1. INTRODUCTION:

Telmisartan¹ (TELM) is a non-peptide AIIRA that binds selectively to AT1 receptors, thereby blocking the physiological actions of angiotensin II. It is white to off-white crystalline powder that is insoluble in water, freely soluble in Methanol and Acetonitrile. It has a number of pharmacological properties that translate into an extended duration of antihypertensive efficacy over the entire 24-hour dosing period and particularly during the final 6 hours of dosing.² This property has the potential to reduce the increased incidence of adverse cardiovascular outcomes that coincide with the morning surge in blood pressure.³

Amlodipine besylate⁴ (AML) is one of the most commonly used long-acting calcium channel blocker (dihydropyridine class) in the management of patients with hypertension and in the treatment of angina.⁵ Amlodipine exhibits plaque stabilization properties and may prevent more number of events of stroke and myocardial infarction (MI) as compared to ARB .^6,7. HCT is a thiazide diuretic which helps to reduce BP by volume depletion. It has a long history of usage and has been tried in combination with many other antihypertensive drugs.⁸

The combined dosage form of telmisartan and amlodipine is available in the market for the treatment of hypertension.⁹ Literature survey reveals that some method was reported for the Multicomponent mode determination of these drugs in combined dosage form. The Aim of present work was to developed simple, accurate and rapid UV spectrophotometric methods for Multicomponent mode of Telmisartan and amlodipine in combined dosage form.

Fig. 1 Chemical structure of Telmisartan

Fig. 2 Chemical structure of Amlodipine besylate

MATERIALS AND METHODS:

Apparatus:

A Shimadzu model 1800 double beam UV-Visible spectrophotometer with spectral width of 1 nm, wavelength accuracy of ± 0.1 nm and a pair of 10 mm matched quartz cell was used to measure absorbance of all the solutions. Spectra were automatically obtained by UV-Probe system software (Ver.2.34).

Solvent used:

Methanol AR grade is used as solvent.

Preparation of standard stock solution:

Standard stock solution of Telmisartan and Amlodipine, Each having concentration of 1000 μg/ml, were prepared in methanol. From these solutions, 100 μg/ml of telmisartan and amlodipine respectively standard solution were prepared by dilution with methanol.

Selection of analytical wavelengths:

Appropriate dilutions were prepared for each drug from the standard stock solution and scanned in the spectrum mode from 200 nm to 400 nm. Amlodipine besylate and Telmisartan showed absorbance maxima at 238 nm and 295 nm respectively.

Preparation of calibration curve for Telmisartan and Amlodipine:

· Calibration curve for the Telmisartan –

Appropriate aliquots was pipetted out from the standard stock solution in to 10 ml volumetric flasks and dilutions were made with methanol to obtain working standard solutions of concentrations 2, 5, 10, 15, 20, 25 μg/ml of TELM.

Absorbance of these solutions was measured at 295 nm using “Multicomponent Mode” of the instrument, and a calibration curve of absorbance against concentration was plotted.

· Calibration curve for the Amlodipine–

Appropriate aliquots was pipetted out from the standard stock solution in to series of 10 ml volumetric flasks and dilutions were made with methanol to obtain working standard solutions of concentrations 2, 5, 10, 20, 30 μg/ml of AML. Absorbances of these solutions were measured at 238 nm using “Multicomponent Mode” of the instrument, and a calibration curve of absorbance against concentration was plotted.

Multi Component mode method

Calibration curves were plotted at 295nm for telmisartan and 238nm for Amlodipine. The regression equations of the two drugs were determined using calibration curves equations. The concentration of test solution was determined from the respective regression equations.

Stability in standard solutions

The standard solutions of Telmisartan and amlodipine were analysed after storing at 0, 1, 2, 4, 5, 6 hours at room temperature after preparation by the developed methods.

Analysis of tablet formulation:

Twenty tablets were weighed and their average weight was determined and finely powdered. The powder equivalent to 40 mg of TELM and 5 mg of AML was accurately weighed and transferred to 100 ml volumetric flask and dissolved in 50 ml methanol and the content was kept in Sonicator for 15 min. The flask was shaken and volume was made up to the mark with methanol to give a solution of 400 μg/ml of TELM and 50 μg/ml of AML. The solution was filtered through whatmann filter paper No. 41 and this solution was used as stock solution, from the above stock solution 0.5 ml of the aliquot was pipetted out and transferred to a 10 ml volumetric flask. The volume was made up to the mark with methanol to obtain a solution with final concentration of 20 μg/ml of TELM and 2.5 μg/ml of AML. Absorbance spectra of each solution against the methanol were measured at 295 nm and 238 nm. The absorbance of each solution was substituted in the Multi component mode equation to calculate the amount of the drug present.

Validation of proposed spectrophotometric method ¹⁰

Intra-day precision

Variation of results within the same day was analyzed. Intraday precision was determined by analyzing TELM and AML individually for three times in the same day at their selected analytical wavelengths.

Inter-day precision

Variation of results between the days was analysed. Inter-day precision was determined by analyzing TELM and AML individually daily once for three days at their selected analytical wavelengths.

Accuracy

Accuracy is the closeness of the test results obtained by the method to the true value. To study the accuracy, 20 tablets were weighed and powered and analysis of the same was carried out. Recovery studies were carried out by addition of the known amount of TELM and AML to the sample at three different concentration levels i.e. 80%, 100%, and 120% of label claim (standard addition method).

Precision

The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of homogenous samples. It is usually expressed as the standard deviation or relative standard deviation (coefficient of variation).

