IAJPS 2016, 3 (2), 86-91
Divya et al
CODEN (USA): IAJPBB
ISSN 2349-7750
ISSN: 2349-7750
INDO AMERICAN JOURNAL OF
PHARMACEUTICAL SCIENCES Available online at: http://www.iajps.com
Research Article
RP-HPLC METHOD DEVELOPMENT AND VALIDTION FOR SIMULTANEOUS ESTIMATION OF TELMISARTAN AND RAMIPRIL IN PHARMACEUTICAL DOSAGE FORMS. M. Divya*, G. Anusha, Dr. H. Padmalatha Department of Pharmaceutical Analysis & Quality Assurance, Gyana Jyothi College of Pharmacy, Uppal depot, Ghatkesar(M), Hyderabad, Rangareddy Dist., Telangana-500039 Abstract: A simple, precise, accurate reverse phase high performance liquid chromatographic method has been developed and validated for the simultaneous estimation of Telmisartan and Ramipril in combined dosage forms. The mobile phase used was a mixture of % triethylamine : acetonitrile(55:45v/v). The elution was carried out at 260 nm. The method was validated in terms of suitability, precisicion , accuracy, linearity, ruggedness and robustness. The method can be successfully used to determine the drug content of marketed formulation. Key Words:Telmisartan,Ramipril, RP-HPLC, development, validation, simultaneous estimation.
Corresponding author:
M.Divya,
QR code
Department of Pharmaceutical Analysis & Quality Assurance, Gyana Jyothi College of Pharmacy, Uppal depot, Ghatkesar(M), Hyderabad, Rangareddy Dist., Telangana-500039. E-mail id: divya.pepper34@gmail.com,
Please cite this article in press as Divya et al, RP-HPLC Method Development and Validation for Simultaneous Estimation of Telmisartan and Ramipril in Pharmaceutical Dosage Forms, Indo Am. J. Pharm. Sci, 2016; 3(2).
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IAJPS 2016, 3 (2), 86-91
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INTRODUCTION:
Telmisartan: Telmisartan is an angiotensin II receptor antagonist (ARB) used in the management of hypertension. Generally, angiotensin II receptor blockers (ARBs) such as telmisartan bind to the angiotensin II type 1 (AT1) receptors with high affinity, causing inhibition of the action of angiotensin II on vascular smooth muscle, ultimately leading to a reduction in arterial blood pressure. Recent studies suggest that telmisartan may also have PPAR-gamma agonistic properties that could potentially confer beneficial metabolic effects. Chemically it is 2-(4-{[4-methyl-6(1-methyl-1H-1,3-benzodiazol-2-yl)-2-propyl-1H1,3-benzodiazol-1-yl]methyl}phenyl)benzoic acid.
Literature survey reveals that some analytical methods like UV-method & few spectrophotometric methods have been reported for the estimation of Telmisartan with Rmipril. The present work reports simple, sensitive, accurate, precise and economical method for determination of Telmisartan and Ramipril by HPLC method in the pure and its tablet formulation. The method was validated by parameters such as linearity,precision, accuracy, LOD&LOQ,robustness,stability and system suitability as per ICH guidelines and USP requirements. Suitable statistical tests were performed on validation data. MATERIALS AND METHODS:
Instruments and Equipements: Phenomenex
Fig.1: Structure of Telmisartan
Ramipril: Ramipril is a prodrug belonging to the angiotensinconverting enzyme (ACE) inhibitor class of medications. It is metabolized to ramiprilate in the liver and, to a lesser extent, kidneys. Ramiprilate is a potent, competitive inhibitor of ACE, the enzyme responsible for the conversion of angiotensin I (ATI) to angiotensin II (ATII). ATII regulates blood pressure and is a key component of the reninangiotensin-aldosterone system (RAAS). Ramipril may be used in the treatment of hypertension, congestive heart failure, nephropathy, and to reduce the rate of death, myocardial infarction and stroke in individuals at high risk of cardiovascular events. Chemically it is (2S,3aS,6aS)-1-[(2S)-2-{[(2S)-1ethoxy-1-oxo-4-phenylbutan-2-yl]amino}propanoyl]octahydrocyclopenta[b]pyrrole-2-carboxylic acid.
Fig.2: Structure of Ramipril
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C18 RP column (5 µm, 25 cm x 4.6 mm ID) with Isocratic mode with UV-Visible Detector (SPDIOA), PUMP (LC-IOAT) and (LC-IOAT Vp) were used. Reagents and chemicals: Tri ethyl amine, Acetonitrile, Methanol of analytical grade. Telmisartan and Ramipril were gift samples. The commercially available Telmisartan Ramipril tablets(Telista-RM) were purchased from the local market.
