Development of RP-HPLC method for separation of atorvastatin calcium, amlodipine besylate and azilsartan medoxomil and its application to analyze their tablet dosage forms

 

Kavita wagh, Sandeep Sonawane*, Santosh Chhaajed, Sanjay Kshirsagar

MET’S Institute of Pharmacy, Bhujbal Knowledge City, Adgaon, Nashik-422003, India

 

ABSTRACT:

A single, simple, accurate and precise RP-HPLC method has been developed for the separation amlodipine besylate in presence of azilsartan medoxomil and atorvastatin calciumand estimation in their respective combined dosage forms. The chromatographic separation was achieved on C18 column (250 × 4.6 mm, 5 µ) using Acetonitrile: 20mM Phosphate buffer (pH 3) 60:40 v/v as a mobile phase at flow rate of 0.8 mL/min. The separation was achieved in isocratic mode and the detection was performed at 242 nm. Further the developed method was validated as per the ICH Q2 (R1) and applied for quantitation of atorvastatin calcium-amlodipine besylate and amlodipine besylate -azilsartan medoxomil in tablet formulations.

 

 

 

 

INTRODUCTION:

Hypertension and hyperlipidemia are common risk factors that frequently co-occur and these two conditions contribute disproportionately to the burden of cardiovascular disease. It was demonstrated that the presence of hypertension with hyperlipidemia have synergistic effect on endothelial dysfunction – a common cellular pathway that have integrated role in many cardiovascular risks[1].The use of atorvastatin calcium, a HMG-CoA reductase inhibitor and amlodipine besylate, a calcium channel antagonist given in combination to prevent such cardiovascular risks.

 

The risk of nonfatal myocardial infarction and fatal congestive heart failure was reduced in patients receiving atorvastatin calcium and amlodipine besylate. Also, amlodipine besylate plus atorvastatin calcium demonstrated greater antihypertensive efficacy than atorvastatin calcium alone [2].

 

Generally, to treat the stage 2 hypertension a combination therapy of angiotensin receptor antagonist with diuretic was common and popular. Recently, combinations of angiotensin receptor blockers with calcium channel blockers showed blood pressure lowering efficiencies similar to the diuretic combinations. In addition, these newer combinations avoid the metabolic side effects of diuretic combinations and provide therapeutic benefit for patients with angina. [3].

 

Azilsartan medoxomil is an angiotensin receptor blocker used in the treatment of hypertension is a prodrug that is hydrolyzed to azilsartan. In 2011, azilsartan medoxomil was approved in the US for the treatment of hypertension [4].

Chemically, atorvastatin calcium is [R-(R*, R*)]-2-(4-Flurophenyl)-β,-δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino) carbonyl]-1 H-pyrrole-1-heptanoic acid is HMG-CoA reductase inhibitor. Amlodipine besylate is benzenesulfonic acid; 3-O-ethyl 5-O-methyl 2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-1, 4-dihydropyridine-3, 5-dicarboxylate, a calcium channel blocker (CCB) andazilsartan medoxomil is (5-methyl-2-oxo-1, 3-dioxol-4-yl) methyl2-ethoxy-3-[[4-[2-(5-oxo-2H-1, 2, 4-oxadiazol-3-yl)phenyl]phenyl]methyl] benzimidazole-4-carboxylate Angiotensin receptor blocker.

 

There are several HPLC methods reported for the quantitation of atorvastatin calcium and amlodipine Besylate [5-9] and few methods for quantitation of amlodipine besylate and azilsartan medoxomil in their combined dosage forms.[10-12]

 

In present study, a single RP-HPLC method has been developed with the aim to separate atorvastatin calcium, azilsartan medoxomil and amlodipine besylate using single mobile phase. Further, the developed method was successfully applied for the quantitation of atorvastatin calcium and amlodipine besylate and for azilsartan medoxomil and amlodipine besylate in combined tablet formulations.

 

MATERIALS AND METHODS:

Chemicals and reagents:

The working standards of atorvastatin calcium, azilsartan medoxomil and amlodipine besylate were received as gift samples from Blue Cross Pharmaceuticals Ltd., Nashik and Glenmark Pharmaceuticals Ltd., Nashik, respectively. HPLC grade acetonitrile, methanol and phosphoric acid were purchased from S D fine-chemicals ltd, Mumbai. Analytical grade Potassium dihydrogen o-phosphate and di-potassium hydrogen phosphate were purchased from HiMedia laboratories Pvt. Ltd, Mumbai. Double distilled water used for the preparation of mobile phases was prepared freshly using double distillation assembly purchased from Borosil, Mumbai. Every time the prepared mobile phase was filtered through 0.45 µ × 47 mm membrane filter papers purchased from Axiva Scichem Biotech, Delhi. The pH meter used for the adjustment of pH was purchased from Sytronics India Pvt. Ltd, Ahmedabad and sonicator from PCI Analytics Pvt. Ltd, Mumbai. Tablets containing 20mg of Azilsartan Medoxomil- 5mg of Amlodipine besylate and 10mgof Atorvastatin calcium- 5 mg of amlodipine besylate were prepared in-house.