Repeatability

Standard solutions of TELM (2, 5, 10, 20, 25 μg/ml) were prepared and absorbance was measured at 295 nm using methanol as the blank. The absorbance of the same concentration solution was measured six times and standard deviation was calculated.

Similarly, standard solutions of AML (2, 5, 10, 15, 20, 25, 30 μg/ml) were prepared and absorbance was measured at 238 nm taking the methanol as the blank. The absorbance of the same concentration solution was measured six times and standard deviation was calculated.

Linearity and Range

The linearity of analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in sample within a given range. The range of analytical method is the interval between the upper and lower levels of analyte that have been demonstrated to be determined within a suitable level of precision, accuracy and linearity.

Limit of detection and limit of quantitation

Detection limit is the lowest amount of analyte in a sample that can be detected, but not necessarily quantitated. The quantitation limit is the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. Detection limit and quantitation limit were determined based on the standard deviation of response and slope of calibration curve.

RESULT AND DISCUSSION:

The absorbance of solution was measured at 295 nm and 238 nm for estimation of TELM and AML respectively (Fig 1 and 2).The calibration curves were constructed by plotting absorbance versus concentration and the regration equation were generated (Fig 3 and 4).

Fig. 3 Overlain spectra of Mixed Standards of Amlodipine besylate and Telmisartan.

Fig. 4 Overlain spectra of the Optimized standard solutions of Amlodipine besylate, Telmisartan and Mixture.

Fig. 5 Calibration curve for TELM at 295 nm.

Fig.6 Calibration curve for AML at 238 nm

Stability in standard solutions

Standard solutions of TELM and AML were found to be stable in methanol up to 6 hours of preparations when stored at room temperature

Validation of the method:

Results of validation studies are summarized in Table 1. The accuracy of the method was confirmed by recovery studies from tablet at 80%, 100% and 120% levels of standard addition and the results are depicted in Table 2. Recovery in the range of 98-102% justifies the accuracy of the method.

Table 1 Statistical data of TELM and AML at 295 nm and 238 nm respectively by simultaneous equation method

Parameter	TELM at 295 nm	AML at 238 nm
Linear Range (μg/ml)	2-25	2-30
Slope	0.0511	0.0335
Intercept	0.0001	0.0047
Correlation coefficient (r²)	0.9994	0.9999
Limit of Detection (μg/ml)	0.0850	0.1552
Limit of Quantitation (μg/ml)	0.2566	0.4695

Table 2 Results of recovery studies

Level	% Recovery
Level	TELM	AML
80%(n=3)	98.95	98.78
100%(n=3)	99.19	98.64
120%(n=3)	99.24	99.09

CONCLUSION:

The result shows that the developed spectrophotometric method is sensitive, precise and specific for Multi-Component Mode UV Spectrophotometric of TELM and AML. Proposed study describes method for the estimation of TELM and AML combination in mixture. The method was successfully used for determination of drugs in their pharmaceutical formulation.

ACKNOWLEDGMENTS:

The authors are thankful to USV Pharmaceutical Ltd. Baddi, India for providing gift sample of Telmisartan and Amlodipine besylate. The authors are very thankful to Director, HOD and Management of IIMT College of Medical Sciences for providing necessary facilities to carry out research work.

REFERENCES:

1. Mahathi, G., 2011, development and validation of Telmisartan and Atorvastatin calcium in combined dosage form by RP-HPLC, International Journal Of Pharmacy and Technology, Vol. 3, Issue 3, pp. 3370-3389.

2. Doshi, N. and Sheth, A., 2010. Simple, accurate and precise RP-HPLC method for the simultaneous determination of amlodipine and telmisartan in marketed formulation, Journal of Chemical and Pharmaceutical Research, Vol. 2, Issue 2, pp. 252-263.

3. Gupta, Y., 2009. Isocratic RP-HPLC-UV method development and validation for the simultaneous estimation of ramipril and Telmisartan in tablet dosage form, Asian Journal of Pharmaceutical and Clinical Research, Vol. 2, Issue 4, pp. 104-111.

4. Maladkar, M., 2012, Triple drug combination of telmisartan, amlodipine and hydrochlorothiazide in the treatment of essential hypertension, Open Journal of Internal Medicine, Vol. 2, PP. 67-71.

5. Cetin, G. and Sungur, S., 1995. A rapid and sensitive method for the spectrophotometric assay of amlodipine was based on the formation of a colored derived during the reaction of the drug, Scientia Pharmaceutica, Vol. 63, Issue 2, pp. 93-98.

6. Mason, R, P,. 2002, Mechanisms of plaque stabilization for the dihydropyridine calcium channel blocker am- lodipine, Review of the evidence Atherosclerosis, Vol. 165, PP. 191.

7. Portu, S. and Mantovani, L, G., 2009, Amlodipine, A pharmacoeconomic review, Journal of Medical Econom- ics, Vol.12, PP. 60-68.

8. Moen, M.D., 2010. Worked on Telmisartan /amlodipine a single-pill combination of Telmisartan, an angiotensin II receptor antagonist, and amlodipine, a dihydropyridine calcium channel antagonist, WHO, Vol. 10, Issue 6, pp. 401-412.

9. Taylor, J.B., and Triggle, D.J., 2006. Comprehensive Medicinal Chemistry, New York, Elsevier Sciences Inc, Vol. 2, pp. 10-15.

10. ICH, Q2 (R1) Validation of analytical procedure: Text and methodology, International Conference on Harmonization, Geneva, Switzerland, 2005.

Received on 01.08.2013 Modified on 19.08.2013

Asian J. Research Chem. 6(10): October 2013; Page 973-976