INTRODUCTION:

Telmisartan is a non peptide molecule. It is chemically described 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^1-5. 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 inhibits 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. Chlorthalidone is thiazide like diuretic, chemically known as 2-Chloro-5-(1-hydroxy-3-oxo-2,3-dihydro-1H-isoindol-1-yl)benzene-1-sulfonamide, with molecular formula C₁₄H₁₁CIN₂O₄S and molecular weight 338.766 g/mol. Chlorthalidone inhibits sodium ion transport across the renal tubular epithelium in the cortical diluting segment of the ascending limb of the loop of henle. By increasing the delivery of sodium to the distal renal tubule, chlorthalidone indirectly increases potassium excretion via the sodium-potassium exchange mechanism. Literature survey revealed that there were few methods reported for the estimation of Telmisartan and Chlorthalidone by RP-HPLC^5-17 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 Chlorthalidone by RP-HPLC in pharmaceutical dosage form^16-20.

Figure 1: Chemical Structure of Telmisartan

Figure 2: Chemical Structure of Chlorthalidone

EXPERIMENTAL:

Instrumentation:

A Shimadzu LC-2010 HPLC system with auto sampler and programmable temperature control and UV detector using LC-SOLUTION software had been used for the work. For normal UV absorbance estimations during method development were done by a Lab India UV-Vis Spectrophotometer UV 3000⁺ with UV-Win software and weighing were done by a Shimadzu precision balance AUX-220.

Chemicals and reagents:

Telmisartan and Chlorthalidone RS were procured from R and K Pharmaceuticals, Vishakapatnam and Ajantha Pharmaceuticals (Mumbai, Maharashtra). Marketed formulation TELLZY-ch 40 (Alembic Pharmaceutical Ltd), with label claim 40mg TEL, 12.5mg CLT, was purchased from local market of Siddipet, Telangana. HPLC grade acetonitrile, water and methanol were procured from Merck (Mumbai, Maharashtra). Potassium dihydrogen orthophosphate and ortho phosphoric acid of analytical grade were used for the studies. The solvents and the mobile phases were filtered using Millipore 0.45 µm filter medium.

Chromatographic Conditions:

CAPCELL C₁₈ (250mm×4.6mm id., 5µm) column was used as a stationary phase. A mixture of acetonitrile, methanol and phosphate buffer pH 3.5, adjusted with ortho phosphoric acid, in the ratio of 45:20:35% v/v was used as mobile phase with a flow rate of 0.8 mL/min. The detection wavelength was set at 230 nm and column temperature at 25°C.

Selection of Detection Wave length:

UV Spectrum of 20µg/mL Telmisartan and Chlorthalidone in diluents(Methanol) was recorded by scanning in the range of 200nm to 400nm individually. From the UV Spectrum, the detection wavelength selected was 230nm. At this wavelength both the drugs shows good absorbance. (Fig. 3).

Preparation of Potassium di hydrogen Orthophosphate buffer:

6.8gm of Potassium di hydrogen Orthophosphate weighed and transfered it into a 1000mL volumetric flask and added to sufficient quantity of distilled water added, sheken and the final volume was made up to 1000mL with distilled water.

Preparation of Mobile Phase:

Acetonitrile and Potassium di hydrogen orthophosphate buffer and methanol were mixed in the ration of 45:35:20 and its pH was maintained 3.5 by using Ortho phosphoric acid. Then the mobile phase was Filtered through 0.45µ membrane, then sonicated for degassing.

Preparation of standard stock solutions:

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

Preparation of mixed working standard solution:

20mL of Telmisartan and 6.25 mL of Chlorthalidone drug solutions were taken from the stock solutions and diluted to 100mL with Mobile Phase in 100mL volumetric flask to get the concentration of 200µg/mL of Telmisartan and 62.5µg/mL of Chlorthalidone.

