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QUANTITATIVE DETERMINATION OF TELMISARTAN,RAMIPRIL, AMLODIPINE BESYLATE AND ATORVASTATINCALCIUM BY HPLCAbdullah A. Elshanawane a , Lobna M. Abdelaziz a , Magda M. Kamal b c & Hani M. Hafez da Medicinal Chemistry Department, Faculty of pharmacy, Zagazig University, Zagazig, Egyptb Nancy University, Francec EIPICO, Egyptd Bachelor degree of Pharmaceutical Science, Zagazig University, Zagazig, EgyptAccepted author version posted online: 05 Feb 2013.

To cite this article: Journal of Liquid Chromatography & Related Technologies (2013): QUANTITATIVE DETERMINATION OFTELMISARTAN, RAMIPRIL, AMLODIPINE BESYLATE AND ATORVASTATIN CALCIUM BY HPLC, Journal of Liquid Chromatography &Related Technologies

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Quantitative determination of Telmisartan, Ramipril, Amlodipine besylate and

Atorvastatin calcium by HPLC

Abdullah A. Elshanawane1, Lobna M. Abdelaziz1, Magda M. Kamal2,3, Hani M. Hafez4

1Medicinal Chemistry Department, Faculty of pharmacy, Zagazig University, Zagazig,

Egypt, 2Nancy University, France, 3EIPICO, Egypt, 4Bachelor degree of Pharmaceutical Science, Zagazig University, Zagazig, Egypt

Quality control department, EIPICO, 10th Ramadan, Egypt Tel.: 020113231458

The manuscript has not been published elsewhere and that it has not been submitted

simultaneously for publication elsewhere.

Abstract

Telmisartan is angiotensin-II-receptor antagonist (ARA II) which is used in treatment of

hypertension alone or in combination with other antihypertensive drugs like Ramipril and

Amlodipine besylate or in combination with anti hyperlipidemic agent like Atorvastatin

calcium. RP- HPLC method was developed for the assay of Telmisartan, Ramipril,

Amlodipine besylate and Atorvastatin calcium. The method was performed by reversed

phase high performance liquid chromatography using a mobile phase consisting of 0.025

M potassium dihydrogen phosphate (pH 6.0): acetonitrile =60:40v/v with detection at

205nm on a BDS Hypersil C18 (250x 4.6 mm, 5µm i.d) at flow rate of 1.5 ml/min in an

isocratic manner.

Analytical run time was 8 min. Method exhibited good linear relationship in

concentration ranges (10-60, 16-96, 10-60, 10-60µg/ml), Recovery percentage (100.06,

100.85, 99.54, 100.8%), LOD (0.58, 0.16, 0.72, 0.3 µg/ml) and LOQ (1.92 0.55, 2.4, 0.98

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µg/ml) for Ramipril, Telmisartan, Amlodipine besylate and Atorvastatin calcium

respectively. Method validation was developed following the recommendations for

analytical method validation of International Conference on Harmonization (ICH) and

Food and Drug Administration (FDA) organizations

KEYWORDS: HPLC, Telmisartan, Ramipril, Amlodipine besylate and Atorvastatin

calcium.

1. INTRODUCTION

Hypertension is the "silent killer" of humans because this disease is usually asymptomatic

until the damaging effects of hypertension such as coronary heart disease and stroke [1].

Treatment of Hypertension is carried out by anti-hypertensive drugs such as calcium

channel blockers like Amlodipine besylate and diuretics: e.g. angiotensin II receptor

antagonists (ARA II) like Telmisartan and angiotensin converting enzyme inhibitors

(ACEI) like Ramipril; so it is recommended to make combination between anti-

hypertensive drugs to lower blood pressure (BP) as possible especially in persistent cases

or very higher BP. In other dosage forms, combination between anti-hypertensive drugs

and anti-hyperlipidemic agent is present to aid in treatment of hypertension by

decreasing of cholesterol biosynthesis then blood lipid level which cause narrowing

blood arteries , vein and vessels.