 

Instrumentation and chromatographic conditions:

JASCO HPLC system equipped with dual PU-2080 plus pumps, multichannel UV-2075 UV detector and injection loop (20 mL capacity), Rheodynemanual loop injection system 7725i was used. Data were collected using Browin Chromatography software (version 1.5). The mobile phase was composed of Acetonitrile:20mM Phosphare buffer (pH 3) 60:40 v/v. Isocratic elution was carried out on a Phenomenex Kinetex C18 column (250 × 4.6 mm, 5µ) at a flow rate of 0.8 mL/min. The detection was performed at 242 nm.

 

Preparation of standard solution:

Quantity equivalent to 10 mg of azilsartan medoxomil, amlodipine besylate and atorvastatin calcium were weighed and transferred to separate 10 mL volumetric flasks and volume was made upto the mark with methanol. The resulting solutions were of 1000 mg/mL of azilsartan medoxomil, amlodipine besylate and atorvastatin calcium, respectively.

 

Calibration curve standards for Amlodipine besylate and Azilsartan medoxomil:

From the standard stock solution of amlodipine besylate and azilsatran medoxomil six aliquots were prepare and diluted with mobile phase to get calibration curve standard with concentration of 2, 4, 6, 8, 10 and 12 µg/mL for amlodipine besylate and 8, 16, 24, 32, 40 and 48 µg/mL for azilsartan medoxomil. 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 Amlodipine besylate and Atorvastatin calcium:

From the standard stock solution of amlodipine besylate and atorvastatin calcium six aliquots were prepared and diluted with mobile phase to get calibration curve standard with concentration of 5, 10, 15, 20, 25 and 30 µg/mL for amlodipine besylate and 10, 20, 30, 40, 50 and 60 µg/mL for atorvastatin calcium. 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 Amlodipine Beslyate and Azilsartan medoxomil in Tablets:

For analysis of tablet formulation, tablet containing 5mg of amlodipine besylate and 20mg of azilsartan medoxomil were prepared in house. Tablets are weighed and finely powered. A tablet powered equivalent to 5 mg of amlodipine besylate and 20 mg of azilsartan medoxomil to 100 mL volumetric flask and shaken with methanol for 10 min. After filtration, the excipients were separated and the volume was made up to the 100 mL with the same solvent. From the stock solution, suitable aliquot was diluted with mobile phase to get concentration of 5 µg/mL of amlodipine besylate and 20 µg/mLof azilsartan medoxomil and subjected to chromatographic analysis.

 

Estimation of Amlodipine Beslyate and Atorvastatin calcium in Tablets:

For analysis of tablet formulation, tablet containing 5 mg of amlodipine besylate and 10 mg of atorvastatin calcium were prepared in house. Tablets are weighed and finely powered. A tablet powered equivalent to 5 mg of amlodipine besylate and 10 mg of atorvastatin calcium to 100 ml volumetric flask and shaken with methanol for 10 min. After filtration, the excipients were separated and the volume was made up to the 100 mL with the same solvent. From the stock solution, suitable aliquot was diluted with mobile phase to get concentration of 5 µg/mL of amlodipine besylate and 10 µg/mL of atorvastatin calcium and subjected to chromatographic analysis.

 

METHOD VALIDATION:

Method was validated as per ICH Q2 (R1) for accuracy, precision, and specificity, limit of detection (DL) and limit of quantification(QL).

 

Limit of Detection (DL) and limit of Quantification (QL):

DL and QL were determined from standard deviation of y-intercept of regression line and slope method.   

 

Accuracy and precision:

Accuracy of method were determined by analyzing the standard samples at three concentration level of 80%, 100%, 120% by standard addition method across the range for amlodipine besylate and azilsartan medoxomil and for amlodipine besylate and atorvastatin calcium. The method precision was established by three replicates of three (80%, 100% and 120%) for intraday 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.

