1. INTRODUCTION:

Analytical method development and validation play important role in the discovery, development and manufacture of pharmaceuticals. Drug ‘analysis’ means identification, characterization and determination of drugs, drug assay refers to determination of drugs in mixtures such as dosage forms and biological fluids. Bulk drugs are obtained by chemical synthesis, biosynthesis, isolation from plants or animals or biotechnological source. The elevation of dosage forms varies with type of dosage form. It includes physical appearance, strength, content uniformity, active ingredient etc. various components present in dosage forms, including the presence of additives, impurities and multiple drug entities such as vitamins present are some of the challenges encountered during the development¹.

Stability study is an integral part of any product development it is important in the pharmaceutical dosage forms. Stability is the ability of a pharmaceutical product to retain its chemical, physical and microbilogical and biopharmaceutical properties within the specified limits throughout its shelf-life. The world is divided into four zones based on the prevailing annual climatic conditions. The period of time during which a drug product, if stored expected too comply with the specification as determined by stabiity studies on a number of batches of the product. Present work aims to represent the stability testing requirements of International Conference on Harmonization².

Telmisartan is an Angiotensin 2 Receptor Blocker. The mechanism of action of telmisartan is as an Angiotensin 2 Receptor Antagonist³. Telmisartan is a benzimidazole derivative and a non-peptide angiotensin II receptor antagonist with antihypertensive property. 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 pressure⁴. 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 injury⁵. Ramipril is a long-acting angiotensin-converting enzyme inhibitor. It is a prodrug that is transformed in the liver to its active metabolite ramiprilat⁶.Ramipril is an Angiotensin Converting Enzyme Inhibitor. The mechanism of action of ramipril is as an Angiotensin-converting Enzyme Inhibitor⁷. Ramipril is a prodrug and nonsulfhydryl angiotensin converting enzyme (ACE) inhibitor with antihypertensive activity. Ramipril is converted in the liver by de-esterification into its active form ramiprilat, which inhibits ACE, thereby blocking the conversion of angiotensin I to angiotensin II. This abolishes the potent vasoconstrictive actions of angiotensin II and leads to vasodilatation. This agent also causes an increase in bradykinin levels and a decrease in angiotensin II-induced aldosterone secretion by the adrenal cortex, thereby promoting diuresis and natriuresis⁸.

Figure1: Chemical structure of Telmisartan

Figure 2: Chemical structure of Ramipril

2. MATERIALS AND METHODS:

2.1. Materials:

Both Temisartan and Ramipril Active pharmaceutical ingredient were procured from Yarrow chemicals, Mumbai, India. The formulation Telma-R tablet containing telmisartan 40mg and Ramipril 5mg were procured from local pharmacy. Solvents like Methanol of analytical grade were obtained from Distilled water are used for the experiment.

2.2. Apparatus:

Digital weighing machine from Shimadzu, Lab India Analytical UV 3092 Double beam UV–Visible Spectrophotometer were used for the experiment.

2.3. Standard stock solution Preparation:

Accurately weighed and transferred into 100mL standard flasks of 100mg of each drug Telmisartan and Ramipril in two separate standard flasks and dissolved in 100mL of methanol to get concentration of 1mg/mL of each. From this stock solution 1mL is pipetted out and diluted with methanol to get working standard solution of 100µg/mL of both drugs.

2.3. λ max determination:

Both Telmisartan and Ramipril standard concentration were prepared of dilutions 10µg/mL, both the solutions were scanned in UV range (200-400nm) against solvent blank. The wavelength spectra of TEM and RAM in methanol are shown in Fig. 3, 4 respectively. The representative spectra revealed that TEM shows a well defined λmax at 255nm whereas RAM shows at 210nm. These two wavelengths were selected for development of simultaneous equation.

2.4. Preparation of calibration curve:

Telmisartan and Ramipril working standard solutions were prepared of dilution 100µg/mL. Different dilution were taken from standard stock solution and diluted with methanol in the concentration of 2µg/mL, 4µg/mL, 6µg/mL, 8µg/mL, 10µg/mL and 12µg/mL solutions respectively. Prepared working solutions of Telmisartan and Ramipril were scanned at 255nm and 210nm respectively. The respective absorbances were recorded and these absorbances were plotted against the concentrations to obtain the respective calibration curves (Fig. 5 and Fig. 6).

