INTRODUCTION:

Telmisartan, a nonpeptide molecule, is chemically 40-[(1,4-dimethyl-20-propyl[2,60-1H-benzimidazol]-10-yl) methyl]-[1,10-biphenyl]-2-carboxylic acid and Amlodipine is chemically 2 - [(2 - aminoethoxy)methyl] - 4 -(2 - chlorophenyl) - 3 -ethoxycarboxyl - 5 -methoxycarbonyl - 6- methyl - 1,4- dihydropyridine. Telmisartan is an angiotensin II receptor antagonist that is highly selective for type 1 angiotensin II receptor. Angiotensin II is the principle pressor agent of the renninangiotensin system, with effects that include vasoconstriction, stimulation of synthesis and release of aldosterone, cardiac stimulation, and renal reabsorbtion of sodium. Amlodipine is a calcium channel blocker (dihydropyridine) used as an anti-hypertensive and in the treatment of angina. Like other calcium channel blockers, amlodipine acts by relaxing the smooth muscle in the arterial wall, decreasing peripheral resistance and hence reducing blood pressure; in angina it increases blood flow to the heart muscle. Telmisartan and amlodipine combination is used to lower blood pressure (hypertensive agent)^1-4.

The literature survey reveals that several methods were reported for the individual estimation of telmisartan and amlodipine. The methods^5-11 for telmisartan in combination with other drugs in plasma, serum and in tablets by UV and LC and^12-19 for the estimation of amlodipine in combination with other drugs in serum and in tablets by UV and LC. None of the reported analytical procedures describe a method for simultaneous determination of telmisartan and amlodipine in combined pharmaceutical dosage form.

In the present study attempts were made to develop a rapid, economical, precise and accurate method for the simultaneous estimation of the ingredients of this combination.

EXPERIMENTAL:

1. Chemicals and Reagents:

Telmisartan and amlodipine standards were obtained from Nicholas Piramal Pharmaceutical (Mumbai, India) and AGIO Pharmaceutical (Pune, India). Acetonitrile HPLC grade was obtained from Qualigens fine Chemicals, Mumbai. Methanol HPLC grade and Triethylamine AR grade were obtained from Thomas Baker Chemicals, Mumbai. Triethylamine AR grade was obtained from Thomas Baker Chemicals, Mumbai. O-Phosphoric acid AR grade was obtained from S d Fine Chemicals, The combination product of telmisartan and amlodipine label claim (telmisartan 40 mg and amlodipine 5 mg). Telma-AM, tablets (Sun Pharmaceutical, Mumbai, India) were purchased from the local market. Double distilled water was used throughout the experiment. Other chemicals used were of analytical or LC grade.

2. Instrumentation and Chromatographic Conditions:

Chromatography was performed with a Analytical AIS 2610 A HPLC system with Analytical AIS 2010 isocratic pump, Variable wavelength UV–visible detector model AIS 2600. Chromatograms and data were recorded by means of Ecomac Chromatography software. Compounds were separated on a Kromasil C18 size 250 mm×4.6 mm, 5 um. The mobile phase was Acetonitrile: Methanol: Triethylamine buffer PH adjusted to 5.0 with O-Phosphoric acid. The flow rate was 1.5 mL/min. 20 µL of sample was injected and the detection wavelength was 237 nm for determination of two drugs. The overlain UV spectra of telmisartan and amlodipine are shown in Fig. 1.

Fig 1. UV Spectra of standard Telmisartan and Amlodipine (8:1) Concentration

3. Standard Stock Solutions:

Telmisartan:

A Standard stock solution of Telmisartan was prepared by dissolving 100 mg of drug in 100 ml of methanol to get a concentration of 1000 µg/ml.

Amlodipine:

A Standard stock solution of Amlodipine was prepared by dissolving 50 mg of drug in 100 ml of methanol to get a concentration of 500 µg/ml.

Mixed Standard Solution:

The standard solutions were prepared by dilution of the stock standard solution with mobile phase to reach a concentration of 400 µg/ml and 50 µg/ml for Telmisartan and Amlodipine respectively.

4. System suitability:

The appropriate system suitability tests should be defined before method validation. For the system suitability test four parameters namely Relative standard deviation, Tailing factor and Theoretical plates and resolution were studied and are as shown in Table 1. The values obtained demonstrate the suitability of the system for the analysis of these drugs in combination. Chromatogram of standard Telmisartan and Amlodipine is as shown in Fig. 2.

