Development and Validation of RP-HPLC Method for simultaneous estimation of Telmisartan, Amlodipine Besylate and Hydrochlorthiazide in their tablet dosage form

 

Ashwini Parmar, Sandeep Sonawane*, Santosh Chhajed, Sanjay Kshirsagar

MET’s Institute of Pharmacy, Bhujbal Knowledge City, Adgaon, Nashik – 422 003, India

 

ABSTRACT:

To treat the second stage high blood pressure, physicians prefers the combination drug therapy consisting of two or more anti-hypertensive agents. Among these, telmisartan with amlodipine besylate, telmisartan with hydrochlorthiazide as well as triple drug therapy consisting of amlodipine besylate, telmisartan and hydrochlorthiazide are commonly prescribed. In the present paper, a simple, accurate and precise RP-HPLC method was developed for the estimation of amlodipine besylate, telmisartan and hydrochlorthiazide in bulk and combined dosage form. All these three drugs were successfully separated and resolved from each other on C18 kinetex column (250 × 4.6 mm, 5 µ), using acetonitrile: 20mM phosphate buffer (pH 3.0) (60:40 %, v/v) as a mobile phase at a flow rate of 1 mL/min. The detection was performed at 258 nm. The developed method was further validated as per ICH Q2 (R1) guidelines.    

 

 

 

 

INTRODUCTION:

Monotherapy treatment is not sufficient to treat hypertensive patients especially for those who are suffering from other disease. It is demonstrated that a majority of hypertensive patients need two or more antihypertensive drugs to lower their blood pressure effectively. fixed-dose combination appears to be a novel and underlying power in overcoming the cardiovascular disease.[1]

 

Telmisartan

 

Telmisartan is an angiotensin II receptor blocker (ARB). It is chemically2-[4-[[4-methyl-6-(1-methylbenzimidazol -2-yl)-2-propylbenzimidazol-1-yl] methyl] phenyl] benzoic acid. It works by blocking a substance in the body that causes blood vessels to tighten. As a result, the blood vessels relax and this decreases blood pressure. When blood pressure is lowered, the amount of blood and oxygen that go to the heart is increased.

 

Amlodipine besylate

 

Amlodipine besylate is a calcium channel blocker (CCB). It affects the movement of calcium into the cells of the heart and blood vessels. As a result, amlodipine relaxes the blood vessels and increases the supply of blood and oxygen to the heart while reducing its workload. It is Chemically 3-ethyl 5-methyl 2-[(2-aminoethoxy) methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate; benzenesulfonic acid.

 

Hydrochlorthiazide

 

Hydrochlorthiazide is a thiazide diuretic which helps to reduce blood pressure by volume depletion. Thiazides achieve their diuretic action via inhibition of the Na⁺/Cl⁻cotransporter (NCC) in the renal distal convoluted tubule. It is chemically(6-Chloro-3, 4-dihydro-2H- 1, 2, 4benzothiadiazine-7-sulfonamide 1,1-dioxide).

 

literature survey revealed methods reported for the determination of Telmisartan with amlodipinebesylate, [2-5], Telmisartan withhydrochlothiazide[6-12], as well triple drug therapy (Telmisartan, amlodipinebesylate with hydrochlorthiazide)[13-15].

 

The aim of the present work was to develop a simple, accurate and precise RP-HPLC method for estimation of amlodipine besylate, telmisartan and hydrochlorthiazide in bulk and combined dosage form.

 

MATERIALS AND METHODS:

Chemical and reagent:

Telmisartan,Amlodipine besylate and hydrochlothiazide were received as gift samples from Blue Cross Pharmaceuticals Ltd., Nashik and Glenmark Pharmaceuticals Ltd., Nashik, respectively. Acetonitrile (HPLC grade), Potassium dihydrogen orthophosphate and ortho phosphoric acid were used throught these expeiments and obtained from SD fine chemical Mumbai,Maharashtra.Freshly prepared double distilled water used was prepared by all Glass Double Distillation assembly,purchased from Borosil,mumbai,Maharashtra and further used after filtering through 0.45 mmembrane filter papers purchased from Millipore (India) Pvt. Ltd., Bengaluru, Karnataka. Tablet containing (40mg of Telmisartan, 5mg of Amlodipine besylate and 12.5mg of Hydrochlorothiazide was purchased from local market.