Amlodipine besylate is chemically described as (3-Ethyl 5-methyl (4RS)-2-[(2-

aminoethoxy) methyl]-4-(2-chlorophenyl)-6-methyl- 1, 4-dihydropyridine-3, 5-

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dicarboxylate benzene sulphonate) [2]. Amlodipine besylate is a calcium-channel blocking

agent; a dihydropyridine derivative with an intrinsically long duration of action and can

be given once daily. Amlodipine besylate is an anti-hypertensive and used prophylaxis of

angina [3]. Ramipril is chemically described as (2S, 3aS, 6aS)-1-[(2S)-2- [[(1S)-1-

(ethoxy-carbonyl)-3-phenylpropyl] amino]-1-oxopropyl] octa hydro cyclo penta [b]

pyrrole -2-carboxylic acid) [2]. Ramipril is ACE inhibitors which used in all grades of

heart failure, Management of a myocardial infarction and diabetic nephropathy. It is the

most appropriate initial drug for hypertension in younger Caucasian patients [3].

Telmisartan is chemically described as 4-[[4-Methyl-6-(1-methyl-1H-benzimidazol-2-

yl)-2-propyl-1H-benzimidazol-1-yl] methyl] biphenyl-2-carboxylic acid [2]. Telmisartan

is an angiotensin II receptor antagonist with highly selectivity for type I angiotensin II

receptor. It is used as an alternative to an ACE inhibitor in the management of

hypertension, heart failure or diabetic nephropathy [3].

Atorvastatin calcium is (3R,5R)-7-[3-(phenylcarbamoyl)-5-(4-fluorophenyl)-2-

isopropyl-4-phenyl-1H-pyrrol-1-yl]-3,5-dihydroxyheptanoic acid, the calcium salt [4].

Atorvastatin calcium is a synthetic lipid-lowering agent. Atorvastatin competitively

inhibits 3-hydroxy-3-methylglutaryl-3-methylglutaryl coenzyme A (HMG CoA)

reductase, an enzyme involved in cholesterol synthesis, especially in the liver. Statins are

more effective at lowering LDL-cholesterol concentration. However, statins reduce

cardiovascular disease events [3]. (Fig. 1)

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It is necessary to develop a validated analytical method for assay of these drugs in

combination with each other in its pharmaceutical preparations. Literature review

revealed that BP described a RP-HPLC method for assay of Amlodipine besylate and

potentiometric titrations for assay of Atorvastatin calcium, Ramipril and Telmisartan [2].

USP described RP-HPLC methods for assay of Atorvastatin calcium and ion pair HPLC

for Ramipril, Amlodipine besylate and Telmisartan [4]. Some methods have been

published for simultaneous determination of two or three of the studied drugs in their

combination with each other [5–14]. Our recent method is characterized by a simplicity,

accuracy, preciseness and sensitivity.

2. EXPERIMENTAL

2.1. Instrumentation

2.1.1. Balance

KERN model 870-13, Instrument Kern Balance, Supplied from Kern, Germany

Model/Type/P 870-13, Serial No: 84444

2.1.2. High Performance Liquid Chromatography

Consisting of instrumental

(a) AGILENT 1200 Quaternary pump.

(b) AGILENT 1200 Diode Array detector (DAD).

(c) AGILENT 1200 Auto sampler (injector).

(d) Column: BDS Hypersil C18 (250x 4.6 mm, 5µm i.d) supplied from Thermo (USA).

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(e) The system equipped by Agilent chemistation PC program.

2.1.3. Ph-Meter

Metrohm (Switzerland).

2.1.4. Stirrer

Fischer scientific UK.

2.2. Chemicals And Reagents

All reagents used were of analytical grade or HPLC grade. Potassium dihydrogen

phosphate, orthophosphoric acid and Sodium hydroxide (NaoH) were supplied by

(Merck, Darmstadt, Germany), HPLC grade Acetonitrile and Methanol were supplied by

(Fischer scientific, U.K.) and Distilled water used in all the experiments, was obtained

from Milli-RO and Milli-Q systems (Millipore, Bedford, MA).

Telmisartan, Ramipril, Amlodipine besylate and Atorvastatin calcium working standard

powders were kindly supplied by Egyptian international pharmaceutical industries

company (EIPICO) (10th Ramadan, Egypt), and were used without further purification.

