INTRODUCTION:
Telmisartan,4-((2-n-propyl-4-methyl-6-(1-methylbenzimidazol – 2 – yl) –benzimidazol –1-yl) methyl) biphenyl-2- Carboxylic acid (Fig.1), blocks the vasoconstrictor and aldosterone secreting effects of angiotensin II by selectively blocking the binding of angiotensin II to the AT1 receptor in many tissues, such as vascular smooth muscle and the adrenal gland. Literature survey revealed that there are many methods like UV1, 2, HPTLC3, RP-HPLC4 and LC–MS/MS5 for determination of Telmisartan. The simultaneous estimation methods are also available for telmisartan in combination like HPTLC6, RP-HPLC7, 8, TLC Densitometry9. The aim of this work is to develop and validate a simple, accurate and low cost analytical method by using UV spectrophotometry for the estimation of telmisartan. The developed method was simple, precise, specific and accurate. The statistical analysis proved that method is reproducible and selective for the analysis of telmisartan in bulk drug and tablet formulations.
Fig. 1: Structure of Telmisartan.
MATERIAL AND METHOD:
Apparatus:
Shimadzu UV-1800 double beam spectrophotometer with 1 cm path length supported by Shimadzu UV-Probe software, version 2.21 was used for spectral measurements with 1 cm matched quartz cells. Shimadzu balance (BL-220H) was used for all weighing.
Pure samples:
Telmisartan was kindly gifted by Cipla Ltd., India.
Marketed Formulation:
TelsartanTM 40 (Dr. Reddy’s Laboratories Ltd. Hyderabad, India) was taken for study which contains Telmisartan- 40mg.
Reagents and Chemicals:
DMF (Merck Ltd., Mumbai, India) and other reagents and chemicals used were of analytical grade. Double Distilled water was used throughout the experiment.
Preparation of standard stock solution:
Standard drug solution of Telmisartan was prepared by dissolving 25mg of drug in 25ml mixture of DMF and 0.1N HCl in the ratio of 1:9 respectively. 10 ml from it was transferred into 100ml of volumetric flask and volume was made up to mark with above same solvent (DMF and 0.1 N HCl mixture in the ratio 1:9) which makes the stock solution of 100 μg/ml concentration.
Determination of λmax:
From the above stock solution, 1 ml. was pipetted out into a 10mL volumetric flask and the volume was made up to the mark with solvent (DMF and 0.1 N HCl mixture in the ratio 1:9) to prepare a concentration of 10 μg/ml. Then the sample was scanned in UV-VIS Spectrophotometer in the range 400-250nm using distilled water as a blank and the wavelength corresponding to maximum absorbance (λmax) was found to be 291nm(Fig.2).
Fig.2: UV Spectrum of Telmisartan.
Preparation of calibration curve:
Aliquots of 0.3, 0.6, 0.9, 1.2, 1.5 ml portion of stock solutions were transferred to 10 ml volumetric flasks, and volume made up to mark with solvent (DMF and 0.1 N HCl mixture in the ratio 1:9 resp.) to produce 3 μg/ml, 6 μg/ml, 9 μg/ml, 12 μg/ml, 15 μg/ml solutions respectively. Then measure the absorbance of these solutions at λmax 291 nm using solvent (DMF and 0.1 N HCl mixture in the ratio 1:9) as blank. Then, the calibration curve was plotted by taking concentration on X-axis and absorbance on Y-axis. The curve showed linearity in the concentration range of 3-15 μg/ml. The correlation coefficient (r˛) was found to be 0.999(Fig.3).
Fig.3: Calibration curve of Telmisartan.
Assay of Telmisartan (TELSARTANTM 40 - 40mg):
Five tablets were weighed and powdered. The amount of tablet powder equivalent 25mg of Telmisartan was taken in a 25ml volumetric flask and it was dissolved in solvent and made up to the mark with solvent. Then the solution was filtered using Whatmann filter paper No.40. From this filtrate, appropriate dilutions were made with solvent to obtain the desired concentration (6, 9 and 12 μg/ml). These solutions were analyzed in UV and the result was indicated by % recovery given in Table 8.