Fig. 2 Chromatogram of standard Telmisartan and Amlodipine

Table 1 System suitability data

Sr. no.	Parameters	Telmisartan	Amlodipine
1	Theoretical plates/meter	6313	10547
2	Resolution	5.083	6.031
3	Asymmetry factor	1.48	1.85
4	% RSD	0.132	0.583

5. Calibration Curve:

The calibration curve solutions contained 320 µg/ml to 480 µg/ml for Telmisartan and 40 µg/ml to 60 µg/ml for Amlodipine. Of each solution, 20 µl was injected in triplicate under the operating chromatographic conditions described above. The peak areas were plotted against the corresponding concentrations to obtain the calibration graphs.

6. Preparation of Sample:

To determine the content of Telmisartan and Amlodipine in Pharmaceutical powder equivalent to 40 mg of Telmisartan was weighed accurately. The powder was transferred to a 100 ml volumetric flask containing 10 ml of methanol, shake for 5 min, to it 50 ml of mobile phase was added and the solution was sonicated for 20 min, allowed the solution to cool to room temperature and then volume made up to the mark with mobile phase, the resulting solution was filtered through Whatmann filter paper No. 41. Filtrate obtained was used as sample stock solution containing 400 µg/ml and 50 µg/ml of Telmisartan and Amlodipine respectively. 20 µL of sample solution was injected in duplicate to HPLC under the conditioned described above.

7. Method Validation:

As per ICH guidelines, the following method validation parameters were checked.

1. Specificity:

The specificity of the HPLC method was determined by complete separation of Telmisartan and Amlodipine in presence of common tablet Excipients like lactose starch etc., by comparing chromatograms of blank, standard and sample solution of same concentration.

2. Precision:

System Precision:

Method precision:-

Method precision was determined with the product. An amount of the product equivalent to 100% of the label claim of Telmisartan and Amlodipine was accurately measured and assayed. (Six replicate samples prepared and analyzed), and percentage relative standard deviation (%RSD) was calculated.

Intra-day Precision:-

Solutions of working standard and sample were repeated thrice in a day and percentage relative standard deviation (%RSD) was calculated.

Inter-day Precision:-

In the inter-day variation studies, injections of standard and sample solutions were made on three consecutive days and percentage RSD were calculated.

3. Limit of Detection and limit of Quantitation:

Limit of Detection (LOD) is the smallest concentration of the analyte that gives the measurable response. LOD was calculated using the following formula;

LOD= 3.3× standard deviation / Slope of calibration curve.

Limit of Quantitation (LOQ) is the smallest concentration of the analyte, which gives a response that can be accurately quantified. LOQ was calculated using the following formula; LOQ = 10 × standard deviation/ Slope of calibration curve.

4. Robustness:

Robustness of the method was determined by injecting the system suitability standard in five replicates by making slight deliberate changes in chromatographic conditions, such as;

1. Wavelength - ±5 nm

2. Flow rate - ± 10%

3. PH of Mobile Phase - ± 0.2 units

4. Mobile Phase composition - ± 2% Organic

Robustness of the method was verified by checking the system suitability requirements.

5. Accuracy:

The accuracy of the method was determined by recovery experiments. The recovery studies were performed by the standard addition method, at 80%, 100%, 120% level, and the percentage recovery was calculated. A known concentration of the working standard was added to the fixed concentration of the pre-analysed tablet solution. Percent recovered was calculated by comparing the area before and after the addition of the working standard. For both the drugs, recovery was performed in the same way. The recovery studies were performed in triplicate.

6. Solution Stability:

In order to demonstrate the stability of both standard and sample solutions, the solutions were analyzed over a period of 24 hrs at a room temperature. Percentage RSD was calculated.

RESULTS AND DISCUSSION:

1. Optimization of HPLC method:

HPLC method was optimized with a view to develop a simple, accurate method for estimation of drug in pharmaceutical formulation and in bulk drug. UV spectra of both Telmisartan and Amlodipine show that 237 is the suitable wavelength for detection of drugs. (Fig.1). Pure drugs were injected and run in different solvent system. It was found that Acetonitrile: methanol: buffer PH 5 in the ratio of 35:15:50 v/v gave an acceptable retention time (Rt-5.8-Amlodipine) and (Rt-11.1-Telmisartan) at the flow rate of 1.5 ml/min. and drugs showed typical peak nature and peaks were symmetrical at 237 nm. (Fig.2) and (Table 1). Tailing factor for both the drugs were less than 2 and the resolution of both the drugs was satisfactory. Ultimately mobile phase consisting of Acetonitrile, Methanol and Buffer (35:15:50, v/v) was selected for validation purpose.