2.3. Pharmaceutical preparations

Caduet tablets 5/10 Pfizer Company (Egypt) contains (5 mg Amlodipine as Amlodipine

besylate and 10 mg Atorvastatin as Atorvastatin calcium) per tablet, B.NO: 0996099.

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Micardis 80 tablets Boehringer Ingelheim Company (Germany) contain 80 mg

Telmisartan per tablet B.NO:104643. Rampicardin 10 tablets EIPICO Company (Egypt)

contains (10 mg Ramipril) per tablet B.NO: 0520032011 (under registration).

2.4. Chromatographic Conditions

Mobile phase: A mixture of potassium dihydrogen phosphate buffer (pH 6.0, 0.025M) -

acetonitrile (60%:40, V/V).

Phosphate buffer (0.025 M) was prepared by dissolving 3.6 g potassium dihydrogen

phosphate in approximately 950 ml distilled water. The pH was adjusted to 6.0 with 1 M

NaoH and water was added to 1000 ml.

Column: BDS Hypersil C18 (250x 4.6 mm, 5µm i.d) supplied from Thermo (USA).

Detector: was set at 205 nm.

Flow rate: 1.5 ml/min.

Column temperature: 40 C˚.

Injection volume: 50 µl.

The mobile phase was filtered through a 0.45 µl Nylon membrane filter (Millipore,

Milford, MA, USA) under vacuum and degassed by ultrasonication (Cole Palmer,

Vernon Hills, USA) before usage.

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2.5. Preparation Of Stock Standard Solutions

Stock standard solutions containing (0.5, 0.8, 0.5, 0.5 mg/ml) of Ramipril, Telmisartan,

Amlodipine besylate and Atorvastatin calcium respectively were prepared by dissolving

(25, 40, 25, 25 mg) of each in methanol in 50 ml volumetric flask respectively. It was

then sonicated for 15 minutes and the final volume of solutions was made up to 50 ml

with methanol to get stock standard solutions.

2.6. Preparation Of Calibration Plot (Working Standard Solutions)

To construct calibration plots, The stock standard solutions were diluted with the mobile

phase(freshly prepared) to prepare working solutions in the concentration ranges (10-

60,16-96 ,10-60, 10-60µg/ml) for Ramipril, Telmisartan, Amlodipine besylate and

Atorvastatin calcium respectively. Each solution (n=5) was injected and

chromatographed under the mentioned conditions above. Linear relationships were

obtained when peak area was plotted against the corresponding concentrations for each

drug. Regression equation was computed.

2.7. Sample Preparation

A composite of ten Rampicardin tablets and Micardis tablets were prepared by grinding

them to a fine, uniform size powder, triturated using mortar and pestle. After calculating

the average tablet weight, amounts of powder equivalent to (10 and 40 mg) for Ramipril

and Telmisartan respectively of each type of tablets were accurately weighed and

transferred separately to 50 ml volumetric flasks respectively.

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A composite of ten Caduet tablets were prepared by grinding them to a fine, uniform size

powder, triturated using mortar and pestle. After calculating the average tablet weight,

amounts of powder equivalent to (5 and 10 mg) for Amlodipine besylate and Atorvastatin

calcium respectively of tablets were accurately weighed and transferred separately to 50

ml volumetric flasks respectively. The solutions were sonicated for 15 min and the

solutions were then filtered. Aliquots of appropriate volume (5 ml) were transferred to 50

ml calibrated flasks and diluted to volume with mobile phase to obtain the mentioned

concentration above. The diluted solutions were analyzed under optimized

chromatographic conditions and chromatogram is depicted in (Fig. 2).

3. RESULTS

3.1. Method Validation

3.1.1. Specificity

Specificity of the method was evaluated by assessing whether excipients present in the

pharmaceutical formulations interfered with the analysis or not [15]. A placebo for each

tablet was prepared by mixing the respective excipients and solutions were prepared by

following the procedure described in the section of sample preparation. The commonly

used tablet excipients did not interfere with the method. The diluent chromatogram shows

that the tablet diluent has negligible contribution after the void volume at the method

detection wavelength of 205 nm.

