INTRODUCTION:

Telmisartan is a non peptide molecule. It is chemically described^1-5 as 2-(4-{[4-Methyl-6-(1-methyl-1H-1,3-benzodiazol-2-yl)-2-propyl-1H-1,3-benzodiazol-1-yl]methyl}phenyl)benzoic acid. Telmisartan is a non peptide angiotensin II receptor antagonist which selectively and insurmountably inhibits angiotensin II AT1 receptor subtype without affecting other systems involved in cardiovascular regulation. Telmisartan blocks the vasoconstrictor and aldosterone secretion effect 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. Its action is therefore independent of the pathways for angiotensin II synthesis. AT2 receptor is found in many tissues. But AT2 is not known to be associated with cardio vascular homeostasis.

Telmisartan has greater affinity (>3,000 fold) for AT1 receptor than for the AT2 receptor. Ramipril’s chemical name is . (2S,3aS,6aS)-1-[(2S)-2-{[(2S)-1-ethoxy-1-oxo-4-phenylbutan-2-yl]amino}propanoyl]-octahydrocyclopenta[b]pyrrole-2-carboxylic acid. Ramipril is an angiotensin converting enzyme (ACE) inhibitor. An inactive prod rug, Ramipril is converted to ramiprilat in the liver and is used to treat hypertension and heart failure, to reduce proteinuria and renal disease in patients with nephropathies, and to prevent stroke, myocardial infarction, and cardiac death in high-risk patients. Ramiprilat, the active metabolite, competes with angiotensin I for binding at the angiotensinconverting enzyme, blocking the conversion of angiotensin I to angiotensin II. As angiotensin II is a vasoconstrictor and a negative-feedback mediator for rennin activity, lower concentrations result in a decrease in blood pressure and an increase in plasma rennin. Ramiprilat may also act on kininase II, an enzyme identical to angiotensin-converting enzyme that degrades the vasodilator bradykinin.

Literature survey^6-11 revealed that there were few methods reported for the estimation of Telmisartan and Ramipril by RP-HPLC and UV Spectrophotometry in the combined dosage form. And also HPLC, Spectroscopic methods have been reported for the estimation of individual drugs and in combination with other drugs. Our study, attempts to develop a simple, precise, accurate, sensitive and economical method for the simultaneous estimation of Telmisartan and Ramipril by UV-Spectrophotometric method in pharmaceutical dosage form.

Figure 1: Chemical Structure of Telmisartan

Figure 2: Chemical Structure of Chlorthalidone

EXPERIMENTAL:

Instrumentation:

Double beam UV Visible - Spectrophotometer, Labindia (Brand), UV 3000⁺with a pair of 1cm matched quartz cells, All weighing was done on Shimadzu electric balance, capacity (220gm), readability (0.001gm) and sonication was done in Digital Ultra Sonicator Citizen.

Table 1: Absorbance values of Telmisartan with 0.1N NaOH.

S.NO.

Concentration(µg/mL)

Absorbance

at 232nm

At 222nm

0.511

0.742

0.994

1.168

1.373

0.365

0.453

0.532

0.607

0.670

Chemicals and Reagents:

Telmisartan and Ramipril RS were procured from R and K Pharmaceuticals, Vishakapatnam. Marketed formulation Telma-R (Glenmark Pharmaceutical Ltd., H.P.), with label claim 40mg TEL, 5mg RAM, was purchased from local market of Siddipet, Telangana. Sodium hydroxide of analytical grade and double distilled water were used throughout the analysis.

Table 2: Absorbance values of Ramipril with 0.1N NaOH

S.NO.

Concentration(µg/mL)

Absorbance

at 222nm

At 232nm

0.058

0.094

0.131

0.172

0.206

0.023

0.035

0.050

0.064

0.079

Preparation of 0.1N NaOH:

4gm. of NaOH was added to sufficient quantity of distilled water and finally volume was made up to 1000mL with distilled water to get the concentration of 0.1N NaOH.

Solubility Testing:

As 0.2M H₂S0₄was already reported as solvent, a trial was made to get an alternate solvent for estimation of Telmisartan and Ramipril by using 0.1N NaOH solution. It has been found that 0.1N NaOH is more suitable as solvent in terms of both solubility and stability.

Preparation of standard stock solutions:

Standard stock solutions of Telmisartan and Ramipril were prepared by dissolving 100mg of each drug in 100mL of 0.1N NaOH individually to get the concentration of 1000µg/mL.

