on Harmonization, Geneva, March, 1994.

8. ICH, Q2B, Hamonised Tripartite Guideline, Validation of Analytical Procedure: Methodology,IFPMA, in: Proceedings of the International Conference on Harmonization, Geneva, March 1996.

9. ICH Guidance on Analytical Method Validation, in: Proceedings of the International Convention on Quality for the Pharmaceutical Industry, Toronto, Canada, and September, 2002.

10. Shah NJ, Suhagia BN, Shah RR, Shah PB. Development and validation of a HPTLC method for the simultaneous estimation of telmisartan and hydrochlorothiazide in tablet dosage form. Indian J Pharm Sci., 69(2), 2007, 202-5.

11. Bankey S, Tapadiya GG, Saboo SS, Bindaiya S, Deepti J,Khadbadi SS. Simultaneous determination of ramipril, hydrochlorothiazide and telmisartan by spectrophotometry. Int J ChemTech Res., 2, 2008, 183-8.

12. Rane VP, Sangshetti JN, Shinde DB. Simultaneous High-Performance Liquid Chromatographic determination of telmisartan and hydrochlorothiazide in pharmaceutical preparation. J. Chromatogr. Sci, 46(10), 2008, 887-91.

13. Chitra P, Ganesa SS, Arumugam K, Suvarna K, Mallayasamy SR, U. N. Determination of telmisartan by HPTLC — A stability indicating assay. J Planar Chromatogr - Mod TLC, 20(6), 2007, 477-81.

14. Sonia .K, Nappinnai.M , Manikandan.K. Stability Indicating RP-HPLC Method for the Estimation of Metformin Hydrochloride and Repaglinide as API and Estimation in Tablet Dosage Form. International Journal of Pharmaceutical Quality Assurance 2016; 7(3); 46-50.

Received on 04.06.2018 Modified on 11.07.2018

Research J. Pharm. and Tech 2018; 11(11): 4825-4828.

DOI: 10.5958/0974-360X.2018.00877.6