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3.1.2. Linearity And Range

The linearity of the method was evaluated by analyzing different concentration of the

drugs. According to ICH recommendations [15] at least five concentrations must be used.

In this study five Concentrations were chosen, in the ranges (10-60, 16-96, 10-60, and 10-

60µg/ml) corresponding to levels of 20-120% w/w of the nominal analytical

concentration for Ramipril, Telmisartan, Amlodipine besylate and Atorvastatin calcium

respectively. The linearity of peak area responses versus concentrations was

demonstrated by linear least square regression analysis. The linear regression equations

were {y = 81.732x - 90.758 (r= 0.9998), y = 261.59x + 337.95 (r= 0.9994), y = 32.153x -

78.694 (r= 0.9996), y = 155.56x - 194.21 (r= 0.9999)} for Ramipril, Telmisartan,

Amlodipine besylate and Atorvastatin calcium respectively. Where Y is the peak area of

standard solution and X is the drug concentration.

3.1.3. Precision

The precision of the assay was investigated by measurement of both repeatability and

Intermediate precision.

3.1.3.1. Repeatability

Repeatability was investigated by injecting 6 determinations at 100% of the test

concentration percentage RSD were calculated in Table 1.

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3.1.3.2. Intermediate Precision

In the inter-day studies, standard and sample solutions prepared as described above, were

analyzed in triplicate on three consecutive days at 100% of the test concentration and

percentage RSD were calculated in Table 2.

3.1.4. Accuracy

Accuracy was assessed using 9 determinations over 3 concentration levels covering the

specified range (80,100 and 120%). Accuracy was reported as percent recovery by the

assay of known added amount of analyte in the sample (Table 3).

3.1.5. Limits Of Detection And Limits Of Quantitation

According to the ICH recommendations [15], determination of limits of detection and

quantitation was based on signal to noise ratio (Table 4).

3.1.6. Robustness

Robustness of an analytical procedure is a measure of its capacity to remain unaffected

by small variations in method parameters and provides an indication of its reliability

during normal usage [15]. Robustness was tested by studying the effect of changing mobile

phase pH by ±0.1, the amount of acetonitrile in the mobile phase by ± 2%, temperature ±

2C,ْ different column and flow rate ± 0.05 ml/min had no significant effect on the

chromatographic resolution of the method.

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3.1.7. Stability Of Analytical Solution

Also as part of evaluation of robustness, solution stability was evaluated by monitoring

the peak area response. Standard stock solutions in methanol were analyzed right after its

preparation by 24 and 48 hours after at 5 c ْand for a day at room temperature. The change

in standard solution peak area response over 2 days was (1.09, 0.43, 0.34 and 0.18%) for

Ramipril, Telmisartan, Amlodipine besylate and Atorvastatin calcium respectively. Their

solutions were found to be stable for 2 days at 5 c ْand for a day at room temperature at

least.

4. DISCUSSION

4.1. Optimization Of Chromatographic Condition

Several trials were carried out to obtain simple, rapid simultaneous determination for

Ramipril, Telmisartan, Amlodipine besylate and Atorvastatin calcium by HPLC method

with simple mobile phase and more available accessories e.g. detectors and column.

Ramipril has maximum absorption at 205 nm so this wave length was selected because

other compounds give good absorption at 205 nm and beyond 220 nm, in which Ramipril

give poor absorption. Phosphate buffer is more suitable than acetate because its cut off

equals to 200 nm but acetate at 230 nm. Hypersil BDS C-18 (25cm) columns exhibited

good separation.