Preparation of working standard solutions:

10mL of each drug solution was taken from the stock solution and diluted to 100mL with 0.1N NaOH in 100mL volumetric flask to get the concentration of 100µg/mL individually.

Determination of λmax:

The working standard dilutions of each drug were scanned from 200nm-400nm to determined the λmax individually. Telmisartan and Ramipril were showed maximum absorbance at 232nm and 222nm respectively.

Validation Parameters:

Linearity Range:

Adequate dilutions were made from working standards to get the concentrations of 4-20µg/mL for both Telmisartan and Ramipril using 0.1N NaOH. Absorbance of these solutions were determined at their corresponding λmax. The measured absorbance was plotted against concentrations.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The detection limit of an individual analytical procedure is the lowest of analyte in a sample which can be detected but not necessarily be quantitated as an exact value. The quantification limit of an analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. It is used particularly for determination of impurities and/degradation studies. For these LOD and LOQ five sets of linear dilutions were prepared and standard graphs were drawn. From the standard graphs standard deviation of the intercept and mean of the slope were calculate, then LOD and LOQ values were calculated using the following formulas.

LOD = 3.3 S_a/b

LOQ = 10 S_a/b

S = Standard deviation of intercept

b = mean of slope of calibration curve

Precision Studies:

System Precision:

A fixed concentration of 12:1.5µg/mL of the each standard drug as mixture from the linearity range was checked for absorbance, then SD and percentage RSD values were calculated.

Method Precision:

A fixed concentration in the ratio of 8:1µg/mL of the marketed formulation from the linearity range was checked for absorbance, then SD and percentage RSD values were calculated.

Intraday Precision:

Dilutions of 4,8,12 µg/mL concentration mixtures were prepared from two drugs. Absorbance of all the dilutions was checked for every one hour for 5hours, then SD and percentage RSD values were calculated.

Inter day Precision:

Dilutions of 4,8,12 µg/mL concentration mixtures were prepared from standard drugs. Absorbance of all the dilutions was checked for 5 days, then SD and percentage RSD values were calculated.

Assay:

Accurately weighed and powered 20 tablets of Telma-R, Manufactured by Glenmark Pharmaceutical Ltd, Himachal Pradesh. It contains 40mg of Telmisartan and 5mg of Ramipril in the ratio of 8:1. A weight equivalent to 50mg Telmisartan of the powdered tablet was taken and to this added 25mL of 0.1N NaOH in 50mL standard volumetric flask. Ultrasonication was down for 30min. and kept overnight for dissolving. Again ultrasonication was done filtered using Whatman filter paper grade 1, then volume was made up to the mark with 0.1N NaOH. The stock solution prepared above contains both the drugs in the ratio of 1:8. 10 mL From the stock solution was taken in the 100mL volumetric flask and the volume was made up to the mark to get the concentration of 100µg/mL of Telmisartan and corresponding concentration of Ramipril (working standard). From working standard following dilutions were prepared for determination of the percentage purity of the marketed formulation. 8:1 µg/mL: 0.8mL of working standard was taken and diluted to 10mL to get concentration of 8µg/mL of Telmisartan and 1 µg/mL of Ramipril. 12:1.5 µg/mL: 1.2mL of working standard was taken and diluted to 10mL to get concentration of 12µg/mL of Telmisartan and 1.5µg/mL of Ramipril. 16:2 µg/mL : 1.6mL of working standard was taken and diluted to 10mL to get concentration of 16µg/mL of Telmisartan and 2µg/mL of Ramipril. Amount of Telmisartan and Ramipril present in the marketed formulation was determined by using the following simultaneous equations.

C_T = A₂ay₁– A₁ay₂/ ax₂ay₁– ax₁ay₂

C_R = A₁ax₂ – A₂ax₁ / ax₂ay₁ – ax₁ay₂

Where C_T = concentration of Telmisartan

C_R = concentration of Ramipril

A₁, A₂ = Absorbance of drug at the selected two wavelengths

ax₁, ax₂ = Absorptivity values of Telmisartan

ay₁, ay₂= Absorptivity of Ramipril

Amount found = Concentration in mg. × Dilution factor × Average weight

% Purity = Amount found / Labeled claim × 100

Recovery Studies (Accuracy):

To ensure the reliability (accuracy) of the method recovery studies were carried out by mixing standard quantity of drug with the pre analysed sample formulation and the contents were reanalyzed by the proposed method. To perform the recovery studies 3 dilutions were prepared using both standard drug and marketed formulation. The dilutions prepared were having concentrations of the marketed formulation in the ratio of 4:0.5 (TEL:RAM) was kept fixed and the standard drugs mixture in the ratio of 8:1(TEL:RAM) was added in 50%, 100%, 150% respectively.