Fig. 5: Calibration curve of TEL

Fig. 6: Calibration curve of CLT

Validation Parameters:

Linearity Range:

Adequate dilutions were made from mixed working standards to get the concentrations of 20-100µg/mL for Telmisartan and 6.25-31.25µg/mL for Chlorthalidone using mobile Phase. Peak Area of these solutions were determined at their corresponding detection wave length. The graph was plotted with the measured Peak Areas against concentrations. (Fig. 5 and Fig. 6, Table. 1)

Table 1: Linearity results for TEL and CLT

S.NO.	TEL Conc. (µg/mL)	TEL Peak Area	CLT Conc.(µg/mL)	CLT Peak Area
1.	20	191948.3	6.25	30427.1
2.	40	370611.6	12.5	58442.2
3.	60	526796.3	18.75	83284.2
4.	80	684260.2	25	108442.1
5.	100	842454.6	31.25	131103.3

Sensitivity:

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. For this five sets of linear dilutions were prepared and injected then Chromatograms were recorded. From the standard graphs standard deviation of the intercept and mean of the slope were calculate, then LOD and LOQ values were determined using the following formulas. (Table. 2)

LOD = 3.3 S_a/b

LOQ = 10 S_a/b

S = Standard deviation of intercept

b = mean of slope of calibration curve

Table 2: LOD and LOQ values for Telmisartan and Chlorthalidone

S.NO.	Drugs	LOD(µg/mL)	LOQ(µg/mL)
1.	Telmisartan	0.30744	0.93164
2.	Chlorthalidone	0.05796	0.17565

Precision Studies:

System Precision:

A fixed concentration of 60:18.75µg/mL ratio of the standard drug mixture from the linearity range was injected 5times , 5 repeated injections of standard drug mixture response factor of drug peak and SD and percentage RSD values were calculated. (Table. 3)

Table 3: Statistical Report of System Precision for TEL and CLT

Injection	Telmisartan		Chlorthalidone
Injection	RT	Peak Area (60µg/mL)	RT	Peak Area (18.75µg/mL)
Injection-1	4.939	526796.3	3.644	83284.2
Injection-2	4.954	527347.8	3.665	83316.2
Injection-3	4.965	525962.5	3.654	83292.1
Injection-4	4.975	527134.7	3.659	83146.9
Injection-5	4.959	526825.4	3.654	83257.4
Mean	4.958	526813.34	3.6552	83259.36
SD	0.00598	235.9326	0.00344	29.640183
%RSD	0.1206	0.04478	0.0943	0.03559

Table 4: Statistical Report of Method Precision for TEL and CLT

Injection	Telmisartan		Chlorthalidone
	RT	Peak Area (60µg/mL)	RT	Peak Area (18.75µg/mL)
Injection-1	4.976	521651.5	3.654	82344.2
Injection-2	4.950	522723.7	3.644	82426.8
Injection-3	4.939	523812.8	3.685	82512.6
Injection-4	4.949	522632.9	3.676	82314.8
Injection-5	4.956	524721.1	3.670	82416.7
Mean	4.954	523108.4	3.665	82403.2
SD	0.00614	528.84931	0.00744	34.643
%RSD	0.1239	0.10109	0.2030	0.04204

Assay (Sample Preparation):

Accurately weighed and powered 20 tablets of “Tellzy-ch 40” manufactured by Alembic Pharmaceutical Ltd., Himachal Pradesh. It contains 40mg of Telmisartan and 12.5mg of Chlorthalidone in the ratio of 3.2:1. A weight equivalent to 50mg Telmisartan of the powdered tablet was taken and to this added 25mL methanol in 50mL standard volumetric flask. Ultrasonication was down for 30min. and kept overnight for dissolving. Again after ultrasonication filtered using Whattman filter paper grade 1, then volume was made up to the mark with methanol. The stock solution prepared above contains both the drugs in the ratio of 3.2 : 1 and 10mL From the stock solution was taken in the 50mL volumetric flask and the volume was made up to the mark to get the concentration of 200µg/mL of Telmisartan and corresponding concentration of Chlorthalidone (working standard) with mobile Phase. From working standard following dilutions were prepared for determination of the percentage purity of the marketed formulation. The 3 mL of working standard was taken and diluted with mobile phase up to 10mL to get concentration of 60µg/mL of Telmisartan and 18.75 µg/mL of Chlorthalidone. 3 repeated injections of this solution response factor of drug peak and SD and percentage RSD values were calculated. (Fig. 7, Table. 5)

Table 5: Assay Results for the Marketed Formulation

Conc. TEL:CLT 60:18.75µg/mL	Peak Area
Conc. TEL:CLT 60:18.75µg/mL	Telmisartan	Chlorthalidone
Injection-1	523146.1	84706.8
Injection-2	512727.2	81624.9
Injection-3	519361.7	80700.9
Avg.	518411.6	82344.2
Amt Estimated(µg/mL)	59.045	18.538
mg/tab	39.6092	12.358
%Purity	98.408	98.8