2.5. Sample preparation:

Twenty tablets were weighed and finely powdered in a mortar. The equivalent amount of 40mg of Telmisartan and 5mg of Ramipril was accurately weighed and transferred into a 100mL clean dry volumetric flask containing 70mL of methanol and sonicated for 5min and the drug was dissolved completely. The volume was made up to the mark with a methanol to get a stock concentration of Telmisartan and Ramipril. The solution was filtered by using whatmann filter paper, discarding first few mL. Pipette out 2.5mL of the stock solution into a 10mL volumetric flask and the volume was made up to the mark with the methanol. One mL above stock solution is pipette out into a 10mL volumetric flask and the volume was made up to the mark with the methanol.

3. RESULTS:

3.1. Linearity:

For the estimation of Telmisartan and Ramipril were found to be 255nm and for Ramipril was found to be 210nm in methanol solvent. The linearity was checked in the concentration range of 2-12µg/mL for both Telmisartan and Ramipril. (Table 1 and Figure 3and 4).

Table 1: Linearity results of Telmisartan and Ramipril

S. No.	Concentration (µg/mL)	Telmisartan Absorbance	Ramipril Absorbance
1	0	0	0
2	2	0.099	0.099
3	4	0.197	0.209
4	6	0.298	0.340
5	8	0.411	0.446
6	10	0.521	0.550
7	12	0.648	0.663
Slope		0.053	0.055
Intercept		-0.011	-0.005
Regression Equation (y)		0.053x-0.011	0.055x-0.005
Correlation Coefficient		0.998	0.999

Figure 3: Linearity graph of Telmisartan

Figure 4: Linearity graph of Telmisartan

3.2. Precision:

Precision of the method was measured in terms of intra-day and inter-day variation (%RSD). For the intra-day precision 0-10 hours with the interval of every two hours and interday precision 1-6 days were choosen and readings were taken for every day for Telmisartan and Ramipril and tabulated in table 2.

3.3. Stability parameter:

An accurately weighed quantity of tablet formulation which is equivalent to 40mg of Telmisartan and 5mg of Ramipril was transferred into 100 mL volumetric flask and kept under following conditions which include Alkaline (0.1 N NaOH), Acidic (0.1 N HCl) reflux for 3 hr, 3% H₂O₂ at 50ºC, heat (60ºC), humidity (75% RH) for 24 hr and after the specified time volume was made upto the mark with distilled water, filtered using Filter paper no. 41. From this stock solution 5 mL portion of the filtrate was pipette out and further diluted with distilled water in a 100 mL volumetric flask upto mark (10 µg/mL). The standard stock solution of two drugs were prepared and compared against a label claim and results were tabulated in table 3.

3.4. Limit of Detection and Limit of quantification:

The limit of detection and limit of quantification for Telmisartan were found to be 0.15µg/mL and 0.32µg/mL respectively. The limit of detection and limit of quantification for Ramipril were found to be 0.35µg/mL and 0.95µg/mL (Table 4) respectively.

3.5. Accuracy:

Accuracy was determined for both drugs by spiking with 80, 100 and 120% of additional pure drug and the mean recovery of the Telmisartan and Ramipril were found to be 99.67% and 99.58% respectively (Table 5).

Table 2: Intra-day and Inter-day precision results of Telmisartan and Ramipril

Intra-day precision				Inter-day precision
S. No	Time (Hours)	Telmisartan Absorbance	Ramipril Absorbance	Time (Days)	Telmisartan Absorbance	Ramipril Absorbance
1	0	0.503	0.546	1	0.506	0.545
2	2	0.502	0.542	2	0.504	0.543
3	4	0.501	0.540	3	0.502	0.544
4	6	0.502	0.544	4	0.501	0.546
5	8	0.502	0.545	5	0.501	0.546
6	10	0.503	0.546	6	0.502	0.547
Mean		0.502	0.543	Mean	0.502	0.545
SD		0.00044	0.00089	SD	0.00013	0.0013
%RSD		0.08	0.16	%RSD	0.25	0.24

Table 3: Results of stability studies parameters

Sample (treated)	Percent Label claim
	Comparison with standard	A (1%, 1cm)
0.1 N NaOH	98.67	99.11
0.1 N HCl	95.19	95.92
60ºC for 2hr	98.79	99.01
Humidity (75% RH)	95.97	96.52