Standard solutions and calibration graphs for chromatographic measurement: Stock standard solutions were prepared by dissolving separately 5 mg of Telmisartan and Ramipril in 10 ml mobile phase (1000 μg/ml). The standard calibration solutions were prepared by appropriate dilution of the stock solution with methanol to reach a concentration range of 10-60μg/ml.20 μl injections were made for each concentration and chromatographed under the optimized conditions. METHOD VALIDATION: Linearity: The standard curve was obtained in the concentration range of 50-150 μg/mL. The linearity was evaluated by linear regression analysis using the least square method. It was found that correlation coefficient and regression analysis are within the limits. Precision: The precision of the assay was determined in terms of intra-day and inter-day precision. The intra-day and inter-day variation in the peak area of drug solution was calculated in terms of coefficient of variation (C.V.) obtained by multiplying the ratio of standard deviation to mean with 100.
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IAJPS 2016, 3 (2), 86-91
Divya et al
ISSN 2349-7750
System suitability: System suitability parameters such as column efficiency (theoretical plates), resolution factor and peak asymmetry factor of the optimized methods were found satisfactory.
Limit of detection (LOD) and limit of quantitation (LOQ): The LOD was 10 mg/mL. The LOQ was 100mg/mol with coefficient of variation of 0.50 % and accuracy of 99.17 %. The LOQ was 500 mg /mol with coefficient of variation of 0.065 % and accuracy of 100.11 %.
RESULTS: Acetonitrile was selected as good separating agent for Robustness: this drug, since it shows maximum recovery; The robustness was checked by changing the flow comparison of recovery with methanol was done. The rate to 0.8 and 1.2 mol/min and the wavelength chromatogram was recorded for the standard 260nm the method suits best. calibration and plasma sample developed Accuracy: chromatographic conditions. The retention time of The accuracy of the HPLC method was assessed by Telmisartan and Ramipril are 5.33 and 7.47min. The adding known amount of drug solution to a drug chromatogram is well resolved without any solution of known concentration and subjecting the interference from one another. Moreover, peaks do samples to the proposed HPLC method. The recovery not show any tailing or fronting and it shows straight studies were replicated 3 times. The accuracy was baseline. The concentration of Telmisartan in rat expressed in terms of recovery and calculated by plasma was determined from the calibration of the multiplying the ratio of measured drug concentration spiked plasma by regression analysis. It shows very to the expected drug concentration with 30 μg/mL so good linearity in the range of 100-500 mg/ml and the as to give the percentage recovery. r2 value was found to be 0.9974. Table 1: Recovery Study Levels
Conc. of Drug added (ng/ml)
Level – I
50
Level –II
100
Level III
150
Amount of drug recovered in plasma sample (ng/ml) CAN
MET
ACN
MET
47.2
39.3
94.4
78.6
95.09
87.21
95.09
87.21
144.54
136.39
96.36
90.2
(% ) Recovery
Calibration curv e 0.4500 0.4000
y = 0.0008x - 0.0032 R
Peak area ratio
0.3500
2
= 0.9974
0.3000 0.2500 0.2000 0.1500 0.1000 0.0500 0.0000 0
200
400
600
Concentration (ng/ml)
Fig 3: Calibration curve
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IAJPS 2016, 3 (2), 86-91
Divya et al
ISSN 2349-7750
Fig 4: Typical chromatogram of Telmisartan and Ramipril Table 2: Accuracy and Precision Studies (Intra - Day) S. No.
Conc. of Drug (mg/ml)
1 2 3 4
50 100 150 200
Mean*Conc.found (mg /ml) 48.75 ± 0.420 99.38 ± 0.221 149.53 ± 0.250 199.35 ± 0.369
Accuracy (%)
Precision (%)
97.50 99.38 99.68 99.68
0.862 0.223 0.167 0.185
Table 3: Accuracy and Precision Studies (Inter - Day) S.No.
Conc. of Drug (mg/ml)
Mean*Conc. found (mg /ml)
Accuracy (%)
Precision (%)
1 2 3 4
50 100 150 200
49.4 ± 0.336 99.5 ± 0.216 149.35 ± 0.310 199.55 ± 0.061
98.80 99.50 99.57 99.77
0.681 0.217 0.208 0.064
Table 4: System Suitability Studies S.No 1 2 3 4 5
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Parameters Theoritical Plate Resolution Factor HETP LOD LOQ
Telmisartan 9614.831
Ramipril 7648.34 7.593
16.22 10mg/ml 100mg/ml
21.49 10mg/ml 1000mg/ml
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Divya et al
Pharmacokinetic Study After a single oral dose of 40 mg/kg of Telmisartan the sample was in measurable amount in plasma up to 5 hours. The pharmacokinetic parameters of the samples were calculated manually and using prism, graph pad version 5, Microsoft excel software’s and results were calculated. The half life (T1/2) of Telmisartan were calculated and found to be 1.314 and 1.433 hours. Area under the curve of and was calculated 1930.65 and 2202.85 mg hr/ml and was calculated to be 184.97 and 419.35
ISSN 2349-7750
mg hr/ml respectively and was calculated to be 2115.622 and 2622.2 mg hr/ml. Elimination rate constant (kel) was calculated for and from the slope of log concentration versus time curve with regression analysis. Elimination rate constant was calculated to be 0.5273 and 0.4836 hr-1.