Method Precision:

A fixed concentration in the ratio of 60:18.75µg/mL of the marketed formulation from the linearity range was injected 5 times, 5 repeated injections of marketed formulation response factor of drug peak and SD and percentage RSD values were calculated. (Table. 4)

Fig. 7: A typical chromatogram showing peaks of TEL and CLT mixed sample solution

Table 6: Accuracy Results for Telmisartan

S.NO.	Amount of TEL in mrktd. Form. (µg/mL)	Amount of STD TEL added µg/mL)	Total amount of TEL (µg/mL)	Peak Area		Total amount of TEL found (µg/mL)	%Recovery
				Peak Area	Avg.
1	30	15(50%)	45	392005.2	393484.73	44.816	99.387
				397216.3
				391232.7
2	30	30(100%)	60	527604.3	526984.9	60.021	100
				523724.7
				529625.8
3	30	45(150%)	75	653567.7	653188.8	74.395	97.98
				654736.1
				65126.6

Table 7: Accuracy Results for Chlorthalidone

S. NO.	Amount of CLT in mrktd. Form. (µg/mL)	Amount of STD CLT added(µg/mL)	Total amount of CLT (µg/mL)	Peak Area		Total amount of CLT found (µg/mL)	%Recovery
				Peak Area	Avg.
1	9.375	4.6875(50%)	14.0625	62147.2	62342.9	14.035	99.711
				62536.1
				62345.6
2	9.375	9.375(100%)	18.75	83451.7	83436.6	18.787	100
				83721.8
				83136.5
3	9.375	14.0625(150%)	23.4375	103914.6	102975.1	23.183	97.2
				102762.8
				102247.9

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 analyzed 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 30:9.375 (TEL:CLT) was kept fixed and the standard drugs mixture in the ratio of 3.2:1(TEL:CLT) was added in 50%, 100%, 150% respectively.

First dilution (45:14.0625):

To 30:9.375(TEL:CLT) µg/mL of marketed formulation 15:4.6875(TEL:CLT) µg/mL standard drugs mixture was added in the 10mL volumetric flask to get the final concentration of 45:14.0625 µg/mL dilution was prepared and injected and its peak area was observed.

Second dilution (60:18.75):

To 30:9.375(TEL:CLT) µg/mL of marketed formulation 30:9.375 (TEL:CLT) µg/mL standard drugs mixture was added in the 10mL volumetric flask to get the final concentration of 60:18.75 µg/mL dilution was prepared and its peak area was observed.

Third dilution (75:23.437):

To 30:9.375(TEL:CLT) µg/mL of marketed formulation 45:14.0625 (TEL:CLT) µg/mL standard drugs mixture was added in the 10mL volumetric flask to get the concentration of 75:23.437 µg/mL dilution was prepared and its peak area was observed. The percentage recovery was determined by using the following formula (Table. 6 and7) % Recovery = Amount Found – Amount Added / Actual Amount × 100

Specificity:

Telmisartan Standard:

Standard stock solution of Telmisartan was prepared by dissolving 50mg of drug in 50mL of methanol to get the concentration of 1000µg/mL. 0.6mL drug solution was taken from the stock solutions and diluted to 10mL with Mobile Phase in 10mL volumetric flask to get the concentration of 60µg/mL of Telmisartan.

Chlorthalidone Standard:

Standard stock solution of Chlorthalidone was prepared by dissolving 50mg of drug in 50mL of methanol to get the concentration of 1000µg/mL. 0.187 mL drug solution was taken from the stock solutions and diluted to 10mL with Mobile Phase in 10mL volumetric flask to get the concentration of 18.75 µg/mL of Chlorthalidone.

Mixed Standard:

Used 60µg/mL dilution from Linearity.

Mixed Sample:

Used assay solution as sample preparation.