First dilution (6:0.75):

To 4:0.5(TEL:RAM) µg/mL of marketed formulation 2:0.25(TEL:RAM) µg/mL standard drugs mixture was added in the 10mL volumetric flask to get the final concentration of 6:0.75 µg/mL and its absorbance value was observed.

Second dilution (8:1):

To 4:0.5(TEL:RAM) µg/mL of marketed formulation 4:0.5 (TEL:RAM) µg/mL standard drugs mixture was added in the 10mL volumetric flask to get the final concentration of 8:1 µg/mL and its absorbance value was observed.

Third dilution (10:1.25):

To 4:0.5(TEL:RAM) µg/mL of marketed formulation 6:0.75 µg/mL standard drugs mixture was added in the 10mL volumetric flask to get the concentration of 10:1.25 µg/mL and its absorbance value was observed.

RESULTS AND DISCUSSION:

Fig.5 : Standard curve of TEL at 232nm

Fig.6 : Standard curve of TEL at 222nm

Fig.7 : Standard curve of RAM at 222nm

Fig.8 : Standard curve of RAM at 232nm

Table 3 : Absorptivity values of Telmisartan and Ramipril

S.NO.	Conc.(µg/mL)	Absorptivity of TEL		Absorptivity of RAM
S.NO.	Conc.(µg/mL)	232nm	222nm	232nm	222nm
1. 2. 3. 4. 5.	4 8 12 16 20	12.775×10^-2 9.2×10^-2 8.2×10^-2 7.3×10-² 6.8×10^-2	9.125×10^-2 5.665×10^-2 4.43333×10^-2 3.79375×10^-2 3.35×10^-2	0.575×10^-2 0.4375×10^-2 0.4166×10^-2 0.4×10^-2 0.39×10^-2	1.45×10^-2 1.175×10^-2 1.0916×10^-2 1.075×10^-2 1.03×10^-2
	Avg.	8.899×10^-2	5.27291×10^-2	0.44483×10^-2	1.1643×10^-2

Limit of Detection (LOD) and Limit of Quantification (LOQ)

Table 4 : LOD and LOQ values for Telmisartan at 232nm

S.No.	Conc. (µg/mL)	Absorbance at 232nm
S.No.	Conc. (µg/mL)	Set 1	Set 2	Set 3	Set 4	Set 5
1 2 3 4 5	4 8 12 16 20	0.511 0.742 0.994 1.168 1.391	0.519 0.749 0.959 1.171 1.410	0.521 0.752 0.0.963 1.175 1.376	0.506 0.756 0.967 1.156 1.371	0.515 0.732 0.999 1.165 1.388
Intercept		0.305	0.300	0.317	0.312	0.306
S.D. of Intercept 0.0029
Avg. of Slope 0.0538
LOD 0.177µg/ml LOQ 0.539 µg/ml

Table 5 : LOD and LOQ values for Ramipril at 222nm

S.No.	Conc.(µg/mL)	Absorbance at 222nm
S.No.	Conc.(µg/mL)	Set 1	Set 2	Set 3	Set 4	Set 5
1 2 3 4 5	4 8 12 16 20	0.058 0.094 0.131 0.172 0.206	0.062 0.099 0.139 0.182 0.212	0.057 0.093 0.132 0.171 0.205	0.058 0.099 0.132 0.181 0.210	0.063 0.095 0.135 0.175 0.215
Intercept		0.02	0.023	0.020	0.02	0.019
S.D. of Intercept 0.0006324
Avg. of Slope 0.007
LOD 0.298µg/ml LOQ 0.903µg/ml

Precision Studies

System Precision

Table 6 : Absorbance values for the System Precision

S.NO.	Conc. (µg/mL)	Absorbance of TEL and RAM
S.NO.	Conc. (µg/mL)	232nm	222nm
1.	12:1.5	1.008	0.708
2.	12:1.5	1.010	0.695
3.	12:1.5	1.001	0.714
4.	12:1.5	1.012	0.691
5.	12:1.5	1.005	0.710

Table 7 : Statistical Report of System Precision for TEL and RAM

S.NO.	Parameter	TEL and RAM
S.NO.	Parameter	232nm	222nm
1.	Mean	1.0072	0.7036
2.	Standard deviation	0.0019235	0.0044721
3.	Percentage Relative Standard deviation	0.19097	0.635