Controlling mobile phase pH has important role when analyzing ionizable compounds

(Ramipril, Telmisartan and Atorvastatin) by reversed phase (RP) HPLC, the retention of

analytes is related to their hydrophobicity. The more hydrophobic the analyte, the longer

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it is retained. When an analyte is ionized, it becomes less hydrophobic and therefore, its

retention decreases. Acids lose a proton and become ionized and carry a negative charge

when pH increases (at pH’s above the analyte’s pKa) so behaves as an extremely polar

molecule and bases gain a proton and become ionized when pH decreases therefore,

when separating mixtures containing acids and/or bases by reversed phase HPLC, it is

necessary to control the pH of the mobile phase using an appropriate buffer in order to

achieve reproducible results [16,17]. Addition of a phosphate buffer at higher pH eliminated

the broad tailing peaks and created rugged conditions suitable for successful assay. In this

study, Amlodipine besylate is ionized compound. Ramipril, Telmisartan and Atorvastatin

may be turn to ionizable compound by changing pH of mobile phase due to presence of

carboxylic group (-COOH) in their chemical structure.As pH=6.0 greater than pKa of

Ramipril, Telmisartan and Atorvastatin by difference equals to 1.1 which enables (-

COOH) to give carboxylate anion (-COO־) carrying negative charge so elute rapidly than

others. Telmisartan also elute more rapidly than other pH. The concentration of the

mobile phase buffer usually has little effect on retention in reversed phase HPLC, just as

long as the buffer concentration is high enough to control pH. A buffer concentration in

the range of 25 to 50 mM is adequate for reversed phase applications [17].

4.2. Application On Pharmaceutical Preparation

The proposed methods were successfully used to determine Ramipril, Telmisartan,

Amlodipine besylate and Atorvastatin calcium respectively in their dosage forms e.g.

Rampicardin® tablets, Micardis® tablets, Caduet® tablets respectively.

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Four replicate determinations were performed. Satisfactory results were obtained for each

compound, in good agreement with label claims (Table 5 and 6). The results obtained

were compared statistically with those from published [5, 12] by use of Student’s t-test (for

accuracy) and the variance ratio F-test (for precision). The results showed that the t and F

values were smaller than the critical values, indicating there were no significant

differences between the results obtained from this method and from published methods

(Table 7).

5.0. CONCLUSION

A simple, accurate, precise, robust and reliable LC method has been established for

simultaneous determination for Ramipril, Telmisartan, Amlodipine besylate and

Atorvastatin calcium in their formulations. The method has several advantages:

1- The first is using HPLC-UV which is the most available instrument in

pharmaceutical analysis in companies.

It is suitable for analysis of antihypertensive agents in their formulations in a single run,

in contrast with previous methods. This makes the method suitable for routine analysis in

quality-control laboratories, other merits are rapid analysis, more sensitive, simple mobile

phase and simple sample preparation.

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REFERENCES

[1] González, L.; López, J. A.; Alonso, R. M.; Jiménez, R. M. Fast screening method for

the determination of angiotensin II receptor antagonists in human plasma by high

performance liquid chromatography with fluorimetric detection. J Chromatogr. A. 2002,

949 (1-2), 49-60.

[2] British Pharmacopoeia, London (2011), with the monographs of the sixth edition of

the European pharmacopoeia monograph http://www.pharmacopoeia.co.uk. Accessed 1

Jan.2011

[3] Ahmed – Jushuf, I. H. et al. British national formulary, 2011, 62, 107,110,113 and

141.

[4] Health wise Knowledgebase (2011) US Pharmacopoeia, Rockville.

http://www.healthwise.org. Accessed 3 Jan 2011

[5] Abdallaha, O. M.; Badawey, A. M. Derivative- Ratio Spectrophotometric, Chemo

metric and HPLC Validated methods for Simultaneous Determination of Amlodipine and

Atorvastatin in Combined Dosage Form. Int. J. Ind. Chem. 2011, 2 (2), 78-85.

[6] Patil, U. P.; Gandhi, S.V.; Sengar, M. R.; Rajmane, V. S. Simultaneous

Determination of Atorvastatin Calcium and Telmisartan in Tablet Dosage Form by

Spectrophotometry. Int. J. Chem. Res. 2009, 1 (4), 970-973.

[7] Panchal, H.; Suhagia, B. Simultaneous Determination of Atorvastatin Calcium and

Ramipril in Capsule Dosage Forms by HPLC and HPTLC. JAOAC Int. 2010, 93 (5),

1450-1457.