Table 4: LOD and LOQ of Telmisartan and Ramipril

Parameter	Telmisartan measured value (µg/mL)	Ramipril measured value (µg/mL)
Limit of detection	0.15	0.35
Limit of quantification	0.32	0.95

Table 5: Recovery studies for Telmisartan and Ramipril

Telmisartan				Ramipril
	80%	100%	120%	80%	100%	120%
Std. conc. (µg/mL)	10	10	10	10	10	10
Conc. added (µg/mL)	8	10	12	8	10	12
Conc. found (µg/mL)	7.98	9.96	11.96	7.96	9.95	11.97
% Recovery	99.75	99.60	99.66	99.50	99.50	99.75
% Mean recovery	99.67				99.58

3.6. Assay:

The percentage purity for the assay of Telmisartan and Ramipril were found to be 99.60% and 100.04% respectively.

Table 6: Assay results of Telmisartan and Ramipril formulations

Formulation		Label claim	Amount found	% Assay
Ramtel	Telmisartan	40mg	39.94mg	99.85
Ramtel	Ramipril	5mg	5.01mg	100.02

4. DISCUSSION:

For the estimation of Telmisartan and Ramipril from bulk samples and their tablet dosage forms was determined by simultaneous equation method by using UV Spectrophotometer. The regression equation of the linearity curve between concentrations of Telmisartan and Ramipril over its absorbances were found to be y=0.053x-0.011and y=0.055x0.005 respectively with a correlation coefficient (r²) of 0.998 for Telmisartan and 0.999 for Ramipril. The % RSD for intra-day precision and inter-day precision for Telmisartan were found to be 0.08% and 0.25% respectively. The % RSD for intra-day precision and inter-day precision for Ramipril were found to be 0.16% and 0.24% respectively. This confirms that the method is sufficiently precise. Accuracy was determined for both drugs by spiking with 80, 100 and 120% of additional pure drug and the % mean recovery of the Telmisartan and Ramipril were found to be 99.67 and 99.58 respectively (Table 5). The percentage purity for the assay of Telmisartan and Ramipril were found to be 99.85% and 100.02% respectively (Table 6). The assay results showed that the proposed method was selective for simultaneous estimation of Telmisartan and Ramipril without interference from the excipients used in tablet dosage form.

5.CONCLUSION:

As there were several methods reported for the determination of Telmisartan and Ramipril but there is no any stability indicating, simultaneous determination, so the recent work is useful for stability indicating, simultaneous determination of Telmisartan and Ramipril in combined dosage form. It is a unique method gives higher and speedy throughput analysis. Stress degradation studies were carried out by conditions such as acidic, alkaline, oxidative, hydrolytic, thermal and photolytic for specificity. By the study of validation parameters, it is confirmed that the results obtained from this analytical method are reliable, within the acceptance criteria and the proposed method is immensely robust as per the ICH guideline.

6. REFERENCE:

1 Sonia. K, Dr. M. Nappinnai. Development and Validation of HPLC and UV-Visible spectrophotometric method for the pharmaceutical dosage form and Biological fluid-Review. European journal of Biomedical and pharmaceutical science. 2016;3 (3):382-391.

2 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):237-246.

3 https://www.accessdata.fda.gov/spl/data/e8086737-020f-442c-a9d8-b7c4d95e1106/e8086737-020f-442c-a9d8-b7c4d95e1106.xmL

4 https://ncit.nci.nih.gov/ncitbrowser/ConceptReport.jsp?dictionary=NCI_Thesaurusandns=NCI_Thesaurusandcode=C47746

5 https://livertox.nlm.nih.gov//Telmisartan.htm

6 https://www.ncbi.nlm.nih.gov/mesh/68017257

7 https://www.accessdata.fda.gov/spl/data/4cbe04ad-123a-4b91-af61-f140c650e95b/4cbe04ad-123a-4b91-af61 f140c650e95b.xmL

8 https://ncit.nci.nih.gov/ncitbrowser/ConceptReport.jsp?dictionary=NCI_Thesaurusandns=NCI_Thesaurusandcode=C29411

Received on 11.03.2018 Modified on 29.04.2018

Research J. Pharm. and Tech 2018; 11(7): 3087-3090.

DOI: 10.5958/0974-360X.2018.00567.X