2. Analysis of the marketed formulation:

The peaks at Rt 5.7 (for Amlodipine) and Rt 12.9 (for Telmisartan) were observed in the chromatogram of the drug samples extracted from tablets The drug content was found to be 98.85% ± 0.695(%RSD of 0.70) and 101.39% ± 0.399(%RSD of 0.39) for amlodipine and telmisartan, respectively and presented in Table 2.

Table 2 Analysis of the marketed formulation by HPLC

Parameters	Amlodipine	Telmisartan
Label claim (mg/tab.)	5	40
Amount found (mg/tab.)	4.94	40.56
Drug content (%)	98.85	101.39
% R.S.D.	0.70	0.39
S.E.	0.163	0.284

3. Linearity

Telmisartan and Amlodipine showed linearity in the concentration range of 320 µg/ml to 480 µg/ml (r2 =0.99996) and 40 µg/ml to 60 µg/ml (r2 =0.99995) for HPLC. For HPLC method the linearity of calibration graphs and adherence of the system to Beer’s law was validated by higher value of correlation coefficient.

4. Precision:

System Precision was determined by six replicate injections and six time measurement of working standard of Telmisartan and Amlodipine and percentage relative standard deviation (%RSD) was found to be 0.40 and 0.69 for Amlodipine and Telmisartan respectively. Method Precision was determined by preparing six replicate samples equivalent to 40 mg of Telmisartan and 5 mg Amlodipine and analyzed as per the proposed method. The percentage assay of each samples and their relative standard deviation were calculated Relative standard deviation (%RSD) was found to be 0.703 and 0.394 for Amlodipine and Telmisartan respectively. In the intra-day studies solutions of working standard and sample were repeated thrice in a day and percentage relative standard deviation (%RSD) was calculated. The % RSD was found to be 1.054 and 0.874 for telmisartan and amlodipine respectively. In the inter-day variation studies, injections of standard and sample solutions were made on three consecutive days and percentage RSD was calculated. The % RSD was found to be 0.622 and 0.858 for telmisartan and amlodipine respectively.

5. Accuracy:

Proposed method when used for extraction and subsequent estimation of Amlodipine and Telmisartan from pharmaceutical dosage form after spiking with additional drug afforded recovery of 98-102%.

6. Robustness:

Each factor selected to examine were changed at three levels (-1,0 and 1). One factor at the time was changed to estimate the effect. Thus, replicate injections (n=5) of mixed standard solution were performed under small changes in chromatographic conditions. Results presented in table indicate that the selected factors remain unaffected by small variation of these parameters.

7. LOD and LOQ:

The LOD and LOQ were found to be 0.0384, 0.116 µg/ml and 0.736, 2.231 µg/ml, respectively for Telmisartan and Amlodipine.

8. Solution Stability:

In order to demonstrate the stability of both standard and sample solutions, the solutions were analyzed over a period of 24 hrs at a room temperature. The results show that for the solutions, retention time and peak area of the Telmisartan and Amlodipine remained almost unchanged (% R.S.D less than 1.5) and no significant degradation within the indicated period was observed, this shows that both solutions were stable for at least 24 hours. The summary of validation parameters of proposed HPLC method is given in Table 3.

Table 3 Summary of Validation

Parameters	HPLC
Parameters	Telmisartan	Amlodipine
Linearity Range (µg/ml)	320-400	40-60
Correlation Coefficient	0.99996	0.99995
Accuracy (% Recovery)	99.72-100.17	99.76-101.26
Limit of Detection (µg/ml)	0.0384	0.736
Limit of Quantitation (µg/ml)	0.116	2.231
Precision % RSD	0.469	0.815
Robustness	Robust	Robust
Specificity	Specific	Specific

CONCLUSION:

The proposed HPLC Method provide simple, accurate and reproducible quantitative analysis for simultaneous determination of Telmisartan and Amlodipine in tablets. This method can be used for routine analysis of the telmisartan.

REFERENCES:

1. Marydele J. O., Ann S., Patricia E. H., John R. O., Jo Ann R. G., Mary A.D., The Merck Index- An Encyclopedia of Chemicals, Drugs and Biologicals, 13th Edition, Merck Research Laboratories-a division of Merck & Co.Inc., Whitehouse Station, NJ, 491, 1628.

2. Sweetman S.C., Martindale, the complete dsrug reference, 34th edition, Pharmaceutical press(a division of Royal Pharmaceutical Society of Great Bretain), 2005, 862, 1010.