[8] Pilli, N.R.; Inamadugu, J. K.; Mullangi, R.; Karra, V. K.; Vaidya, J. R.; Seshagiri, J.

V. Simultaneous determination of Atorvastatin, Amlodipine, Ramipril and Benazepril in

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human plasma by LC-MS/MS and its application to a human pharmacokinetic study.

Biomed Chromatogr. 2011, 25 (4), 439–449.

[9] Damle, M. C.; Patole, S. M.; Potale, L. V.; Khodke, A. S. A validated HPLC

method for analysis of atorvastatin calcium, Ramipril and aspirin as the bulk drug and in

combined capsule dosage forms. Int. J. Pharm. Sci. Rev. and Res. 2010, 3 (4), 40-45.

[10] Patel, V. A.; Patel, P. G.; Chaudhary, B. G.; Rajgor, N. B.; Rathi, S. G.

Development and Validation of HPTLC Method for the Simultaneous Estimation of

Telmisartan and Ramipril in Combined Dosage Form. Int. J. Pharm. Biol. Res. 2010,

1(1), 18-24.

[11] Patil, K. R.; Rane, V. P.; Sangshetti, J. N.; Shinde, D. B. A Stability-Indicating LC

Method for the Simultaneous Determination of Telmisartan and Ramipril in Dosage

Form. Chromatographia. 2008, 67 (7-8), 575-582.

[12] Gupta, Y.; Nagar, S.; pura, G.; road, T.; Rajasthan, J. Isocratic RP-HPLC-UV

method development and validation for simultaneous estimation of Ramipril and

Telmisartan in tablet. Asian J. Pharm. and Clinical Res. 2009, 2 (4), 104-111.

[13] Vekariya, N. R.; Patel, G. F.; Bhatt, H. S.; Patel, M. B.; Dholakiya, R. B.; Ramani,

G. K. Application of TLC-densitometry method for simultaneous estimation of

Telmisartan and Amlodipine besylate in pharmaceutical dosage forms. Int. J. Pharm. Res.

2009, 1 (4), 1644-1649.

[14] Kurade, V. P.; Pai, M. G.; Gude, R. RP-HPLC Estimation of Ramipril and

Telmisartan in Tablets. Ind. J. Pharm. Sci. 2005, 71 (2), 148–151.

[15] ICH Harmonized Tripartite Guideline, Validation of Analytical Procedures: Text

and Methodology Q2 (R1).

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[16] Heyrman, A. N.; Henry, R. A. http://www.hplcsupply.com /pdf/App_9.pdf.

[17] Preparing Buffered Mobile Phases for Reversed Phase HPLC, http://www.mac-

mod.com/pdf/TR-BufferedMP.pdf.

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Table 1. Repeatability of Ramipril, Telmisartan, Amlodipine besylate and Atorvastatin

calcium respectively

Drug name Average µg/ml Average % RSD

Ramipril 9.91 99.1 0.65%

Telmisartan 80.84 101.0 0.58%

Amlodipine besylate 4.99 99.96 0.41%

Atorvastatin calcium 9.99 99.95 0.15%

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Table 2. Intermediate precision of Ramipril, Telmisartan, Amlodipine besylate and

Atorvastatin calcium respectively

Drug name 1st day

µg/ml

2nd day

µg/ml

3rd day

µg/ml

pooled

average

pooled

average %

RSD

Ramipril 9.91 9.73 10.05 9.90 99.0 1.62%

Telmisartan 80.85 80.79 81.19 80.94 101.18 0.26%

Amlodipine besylate 4.99 4.99 4.98 4.99 99.80 0.20%

Atorvastatin calcium 9.99 10.02 10.04 10.02 99.45 0.23%

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Table 3. Recovery results for standard solution plus excipients for Ramipril, Telmisartan,

Amlodipine besylate and Atorvastatin calcium respectively

Drug name

Recovery at

80% conc.

(%)

Recovery at

100% conc.

(%)

Recovery at

120% conc.