Method Precision:

Table 8 : Absorbance values for Method Precision

S.NO.	Conc. (µg/mL)		Absorbance of TEL and RAM
S.NO.	Conc. (µg/mL)		232nm	222nm
1.		8:1	0.701	0.480
2.		8:1	0.714	0.489
3.		8:1	0.694	0.485
4.		8:1	0.725	0.475
5.		8:1	0.705	0.487

Table 9 : Statistical Report of Method Precision for TEL and RAM

S.NO.	Parameter	TEL and RAM
S.NO.	Parameter	232nm	222nm
1.	Mean	0.708	0.4832
2.	Standard deviation	0.0052535	0.0025298
3.	Percentage Relative Standard deviation	0.74201	0.523

Table 10 : Intraday Precision values for TEL and RAM at 232nm

S.NO.	Conc.(µg/mL)	ABS at 232nm
	Conc.(µg/mL)	S1	S2		S3	S4	S5
1.	4:4	0.397		0.391	0.393	0.389	0.385
2.	8:8	0.724		0.729	0.725	0.719	0.726
3.	12:12	0.976		0.971	0.985	0.980	0.975

Table 11 : Intraday Precision values for TEL and RAM at 222nm

S.NO.	Conc.(µg/mL)	ABS at 222nm
S.NO.	Conc.(µg/mL)	S1	S2	S3	S4	S5
1.	4:4	0.372	0.379	0.365	0.375	0.369
2.	8:8	0.596	0.599	0.585	0.582	0.591
3.	12:12	0.717	0.720	0.715	0.725	0.721

Table 13 : Inter day Precision

S. No	Conc. (µg/mL)	ABS at 232nm
S. No	Conc. (µg/mL)	S1	S2	S3	S4	S5
1.	4:4	0.481	0.477	0.485	0.489	0.479
2.	8:8	0.751	0.759	0.745	0.741	0.755
3.	12:12	0.984	0.989	0.979	0.986	0.980

Table 14 : Inter day Precision

S. No.	Conc. (µg/mL)	ABS at 222nm
S. No.	Conc. (µg/mL)	S1	S2	S3	S4	S5
1.	4:4	0.405	0.409	0.403	0.413	0.415
2.	8:8	0.580	0.589	0.575	0.583	0.577
3.	12:12	0.700	0.708	0.712	0.701	0.710

Table 15 : Statistical Report of Inter day Precision for TEL and RAM

S.NO	Mean		Standard deviation		%RSD
S.NO	232nm	222nm	232nm	222nm	232nm	222nm
1.	0.4822	0.409	0.0021447	0.0022803	0.444	0.5575
2.	0.7502	0.5808	0.0032557	0.0024494	0.43397	0.4217
3.	0.9836	0.7062	0.0018439	0.0024083	0.18746	0.3410

Fig.9 : Asaay Spectrum of Telmisartan and Ramipril

Fig.10 : Accuracy Spectrum of Telmisartan and Ramipril

Table 16 : Assay Results for the Marketed Formulation

S.NO.	Conc. (µg/mL) TEL:RAM	ABS at (nm)		Amount estimated(µg/mL)		% Purity(w/w)
S.NO.	Conc. (µg/mL) TEL:RAM	232	222	232	222	232	222
1.	8:1	0.742	0.449	8.285	1.040	103.5	104
2.	12:1.5	1.125	0.680	12.5	1.4935	104.5	99.5
3.	16:2	1.365	0.827	15.235	2.02	95.22	101.3

Table 17 : Accuracy Results for Telmisartan

S.NO.	Amount of TEL in mrkd. Form. (µg/mL)	Amount of STD TEL added(µg/mL)	Total amount of TEL (µg/mL)	ABS.	Total amount of TEL found (µg/mL)	%Recovery
1	4	2(50%)	6	0.557	6.219	105
2	4	4(100%)	8	0.741	8.27	105
3	4	6(150%)	10	0.882	9.846	96.1

Table 18 : Accuracy Results for Ramipril

S.NO.	Amount of RAM in mrkd. Form. (µg/mL)	Amount of STD RAM added(µg/mL)	Total amount of RAM (µg/mL)	ABS.	Total amount of RAM found (µg/mL)	%Recovery
1	0.5	0.25(50%)	0.75	0.337	0.775	105
2	0.5	0.5(100%)	1	0.448	0.995	99.12
3	0.5	0.75(150%)	1.25	0.534	1.268	103