3. Europian pharmacopoeia 5.0, 5th edition, Volume 2, published in accordance with the convention on the elaboration of European Pharmacopoeia (European treaty series no. 50), Council of Europe, Strasbourg Cedex, France, 2004, 981-982.

4. Indian Pharmacopoeia, 5th edition, Volume 2, Controller of Publications, Govt. of India, Ministry of health and Family Welfare, New Delhi, 2007, 554.

5. Palled M.S., Chatter M., Rajesh P.M., Bhat A.R. Difference spectrophotometric determination of telmisartan in tablet dosage forms. Indian Journal of Pharmaceutical Sciences, 2006, 68(5), 685-686.

6. Torrealday N., Gonzalez L., Alonso R. M., Jimenez R. M., Lastra E. Experimental design approach for the optimisation of a HPLC-fluorimetric method for the quantitation of the angiotensin II receptor antagonist telmisartan in urine. Journal of Pharmaceutical and Biomedical Analysis, 2003, 32(4-5), 847-857.

7. Gonzalez L., Alonso R.M., Jimenez R.M., Akesolo E. Application of capillary zone electrophoresis to the screening of some angiotensin II receptor antagonists. Electrophoresis, 2002, 23(2), 223-229.

8. Cagigal E., Gonzalez L., Alonso R.M., Jimenez R.M. pK_a determination of angiotensin II receptor antagonists (ARA II) by spectrofluorimetry, Journal of Pharmaceutical and Biomedical Analysis, 2001, 26(3), 477–486.

9. Hempen C., Schwarz L., Kunz U., Karst U. Determination of telmisartan in human blood plasma: Part I: Immunoassay development , Analytica Chemical Acta2006, 560(1-2), 35-40.

10. Hempen C., Schwarz L., Kunz U., Karst U. Determination of telmisartan in human blood plasma: Part II: Liquid chromatography-tandem mass spectrometry method development, comparison to immunoassay and pharmacokinetic study , Analytica Chemical Acta, 2006, 560(1-2), 41-49.

11. Maotian X., Junfeng S., Yaodong L. Rapid determination of telmisartan in pharmaceutical preparations and serum by linear sweep polarography, Journal of Pharmaceutical and Biomedical Analysis, 34(3), 2004, p. 681-687.

12. Kasture A.V., Ramteke M. Simultaneous UV-spectrophotometric method for the estimation of atenolol and amlodipine besylate in combined dosage form, Indian Journal of Pharmaceutical Sciences, 2006, 68(3), 394-396.

13. Basavaiah K., Chandrasekhar U. Prameela C. Sensitive spectrophotometric determination of amlodipine and felodipine using iron(III) and ferricyanide, II Farmaco, 2003, 58 (2), 141-148.

14. Valiyare G.R., Chandra A., Apte S.k., Mahadik A.A. HPLC determination of amlodipine, losartan and ramipril in pharmaceutical formulation, Indian Drugs, 2005, 42(5), 309-312.

15. Rajeswari K .R., Sankar G.G., Rao A.L., Seshagirirao J.V. RP-HPLC method for the simultaneous determination of Atorvastatin and Amlodipine in tablet dosage form, Indian Journal of Pharmaceutical Sciences, 2006, 68(2), 275-277.

16. Shah D.A., Bhatt K.K., Shankar M.B., Mehta R.S., Gandhi T.R., Baldania S.L. RP-HPLC determination of atorvastatin calcium and amlodipine besylate combination in tablets, Indian Journal of Pharmaceutical Sciences, 2006, 68(6), 796-799.

17. Dongre V.G., Shah S B., Karmuse P.P., Phadke M., Jadhav V.K. Simultaneous determination of metoprolol succinate and amlodipine besylate in pharmaceutical dosage form by HPLC. Journal of Pharmaceutical and Biomedical Analysis, 2008, 46(3), 583-586.

18. Mohammadi A., Rezanour N., Ansari M., Ghorbani F., Hashem M., Walker R. A stability-indicating high performance liquid chromatographic (HPLC) assay for the simultaneous determination of atorvastatin and amlodipine in commercial tablets. Journal of Chromatography B, 2007, 846(1-2), 215-221.

19. Abdel-Wadood H.M., Mohamed N A., Mahmoud A.M. Validated spectrofluorometric methods for determination of amlodipine besylate in tablets. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2008, 70(3), 564-570.

Received on 25.05.2010 Modified on 19.06.2010

Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1227-1230

HPLC Method Development for Telmisartan and Amlodipine