(%)

Average

Recovery

(%)

RSD

Ramipril 100.6 100.0 99.55 100.06 0.20%

Telmisartan 101.59 101.3 99.68 100.85 1.10%

Amlodipine besylate 99.56 99.74 99.31 99.54 0.39%

Atorvastatin calcium 101.0 100.5 100.9 100.8 0.39%

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Table 4. Calibration data was resulted from method validation of Ramipril, Telmisartan,

Amlodipine besylate and Atorvastatin calcium respectively

Item Ramipril Telmisartan Amlodipine

besylate

Atorvastatin

calcium

Linear range (µg/ml) 10-60 16-96 10-60 10-60

Detection limit (µg/ml) 0.58 0.16 0.72 0.3

Quantitation limit (µg/ml) 1.92 0.55 2.4 0.98

Regression data

N 5 5 5 5

Slope (b) 81.732 261.59 32.153 155.56

Intercept (a) 90.758 337.95 78.694 194.21

Correlation coefficient 0.9998 0.9994 0.9996 0.9999

Standard error of

regression

0.68 1.68 0.93 0.55

(Y = a + bC, where C is the concentration of the compound (µg/ml) and Y is the drug peak area)

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Table 5. Results from determination of Ramipril, Telmisartan, Amlodipine besylate and

Atorvastatin calcium respectively in their dosage forms by proposed method

Product

name

Rampicardin

10 tablets

Micardis

tablets

Caduet tablets

Drug

name

Ramipril

(%)

Telmisartan

(%)

Amlodipine

besylate (%)

Atorvastatin

calcium (%)

Test 1 102.5 102.69 96.23 98.78

Test 2 103.26 100.59 96.59 98.3

Test 3 102 100.49 95.94 96.98

Test 4 103.2 100.82 98.49 97.52

SD 0.6 1.04 1.14 0.80

Average 102.74 101.10 96.80 97.89

R.S.D 0.58 1.03 1.17 0.81

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Table 6. Results from determination of Ramipril, Telmisartan, Amlodipine besylate and

Atorvastatin calcium respectively in their dosage forms by reported (published) method.

Product

name

Rampicardin

10 tablets

Micardis

tablets

Caduet tablets

Drug name Ramipril (%) Telmisartan

(%)

Amlodipine

besylate (%)

Atorvastatin

calcium (%)

Test 1 102.62 103.12 97.18 98.35

Test 2 102.59 103.14 96.98 97.82

Test 3 102.54 100.83 96.15 97.63

Test 4 101.87 100.91 96.11 98.46

SD 0.36 1.31 0.55 0.40

Average 102.4 102.00 96.60 98.06

R.S.D 0.35 1.28 0.57 0.41

Reported

method No

12 12 5 5

No. of experiments (n) is 4.

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Table 7. Statistical comparison of the proposed and published methods for determination

of Ramipril, Telmisartan, Amlodipine besylate and Atorvastatin calcium respectively in

their dosage forms by reported method (T- student test) and (F –test for variance).

Drug name Recovery ± SD Calculated

t- values

Calculated

F- values Proposed

methods

Reference

method

Rampicardin 10

tablets

Ramipril

(%)

102.74±0.60 102.4±0.36 0.81 2.82

Micardis tablets Telmisartan

(%)

101.10±1.04 102.00±1.31 1.49 0.63

Caduet tablets Amlodipine

besylate

(%)

96.8 ±1.14 96.60±0.55 0.28 4.29

Atorvastatin

calcium (%)

97.89±0.80 98.06±0.40 0.47 3.95

(Where the Tabulated t-values and F -ratios at p = 0.05 are 2.77 and 6.39) No. of experiments (n) is 4

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Figure 1. Structures of a- Amlodipine besylate b- Atorvastatin calcium c- Ramipril d-

Telmisartan

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Figure 2. Typical HPLC chromatogram of Ramipril (2.6 min), Telmisartan(4.18 min),

Amlodipine besylate (5.5 min) and Atorvastatin calcium (7.3 min) respectively on BDS

Hypersil C-18 (25cm) Column and mobile phase consisted of (A) acetonitrile and (B)

phosphate buffer pH=6.0 in ratio 40:60% at flow rate =1.5 ml/min by an isocratic

technique.

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