Table 19 : Validation results for UV Method (Telmisartan and Ramipril)

S.NO.	Parameters		Telmisartan	Ramipril	Acceptance Criteria
1.	Linearity		R ²= 0.996	R²= 0.999	Correlation coefficient (R²= 0.996-0.999)
2.	Precision	System	%RSD = 0.19097	%RSD = 0.635	RSD<2%
		Method	%RSD = 0.74201	%RSD = 0.523
		Intra day	%RSD = 0.222-0.5115	%RSD = 0.23664-0.6473
		Inter day	%RSD = 0.1874-0.444	%RSD = 0.341-0.557
3.	Assay		95.22-103.5%	99.5-104	95-105%
4.	Accuracy		96.1-105%	99.12-105%	95-105%
5.	LOD		0.177µg/mL	0.2981µg/mL	-
6.	LOQ		0.539µg/mL	0.9034µg/mL	-

CONCLUSION:

Literature survey indicates that the methods for the determination of TEL and RAM (UV) were less sensitive and costlier. So the present work aimed for the development of sensitive, economical and simpler methods for the TEL and RAM by UV in pharmaceutical dosage form. The UV Spectrophotometric method, with 0.1N NaOH was proved to be simple, precise, accurate and sensitive from the results of validation and it is suitable method for the simultaneous estimation of TEL and RAM in the pharmaceutical dosage form. Finally, it can be concluded that the method for quantitation of TEL and RAM (UV), in their pharmaceutical dosage form can be applied for the routine analysis because of simplicity, accuracy and Preciseness.

REFERENCES:

1. United States Pharmacopoeia National Formulary. 2008, P.No. 3156,2334.

2. Indian Pharmacopoeia Ministry of Health and Family Welfare, Government of India, New Delhi, 2007, Vol 2, P.No. 151,159,318,1194,1648.

3. Martindale 35^th edition, The Complete Drug Reference, P.No. 1266.3.

4. Merck index, 14^th edition, 2003, P.No. 5938. Popat B. Mhite, Ramdas .B., Spectrophotometric simultaneous determination of Ramipril and Telmisartan in combined tablet dosage form., Oriental J. of Chemistry., 2009, V-25(1), P.No.137-141.

5. S. Bankey, G.G Tapadlya, S.S Saboo, S. Bindaiya, Simultaneous determination of Ramipril, Hydrochlorothiazide and Telmisartan by Spectrophotometry, Int. J. of Chem Tech Research., 2012, Vol-1, P.No. 183-188.

6. Naman Doshi, Avani Sheth, Tejas Patel, J B Dave and CN Patel., Spectrophotometric absorption factor method development and validation for estimation of Rosuvastatin Calcium and Telmisartan in solid dosage forms., Journal of Chemical and Pharmaceutical Research, 2010, 2(3), P.No. 15-24.

7. Suhas S. Agey, Ashok Peepliwal, Simultaneous estimation of Telmisartan and Amlodipine in bulk and tablets by UV and RP-HPLC Method., Journal of Advances in Pharmacy and Healthcare Research.,2011, V-1,7 P.No. 67-73.

8. Priyanka R Patil, Sachin U Rakesh, Pandurang., Simultaneous estimation of Ramipril and Amlodipin by UV Spectrophotometric method, Reach J. of Pharm and Tech., April-June 2009, 2(2). P.No. 304-307.

9. Thamake SL, Jadhav SD, Development and validation of method for simultaneous estimation of Atorvastatin calcium and Ramipril from capsule dosage form by Order derivative spectroscopy., Asian J. Research Chem, 2009, 2(1), P.No.52-53.

10. Vrushali Tambe, Vijaya Vichare, Ujjawala Kandekar and Shashikant dhole, Novel UV Spectrophotometric methods for estimation of Ramipril and Hydrochlorothiazide by simultaneous equation and area under curve method. International Journal of Applied Pharmaceutics, 2010, V-2, P.No. 20-22.

Received on 06.11.2015 Accepted on 24.11.2015

Asian J. Pharm. Ana. 5(4): October- December, 2015; Page 187-194

DOI: 10.5958/2231-5675.2015.00030.7

S.NO	Mean		Standard deviation		%RSD
S.NO	232nm	222nm	232nm	222nm	232nm	222nm
1.	0.391	0.372	0.002	0.0024083	0.5115	0.6473
2.	0.7246	0.5906	0.0016124	0.0031937	0.222	0.540
3.	0.9774	0.7196	0.0023664	0.0017029	0.24211	0.23664