Blank Preparation:

Used diluents as blank solution. These dilutions were injected and the chromatograms showed no interfering peaks at the retention time of any of the drugs in any chromatogram which indicates the specificity of the method. (Table. 8)

Table 8: data of Specificity

S.NO.	Sample Name	TEL		CLT
S.NO.	Sample Name	RT	Area	RT	Area
1.	Blank	0	0	0	0
2.	Telmisatan Standard	4.947	525963.7	-	-
3.	Chlorthalidone Standard	-	-	3.665	83269.4
4.	Mixed Standard	4.937	526796.3	3.640	83284.2
5.	Mixed Sample	4.980	523146.1	3.649	84706.8

Table 9: Robustness

S.NO.	Parameters	TEL		CLT
S.NO.	Parameters	RT	Area	RT	Area
1.	Standard
2.	Flow-0.6mL/min	6.687	697713.4	4.883	113411.6
3.	Flow-1mL/min	4.054	429843.1	2.954	67548.3
4.	Mobile Phase pH 3	4.563	572326.2	3.752	103127.6
5.	Mobile Phase pH 4	6.125	520590.4	3.647	107530.1

Table 10: Ruggedness Results

S.No.	Injection	CLT		TEL
S.No.	Injection	RT	Peak Area	RT	Peak Area
1.	Analyst A Injection 1	3.652	83281.7	4.970	525962.5
2.	Inection 2	3.660	83279.9	4981	525569.9
3.	Injection 3	3.659	83285.8	4.975	525874.8
4.	Analyst B Injection 1	3.667	83256.1	4.985	525574.9
5.	Injection 2	3.650	83268.5	4.973	525796.8
6.	Injection 3	3.657	83279.4	4.983	525673.4
Average		3.657	83275.23	4.9778	525742.05
Standard deviation		0.00248	4.4871	0.00244	66.2026
%RSD		0.068	0.00538	0.0492	0.0125

Table 11: System suitability parameters

S.NO.	Parameters		Telmisartan	Chlorthalidone	Acceptance Criteria
1.	Retention Time		4.937	3.64	-
2.	Resolution		5.82	0.0	More than 2
3.	Tailing factor		1.35	1.28	Less than 2
4.	Theoretical Plates		6141.47	5648.33	More than 2000
5.	Linearity		0.999	0.998	Correlation coefficient (R²= 0.996-0.999)
6.	Precision	System	0.04478	0.03559	RSD<2%
6.	Precision	Method	%RSD 0.10109	%RSD 0.04204	RSD<2%
7.	Assay		98.408	98.8	95-105%
8.	Accuracy		97.98-100	97.2-100	95-105%
9.	LOD		0.3074	0.0579	-
10.	LOQ		0.9316	0.17565	-
11.	Ruggedness		%RSD 0.0125	%RSD 0.00538	RSD<2%

Robustness:

The robustness of test method is demonstrated by carrying out intentional method variations like mobile phase flow changes, mobile phase pH variations etc. (Table. 9)

Ruggedness:

The ruggedness of test method was demonstrated by carrying out precision study with different analysts and on different days. (Table. 10)

RESULTS AND DISCUSSION:

Linearity and Range

Sensitivity

System Precision

When Specificity is performed by the blank, Telmisartan standard, Chlorthalidone standard, Mixed standard, Mixed Sample no impurities were found. Keeping the ratio of mobile phase constant and the chromatograms of drug solution were recorded with different flow rates such as 0.6mL/min, 0.8mL/min, 1mL/min. At the flow rate of 0.8mL/min, the peaks were sharp with good resolution and found to be satisfactory. So 0.8mL/min flow rate was kept constant throughout the analysis.

CONCLUSION:

Literature survey indicates that the methods for the determination of TEL and CLT (RP-HPLC) were less sensitive and costlier. So the present work aimed for the development of sensitive, economical and simpler methods for the TEL and CLT by RP-HPLC in pharmaceutical dosage form. This RP-HPLC method was developed using the mobile phase of Acetonitrile and KH₂PO₄ Buffer and Methanol were mixed in the ration of 45:35:20 v/v and its pH was maintained 3.5 by using Ortho phosphoric acid. Within 5min of Run time TEL and CLT both drugs were eluted which reduce the solvent usage. The results of the validation parameters showed that the method is simple, sensitive, accurate and precise. Finally, it can be concluded that the method for quantitation of TEL and CLT (RP-HPLC), 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.

5. Bhimashankar Haridas Surwase, Amit Suryakant Tapkir, Shailaja bhanudas Jadhav and Pravin Digamber Chaudhari, Development and validation of RP-HPLC method for simultaneous estimation of telmisartan and chlorthalidone in bulk and tablet dosage form., Der Pharmacopea Letter, 2013, 5(1), P.No. 149-154.

6. Sahoo S, Mishra SK, Panda PK, HPLC Method development for simultaneous estimation of Telmisartan and Chlorthalidone in Tablet dosage form, International journal for Pharmaceutical Research Scholars, 2012, Vol 1, P.No. 1-4.