 

Specificity:

To evaluate the specificity of the proposed method, blank tablets were chromatographed, absence of peaks in the chromatographic run at retention time of the amlodipine besylate and azilsartan medoxomil and amlodipine besylate and atorvastatin calciumwas taken as indication of specificity.

 

RESULT AND DISCUSSION:

Various mobile phases were tried to get separation of amlodipine besylate, azilsartan medoxomil and atrovastatin calcium. It was observed that a mobile phase with composition of Acetonitrile: 20 mM phosphate buffer (pH3) 60:40 v/v was gave appropriate resolution of Amlodipine besylate, azilsartan medoxomil and atrovastatin calcium. The optimum wavelength for detection was 242 nm. The retention time of amlodipine besylate, azilsartan medoxomil and atrovastatin calcium were observed at 3.04 min, 4.23 min, 5.79 min respectively (fig

 

Fig.1: Representative Chromatogram of amlodipine besylate (3.04 min), azilsartan medoxomil (4.23 min) and atorvastatin calcium (5.79 min).

 

 

I]Amlodipine Besylate and Azilsartan Medoxomil:

In calibration studies, it was observed that amlodipine besylate was linear in the range of 2-12 µg/mL and azilsartan medoxomil in the range of 8-48 µg/mL. The calibration curves with their respective calibration curve equation and regression are depicied in figure 2 and figure 3, respectively.

 

 

Fig. 2: Calibration of Amlodipine Besylate

 

Fig. 3: Calibration of Azilsartan Medoxomil

Limit of detection (DL) and limit of quantification (QL):

The result obtained for DL and QL are summarized in Table 1, for amlodipine besylate and azilsartan medoxmil respectively.

 

Table 1: DL and QL for amlodipine besylate and Azilsartan medoxomil

Parameter	Amlodipine Beslyate	Azilsartan Medoxomil
DL	0.16	0.12
QL	0.49	0.36

 

Accuracy and Precision Studies:

The results obtained for accuracy and precision are summarized in Table 2 and Table 3, for amlodipine besylate and azilsartan medoxomil, respectively. Mean values of concentration added were close to the concentration added and low values of %RSD indicates the acceptable accuracy and precision of the method.

 

 

Table 2: Accuracy and Precision study for Amlodipine Besylate

concentration added (µg/mL)	Amount Found (µg/mL)				MEAN	± SD	%RSD	%RECOVERY
4+3.2 7.2µg/mL 80%	DAY 1	7.04	7.06	7	7.033333	0.030551	0.434367	97.68518519
	DAY2	7.04	7.04	7.09	7.056667	0.028868	0.409081	98.00925926
	DAY3	7	7.04	7.09	7.043333	0.045092	0.640215	97.82407407
4+4 8µg/mL 100%	DAY 1	7.81	7.9	7.81	7.84	0.051962	0.662775	98
	DAY 2	7.9	7.81	7.71	7.806667	0.095044	1.21747	97.58333333
	DAY 3	7.81	7.9	7.8	7.836667	0.055076	0.702795	97.95833333
4+4.4 8.8µg/mL 120%	DAY 1	8.5	8.6	8.5	8.533333	0.057735	0.676582	96.96969697
	DAY 2	8.7	8.7	8.6	8.666667	0.057735	0.666173	98.48484848
	DAY 3	8.7	8.6	8.7	8.666667	0.057735	0.666173	98.48484848

 

Table 3: Accuracy and Precision study for Azilsartan Medoxomil

Concentration added (µg/mL)	Amount Found(µg/mL)				MEAN	±SD	%RSD	%RECOVERY
16+12.8 28.8µg/mL 80%	DAY 1	27.74	27.64	27.84	27.74	0.1	0.36049	96.31944444
	DAY 2	27.49	27.5	27.84	27.61	0.199249	0.721654	95.86805556
	DAY 3	27.74	27.5	27.89	27.71	0.196723	0.709936	96.21527778
16+16 32µg/mL 100%	DAY 1	31.98	31.89	31.98	31.95	0.051962	0.162634	99.84375
	DAY 2	31.8	31.76	31.81	31.79	0.026458	0.083226	99.34375
	DAY 3	31.8	31.76	31.81	31.79	0.026458	0.083226	99.34375
16+19.2 35.2µg/mL 120%	DAY 1	34.12	34.16	34.12	34.13333	0.023094	0.067658	96.96969697
	DAY 2	34.12	34.16	34.16	34.14667	0.023094	0.067632	97.00757576
	DAY 3	34.13	34.13	34.19	34.15	0.034641	0.101438	97.01704545

 

Specificity:

When blank tablets were analyzed as per the mentioned chromatographic conditions, no peak was obtained at the times of amlodipine besylate and azilsartan medoxomil.