METHOD VALIDATION:
Validation is a process of establishing documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics.
The method was validated as per ICH guidelines for different parameters like Linearity, Accuracy, Precision, Robustness, Ruggedness, Limit of Detection (LOD) and Limit of Quantification (LOQ) [Table 1].
Linearity:
Various aliquots were prepared from the stock solution (100μg/ml) ranging from 3-15 μg/ml. The samples were scanned in UV-VIS Spectrophotometer using solvent (DMF+0.1N HCl in the ratio 1:9) as blank. Linearity was found in the concentration range of 3-15μg/ml (Table 2). The correlation co efficient was found to be 0.999. Optical characters were mentioned in Table 3.
Accuracy:
The accuracy of the method was determined by preparing solutions of different concentrations that is 50%, 100% and 150% in which the amount of marketed formulation (TELSARTANTM) was kept constant (6 mg) and the amount of pure drug was varied that is 50%, 100% and 150% respectively. The solutions were prepared in triplicates and the accuracy was indicated by % recovery (Table 4).
Precision:
Precision of the method was demonstrated by intra-day and inter-day variation studies. In intra-day variation study, six solutions of same concentration that is 9μg/ml were prepared and analysed three times in a day i.e. morning, afternoon and evening and the absorbances were noted. The result was indicated by % RSD (Table 5). In the inter-day variation study, six solutions of same concentration (9 μg/ml) were prepared and analysed three times for three consecutive days and the absorbances were noted. The result was indicated by % RSD (Table 6).
Robustness:
Robustness of the method was determined by carrying out the analysis at different wavelengths (i.e. 291±0.5 nm). The respective absorbances were noted and the result was indicated by % RSD (Table 7).
Ruggedness:
Ruggedness of the method was determined by carrying out the analysis by two different analysts and the respective absorbances were noted. Result was indicated by % RSD (Table 10).
Limit of Detection (LOD):
The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample. The LOQ was calculated using the formula involving standard deviation of response and slope of calibration curve (Table 9).
LOD =
Limit of Quantification (LOQ):
The LOQ is the concentration that can be quantified reliably with a specified level of accuracy and precision. The LOQ was calculated using the formula involving standard deviation of response and slope of calibration curve (Table 9).
LOQ =
RESULTS AND DISCUSSION:
The developed method was found to be precise as the %RSD values for intra-day and inter-day were found to be less than 2%. Good recoveries (99.99% to 100.48%) of the drug were obtained at each added concentration, which indicates that the method was accurate. The LOD and LOQ were found to be in sub-microgram level, which indicates the sensitivity of the method. The method was also found to be robust and rugged as indicated by the %RSD values which are less than 2%.
The results of assay show that the amount of drug was in good agreement with the label claim of the formulation as indicated by % recovery (99.87%). Summary of validation parameters of proposed spectrophotometric method.
TABLE 1: SUMMARY OF VALIDATION
|
Parameter |
Result |
|
Precision indicated by %RSD Accuracy indicated by average % recovery Limit of detection (LOD), μg ml-1 Limit of quantitation (LOQ), μg ml-1 Linear regression equation Robustness indicated by %RSD Ruggedness indicated by %RSD Assay indicated by % purity |
0.59, 0.61 100.27 + 0.872% 0.44 µg/ml 1.32 µg/ml y=0.058x+0.008 0.20 0.59, 0.74 98.4 – 100.4 % |
TABLE 2: LINEARITY OF TELMISARTAN
|
S.No. |
Concentration (µg/ml) |
Absorbance |
|
1. 2. 3. 4. 5. |
3 6 9 12 15 |
0.188 0.362 0.535 0.712 0.872 |
TABLE 3: OPTICAL CHARACTERISTIC OF TELMISARTAN
|
Optical characteristics |
Result |
|
Beer’s law limit (µg/ml) 10-60 Correlation coefficient (r2) Regression equation y=0.015x+0.002 Slope (a) 0.015 Intercept (b) 0.002 |
3-15 0.999 y=0.058x+0.008 0.058 0.008 |