 

Instrumentation and chromatographic condition:

Chromatographic separation was carried out using an HPLC system (JASCO Corporation, Tokyo, Japan) equipped with dual PU 2080 plus pumps, multichannel UV detector, UV-2075 and injection loop (20 µL capacity), Rheodyne manual loop injector 7725i.Borwin Chromatography software (version 1.5) were used for data collection. The mobile phase was composed of acetonitrile: 20mM Phoshpate buffer (pH 3) 60:40 %v/v. Isocratic elution was carried out on a Phenomenex Kinetex C18 column (250´4.6mm,5m) at a flow rate of 1 mL/min. The wavelength was fixed at 258nm.

 

Preparation of standard stock solution:

Accurately weighed and transfer 10 mg of Telmisartan, Hydrochlorthiazide and Amlodipine besylate in separate 10 mL of clean dry volumetric flasks and made up to the volume with methanol. The resulting solutions were of 1000µg/mL of Telmisartan, Hydrochlorthiazide and Amlodipine besylate respectively.

 

Calibration curve standards for Telmisartan and Hydrochlorthiazide.

From the standard stock solution of telmisartan and hydrochlorthiazide six aliquots were prepare and diluted with mobile phase to get calibration curve standard with concentration of 10, 20, 40, 60, 80, 100, µg/mL for telmisartan and hydrochlorthiazide 3, 6, 12,18, 24, 36 µg/mL for These calibration curves were analyzed in triplicates and then mean peak area were plotted on y-axis against respective concentration on x-axis. The intercept, slope and co-efficient of regression were determined.

 

Calibration curve standards for Telmisartanand Amlodipine besylate:

From the standard stock solution of telmisartan and amlodipine besylate, six aliquots were prepare and diluted with mobile phase to get calibration curve standard with concentration of 8, 16, 24, 32, 40, 48 µg/mL for telmisartan and for amlodipine besylate1, 2, 3, 4, 5, 6 µg/mL. These calibration curves were analyzed in triplicates and then mean peak area were plotted on y-axis against respective concentration on x-axis. The intercept, slope and co-efficient of regression were determined.

 

Estimation of Telmisartan and Hydrochlorthiazide in Tablets:

Twenty tablets were weighed and taken into mortar and crushed to uniform powdered. And then quantity of powder equivalent to 40mg of Telmisartan and 12.5mg of Hydrochlorthiazide was transferred to 100ml volumetric flask and made up the volume upto mark with acetonitrile and shaken for 10min which gives 100µg/mL solution. From the above solution suitable aliquot was diluted with mobile phase to get concentrations of 40µg/mL for Telmisartan, and 12.5 µg/mL Hydrochlorthiazide respectively and subjected to chromatographic analysis under mentioned chromatographic condition.

 

Estimation of Telmisartan and Amlodipine besylate in Tablets:

Twenty tablets were weighed and taken into mortar and crushed to uniform powdered. And then quantity of powder equivalent to 40mg of Telmisartan, and 5mg of Almodipine besylate, was transferred to 100ml volumetric flask and made up the volume up to mark with acetonitrile and shaken for 10min which gives 100µg/mL solution .From the above solution suitable aliquot was diluted with mobile phase to get concentrations of 40µg/mL for Telmisartan, and 5µg/mL for Amlodipine besylate respectively and subjected to chromatographic analysis under mentioned chromatographic condition.

 

Method Validation :

The developed HPLC method was validated in according to ICH Q2(R1) guidelines for validation of analytical procedure for different validation parameters. The method was validated for its linearity, accuracy, precision specificity, robustness [16]

 

Accuracy and Precision:

Accuracy was performed by preparing three different concentrations (corresponding to 80%,100%,120% of the sample solution) by addition of known amounts of standard solution across the analytical range for Telmisartan and Amlodipine besylate and for Telmisartan and Hydrochlorthiazide. Three replicates were prepared for the intra-day precision and on three successive days for the intermediate precision. The percent recovery of added concentration and % RSD were taken as measures of accuracy and precision, respectively. Also, the results obtained were subjected to one way ANOVA and the between – day mean square compared to the within – day mean square by F – test.

 

 

Specificity:

The specificity studies were performed by preparing the placebo containing all excipients except the drug. According to that sample solution was prepared. Absence of peaks in the chromatographic run was taken as indication of specificity.

 

RESULT AND DISCUSSION:

Various mobile phases were tried, and a mobile phase with composition of acetonitrile: 20mM phosphate buffer (pH 3) 60: 40 %v/v, flow rate 1mL/min was found to resolve Telmisartan, Amlodipine besylate and Hydrochlorthiazide. The optimum wavelength for detection was 258 nm. The retention time of Telmisartan, Amlodipine besylate and Hydrochlorthiazide were observed at 2.633, 3.225 and 4.883 mins, respectively (Figure 1)

 

Figure 1: Representative Chromatogram of Hydrochlorthiazide (2.633), Amlodipine besylate (3.225min) and Telmisartan (4.883min).                  