Table.5: Plasma concentration of Telmisartan of two products
Time (hr)
Blood Conc. (mg/ml) Telsite 00 104.78 188.56 220.3 291.47 331.84 987.385 425.68 310.52 256.87 201.05 97.538
0 0.416 0.75 1 1.5 2 2.5 3 3.5 4 4.5 5
Telvas 00 148.65 214.47 267.42 314.56 764.87 1169.234 624.56 412.78 332.12 248.78 202.8
Table.6: Pharmcokinetic parameters of two products S.No.
Parameters
Telsite (reference product)
Telvas (test product)
1
AUC 0 – t (mg. hg/ml)
1930.65
2202.85
2
AUC 0 – ∞ (mg. hr/ml)
2115.62
2622.2
3
C max (mg /ml)
987.39
1169.23
4
T max (hrs)
1.621
1.651
5
K eli (hrs -1)
0.5273
0.4836
6
T ½ (hrs)
1.314
1.433
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IAJPS 2016, 3 (2), 86-91 The pharmacokinetic parameter such as Cmax, Tmax,, , Kel and T1/2 are compared for the bioequivalence study by statistical analysis. The p value obtained from one-way ANOVA and t-test were found to be 0.7928. Hence there is no significant difference between two products. DISCUSSION: A rapid, sensitive and selective HPLC method for the determination of Telmisartan in rat plasma was developed and validated. Sample preparation was assured by one-step protein precipitation method. Separation occurred on a Phenomenex C18 RP column (5 µm, 25 cm x 4.6 mm ID) with a mobile phase of 0.4 % triethylamine (pH 7.0) and acetonitrile(55:45% v/v) and detection at 295 nm. The standard curve was linear (r > 0.9974) over the concentration range of 100 – 500 mg/ml. The lower limit of quantification for Telmisartan was 10.0 mg/ml using 20 µl plasma samples. This method was successfully applied to the bioequivalent study of Telmisartan in rats for two formulation, reference (pantocid) and test (pantop). For bioequivalence study, parameters like Cmax, Tmax,, , Kel and T1/2 are compared by statistical analysis. The maximum concentration (Cmax) obtained in two brands Telsite and Telvas were 987.39 and 1169.23 mg/ml respectively. The half life (T 1/2) of Telmisartan were calculated and found to be 1.621 and 1.651hours. Area under the curve was calculated as 1930.65 and 2202.85 mg hr/ml and mg hr/ml respectively and was calculated to be 2115.62 and 2622.2 mg hr/ml. Elimination rate constant (kel) was calculated for the slope of log concentration versus time curve with regression analysis. Elimination rate constant was found to be 0.5273 and 0.4836 hr-1. The p value obtained from one-way ANOVA and t-test were found to be 0.7328, which shows there is no significant difference between two products. The statistical comparison of AUC0-t, AUC0-∞,Tmax and Cmax clearly indicated no significant difference in the two brands of Telsite and Telvas 40-mg capsules. 90% confidence intervals for the mean ratio (T/R) of AUC0-t, AUC0-∞ and Cmax were entirely within the Food and Drug Administration acceptance range. The method has proven to be simple, specific, sensitive, precise and accurate and is suitable for Telmisartan quantification in plasma samples from
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Divya et al
ISSN 2349-7750
bioequivalence, and pharmacokinetic studies in healthy albino rats. It was successfully applied to a bioequivalence study of two drug products containing Telmisartan. CONCLUSION: The proposed HPLC method was found to be simple, rapid, sensitive, precise and accurate for the estimation of Telmisartan & Ramipril in pharmaceutical formulations. Hence, this method can easily and conveniently adopt for routine quality control analysis in bulk drug and its pharmaceutical formulations. ACKNOWLEDGEMENT: The authors are thankful to Gyana Jyothi College of Pharmacy , Hyderabad for their valuable guidance , innovate advice, technical and moral support given to us throughout the entire course to carry out this project work. REFERENCES: 1.Pal, T.K., Gamesman, M., Bioavailability and Bioequivalence in Pharmaceutical technology, CBS Publication and Distributors, New Delhi. 01 – 51. 2.Shargel L.,Wu-pong S., Andrew B.C., Applied Biopharmaceutics and Pharmacokinetics,5th edition, Mc Graw Hill Companies, US, 453 – 498. 3. Detailed drug profile from the web page of Wyeth laboratories,Germany,www.fda.gov/cder/foi/label/20 00/20987lbl.pdf 4.N.V.S. Ramakrishna, K.N. Vishwottam, S. Wishu, M. Koteshwara, S. Suresh Kumar,High-performance liquid chromatography method for the quantification of pantoprazole in human plasma. Journal of Chromatography B, 2005;822: 326–329. 5.Snyder, L.R., Kirkland, J.J., Practical HPLC method development, 2 nd edition. A WilleyInterscience Publication, USA, 643 – 712. 6.Sethi, P.D., Quantitative Analysis of Drugs in Pharmaceutical Formulation., 3 rd edition, CBS Publisher and Distributors, 1997, 3. 7.Chung Chow Chan., Y.C. Lee, Herman Lam., Xue – Ming Zhang, Analytical Method Validation and Instrument Performance Verification, Pg. No: 35-45. 8.Validation oaf analytical procedure: Methodology, ICH Harmonized Tripartite Guideline. 1996;1-8
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