7. Kreny E. Parmar, Nikita D. Patel, Stability indicating RP-HPLC Method for simultaneous determination of Telmisartan and Chlorthalidone in bulk and pharmaceutical Dosage form., IJPTR, Oct-Dec 2013, Vol-5, P.No. 1728-1735.

8. Kreny E. Parmar, R.S Mehta, Nikita D. Patel, Development and Validation of HPTLC method for simultaneous determination of Telmisartan and Chlorthalidone in bulk and Pharmaceutical dosage form. International Journal of Pharmacy and Pharmaceutical Sciences. 2013, Vol-5.

9. Kreny E. Parmar, R.S Mehta, First order derivative spectrophotometric method for simultaneous estimation of Telmisartan and Chlorthalidone in bulk and pharmaceutical dosage form, International Research Journal of Pharmacy, 2013, 4(1), P.No. 224-228.

10. Bhimashankar Haridas Surwase, Amit Suryakant Tapkir, Shailaja bhanudas Jadhav and Pravin Digamber Chaudhari, Development and validation of UV Spectrophotometric Method for simultaneous determination of Telmisartan and chlorthalidone in its pure and pharmaceutical dosage forms., Int. J. Pharm. Res. Sci., 2014, 2(4), P.No. 267-272.

11. Mohamed S. Elgawish, Samia M. Mostafa, Abdalla A. Elshanawane, Simple and rapid HPLC method for simultaneous determination of atenolol and Chlorthalidone in spiked human plasma, Saudi Pharmaceutical Journal 2011, 19, P.No. 43-49.

12. P. Hari Prasad, P.M. Patei, D. Vijaysree, Y. Suresh Reddy and B. Ranjith kumar, Der Pharma Chemica, 2013, 5(5), P.No. 139-143.

13. P. Sravani, S. Rubesh kumar, N. Duganath and N. Devanna., Method development and Validation for the simultaneous estimation of Azilsartan and Chlorthalidone by RP-HPLC in Pharmaceutical dosage form., International Journal of Pharma Sciences., 2014 Vol-4, P.No. 725-729.

14. Kalaiselvi P and Lalitha K G., Development and validation of RP-HPLC method for the simultaneous estimation of Chlorthalidone and Irbesartan in pharmaceutical dosage form., Pharmacophore 2014, Vol 5(2), P.No. 279-286.

15. Reema H. Rupareliya and Hitendra S. Joshi, Stability indicating simultaneous validation of Telmisartan and Cilnidipine with forced degradation behavior study by RP-HPLC in Tablet dosage form. Int. J. of Pharm Tech Research, 2010, P.No. 1-5.

16. G.S Kumar, V. Ramya, Sumanta, Mondal, Sai Pavan Kumar, Development and validation of RP-HPLC method for simultaneous estimation of Atenolol and Chlorthalidone from pharmaceutical formulation, International Research Journal of Pharmacy, 2012, 3(10). P. No. 215-219.

17. Suresh kumar GV, Rajendraprasad Y, Chandrashekar SM, Development and validation of reverced-phase HPLC method for simultaneous estimation of Atorvastatin calcium and Telmisartan in tablet dosage form., Int. J. of Pharm Tech Research,2010, V-2, P. No. 463-470.

18. R. Vijayamirtharaj, J. Ramesh, B. Jayalakshmi and Hanas Bin Hashim, Development and validation of RP-HPLC method for the simultaneous estimation of Telmisartan and Atorvastatin calcium in tablet dosage forms., Int. J. of Comprehensive Pharmacy. 2010, 4(3),. P.No. 1-4.

19. J. Kavitha, J.S.K. Nagarajan, S. Muralidharan and B. Suresh, Development and validation of RP-HPLC method for simultaneous estimation of Telmisartan and Hydrochlorothiazide in tablets, Int. J. of Pharmacy and Pharmaceutical Sciences. 2011, Vol-3. P. No. 113-115.

20. Ilango K., Shiji Kumar P.S., Simultaneous estimation of Telmisartan and Hydrochlorothiazide in Pharmaceutical dosage form., Asian J. of Pharmaceutical and Health Sciences.2013, P.No. 12-15.

Received on 14.10.2015 Accepted on 10.11.2015

Asian J. Pharm. Ana. 5(4): October- December, 2015; Page 171-177

DOI: 10.5958/2231-5675.2015.00027.7