 

I] Telmisartan and Hydrochlorthiazide:

In calibration studies, it was found that Telmisartan was linear in the range of 10-100µg/mL and Hydrochlothiazide 3-36 µg/mL. The calibration curves with their respective calibration curve equations and regressions are depicted in Figure 2, and Figure 3 respectively.

 

The results obtained for accuracy and precision are summarized in Table 1 and Table 2, for Telmisartan and Hydrochlorthiazide, respectively. The drug content was found to be 98.8 ± 1.94 of the added amount. Mean values of concentration found were close to the concentration added and low values of % RSD indicates the acceptable accuracy and precision of the method.

 

Figure 2: Calibration curve of Telmisartan

 

Figure 3: Calibration curve of Hydrochlorthiazide

 

Table 1: Accuracy and Precision studies of Telmisartan.

Concentration added (µg/mL)	Amount Found ((µg/mL)			Within mean square	Between mean square	F-value
	Day 1	Day 2	Day 3
(40+32) 72(µg/mL) 80%	71.89	71.67	71.28	0.1417	0.0703	0.496
	71.92	71.82	71.23
	71.78	72.13	72.3
Mean	71.86333333	71.87333333	71.60333333
SD	0.073711148	0.234591844	0.603848767
%RSD	0.10257129	0.326396221	0.843324939
(40+40) 80(µg/mL) 100%	79.78	79.97	79.56	0.045844444	0.046044444	1.004
	79.82	80.13	80.17
	80.1	80.2	79.89
Mean	79.83333333	80.1	79.87333333
SD	0.061101009	0.117898261	0.305341339
% RSD	0.076535711	0.14718884	0.382281954
(40+48) 88(µg/mL) 120%	87.65	87.92	87.67	0.009866667	0.0112	1.135
	87.9	87.9	87.8
	87.89	87.86	87.85
Mean	87.77666667	87.89333333	87.77333333
SD	0.120554275	0.030550505	0.092915732
%RSD	0.13734205	0.034758614	0.10585872

 

Table 2: Accuracy and Precision studies of Hydrochlorthiazide.

Concentration added (µg/mL)	Amount Found ((µg/mL)			Within mean square	Between mean square	F-value
	Day 1	Day 2	Day 3
(12+9.6) 21.6(µg/mL) 80%	20.53	20.64	20.73	0.022077778	0.010077778	0.4564
	20.42	20.43	20.47
	20.31	20.52	20.32
Mean	20.42	20.53	20.50666667
SD	0.11	0.105356538	0.207444772
%RSD	0.538687561	0.513183329	1.011596741
(12+12) 24(µg/mL) 100%	23.71	23.9	23.57	0.010822222	0.011811111	1.091
	23.73	23.85	23.69
	23.65	23.65	23.8
Mean	23.69666667	23.8	23.68666667
SD	0.04163332	0.132287566	0.115036226
%RSD	0.175692727	0.555830107	0.485658146
(12.14.4) 26.4(µg/mL) 120%	25.8	25.99	25.74	0.008011111	0.005511111	0.6879
	25.73	25.7	25.77
	25.62	25.72	25.8
Mean	25.71666667	25.70333333	25.77
SD	0.090737717	0.015275252	0.03
%RSD	0.35283623	0.059429071	0.116414435

Also, when the results of intra-day and inter-day were subjected to one-way ANOVA and F values were calculated at each QC level, the F values were found to be less than the tabulated F values. This indicated that there was no significant difference between intra-and inter-day variability, suggesting good intermediate precision.

 

When blanked tablets were analyzed as per the mentioned chromatographic conditions, no peak was obtained at the retention times of Telmisartan and Hydrochlorthiazide which indicates that the method was specific for the analysis of analytes in their dosage form. The results of specificity are depicted in Figure 4.

 

Figure 4: Representative Chromatogram of specificity.

 

Analysis of formulation:

Represented chromatogram for formulation of Telmisartan and Hydrochlorthiazide is shown in figure 5. The chromatogram of the drug samples did not show a change in the retention time. There were no interferences from excipients, which are commonly present in the solution for drugs.

 

Figure 5- Representative chromatogram of formulation.

 

II] Telmisartan and Amlodipine besylate:

In calibration studies, it was found that Telmisartan was linear in the range of 8-48µg/mL and Amlodipine besylate 1-6 µg/mL. The calibration curves with their respective calibration curve equations and regressions are depicted in Figure 6 and 7.

 

The results obtained for accuracy and precision are summarized in Table 3 and Table 4, for Telmisartan and Amlodipine besylate, respectively. The drug content was found to be 98.52 ± 1.94 of the added amount. Mean values of concentration found were close to the concentration added and low values of % RSD indicates the acceptable accuracy and precision of the method.

 

Also, when the results of intra-day and inter-day were subjected to one-way ANOVA and F values were calculated at each QC level, the F values were found to be less than the tabulated F values. This indicated that there was no significant difference between intra-and inter-day variability, suggesting good intermediate precision.

 

Robustness:

To evaluate the robustness of the method, a small but deliberate change was done with respect to the flow rate, composition of mobile phase. No significant change was observed with respect to the system suitability. Hence, the method was concluded as robust.

 

Analysis of Formulation:

Figure 8 depicts the chromatogram of telmisartan and amlodipine besylate.

 

Specificity:

Figure 9 represents the chromatogram of specificity. As no interference is observed at the retention time of the drugs indicates the specificity of the method. 

 

Figure 6: Calibration curve of Telmisartan

 

 

Figure 7: Calibration curve of Amlodipine besylate

 

Table 3: Accuracy and Precision studies of Telmisartan.

Concentration added (µg/mL)	Amount Found ((µg/mL)			Within mean square	Between mean square	F-value
	Day 1	Day 2	Day 3
(40+32) 72(µg/mL) 80%	71.89	71.67	71.28	0.1417	0.0703	0.496
	71.92	71.82	71.23
	71.78	72.13	72.3
Mean	71.86333333	71.87333333	71.60333333
SD	0.073711148	0.234591844	0.603848767
%RSD	0.10257129	0.326396221	0.843324939
(40+40) 80(µg/mL) 100%	79.78	79.97	79.56	0.045844444	0.046044444	1.004
	79.82	80.13	80.17
	80.1	80.2	79.89
Mean	79.83333333	80.1	79.87333333
SD	0.061101009	0.117898261	0.305341339
%RSD	0.076535711	0.14718884	0.382281954
(40+48) 88(µg/mL) 120%	87.65	87.92	87.67	0.009866667	0.0112	1.135
	87.9	87.9	87.8
	87.89	87.86	87.85
Mean	87.77666667	87.89333333	87.77333333
SD	0.120554275	0.030550505	0.092915732
%RSD	0.13734205	0.034758614	0.10585872

 

Table 4: Accuracy and Precision studies of Amlodipine besylate.

Concentration added (µg/mL)	Amount Found ((µg/mL)			Within mean square	Between mean square	F-value
	Day 1	Day 2	Day 3
(5+4)     9(µg/mL) 80%	8.92	8.9	8.78	0.006555556	0.005033333	0.7677
	8.9	8.81	8.69
	8.8	8.87	8.92
Mean	8.873333333	8.86	8.796666667
SD	0.064291005	0.045825757	0.115902258
%RSD	0.724541755	0.517220733	1.31757019
(5+5) 10(µg/mL) 100%	9.7	9.89	9.61	48.29106667	95.5567	1.9787
	9.88	9.76	9.76
	9.9	9.81	9.82
Mean	9.826666667	9.82	9.73
SD	0.110151411	0.065574385	0.108166538
%RSD	1.120943802	0.667763597	1.111680763
(5+6) 11(µg/mL) 120%	10.67	10.7	10.33	0.015677778	0.012844444	0.81927
	10.5	10.65	10.64
	10.72	10.54	10.58
Mean	10.63	10.63	10.51666667
SD	0.115325626	0.081853528	0.164418166
%RSD	1.084907111	0.770023779	1.5634057

 

Figure 8- Representative chromatogram of formulation

 

Figure 9: Representative Chromatogram of specificity.

 

CONCLUSION:

A single, simple, accurate and precise RP-HPLC method has been developed, optimized and validated for simultaneous estimation of amlodipine besylate, telmisartan and hydrochlorthiazide, Telmisartan in tablet dosage forms. This method reduces overall assay development time and solvents. All the parameters of validation are within the acceptable range. The developed method was specific as there was no any interfering peak at retention time of drugs. Hence the developed method was accurate and can be used for routine analysis of amlodipine besylate, telmisartan and hydrochlorthiazide in tablet dosage forms.

 

ACKNOWLEDGEMENT:

Authors are thankful to the trustees and management of MET’S Institute of Pharmacy, Bhujabal Knowledge city, Nashik for providing necessary analytical facilities and Glenmark Pharmaceutical Ltd., Nashik for providing gift samples of amlodipine besylate.

 

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Received on 31.05.2017       Accepted on 29.07.2017     

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