 

Analysis of formulation:

Represented chromatogram for formulation of amlodipine beslyate and azilsartan medoxomil Respectively. 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.

 

Fig. 4 - Representative Chromatogram of specificity

 

Fig. 5- Representative chromatogram of formulation

 

 

II] Amlodipine Besylate and Atrovastatin Calcium

In calibration studies, it was observed that amlodipine besylate was linear in the range of 5-30 µg/mL and atorvastatin calcium in the range of 10-60 µg/mL. The calibration curves with their respective calibration curve equation and regression are depicied in figure 4 and figure 5, respectively.

 

Fig. 6: calibration of Amlodipine Besylate

 

Fig. 7: Calibration of Atorvastatin calcium

 

Limit of detection (DL) and limit of quantification (QL)

The result obtained for DL and QL are summarized in Table 4, for amlodipine besylate and atrovastatin calcium respectively.

Table 4: DL and QL for amlodipine besylate and atrovastatin calcium

Accuracy and Precision studies

The results obtained for accuracy and precision are summarized in Table 3 and Table 4, for amlodipine besylate and atorvastatin calcium, respectively. Mean values of concentration added were close to the concentration added and low values of %RSD indicates the acceptable accuracy and precision of the method.

 

 

Table 5: Accuracy and Precision study for Amlodipine Besylate

 

Table 6: Accuracy and Precision study for Atorvastatin calcium

 

Fig. 8 - Representative Chromatogram of specificity

Specificity:

When blank tablets were analyzed as per the mentioned chromatographic conditions, no peak was obtained at the times of amlodipine besylate and atorvastatin calcium.

 

Analysis of formulation

Represented chromatogram for formulation of amlodipine beslyate and atorvastatin calcium respectively. 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.

 

Fig. 9 - Representative Chromatogram of formulation

 

CONCLUSION:

A single, simple, accurate and precise RP-HPLC method has been developed, optimized and validated for simultaneous estimation of amlodipine besylate, azilsartan medoxomil and atorvastatin calcium 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, azilsartan medoxomil and atorvastatin calcium 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 Blue Cross Pharmaceuticals Ltd., Nahik and Glenmark Pharmaceutical Ltd., Nashik for providing gift samples of amlodipine besylate, azilsartan Medoxomil, atorvastatin calcium.

 

REFERENCES:

1.     Mason, R.P., A Rationale for Combined Therapy with a Calcium Channel Blocker and a Statin: Evaluation of Basic and Clinical Evidence. Current Drug Targets - Cardiovascular and Hematological Disorders, 2005. 5(6): p. 489-501.

2.     McKeage, K. and M.A.A. Siddiqui, Amlodipine/Atorvastatin Fixed-Dose Combination. American Journal of Cardiovascular Drugs, 2008. 8(1): p. 51-67.

3.     Weber, M.A., et al., Effects of combining azilsartan medoxomil with amlodipine in patients with stage 2 hypertension. Blood Pressure Monitoring, 2014. 19(2): p. 90-97.

4.     Vasiliou, S., Azilsartan medoxomil for the treatment of hypertension. Drugs Today (Barc), 2011. 47(9): p. 647-51.

5.     M. Madhu, S.L., C. Madhusudhana Chetty, Y. Pradeepkumar, Y. Hrushikeshreddy, V. Jaya Sankar Reddy, Analytical method development and validation of simultaneous determination of atorvastatin calcium and amlodipine besilate in tablet dosage form by RP-HPLC Pharmaceutical Sciences, 2011. Vol.2(Issue 2,): p. 149-160.

6.     Mohammadi, A., et al., 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): p. 215-221.

7.     Ahmed, M., Y. Manohara, and M. Ravi, RP-HPLC method development and validation for simultaneous estimation of atorvastatin calcium and amlodipine besylate. Int. J Chem. Tech Res, 2012. 4(1): p. 337-45.

8.     Chaudhari, B.G. and A.B. Patel, Simultaneous spectrophotometric estimation of atorvastatin calcium and amlodipine besylate in tablet dosage forms. International Journal of Chem. Tech Research, 2010. 2(1): p. 633-639.

9.     Juyal, V., et al., Method development and its validation for simultaneous Estimation of atorvastatin and amlodipine in combination in tablet dosage form by UV spectroscopy, using multi-component Mode of analysis. Journal of Pharmacy Research Vol, 2008. 1(2).

10.  Modi J. G. and Patel J. K., Stability indicating RP-HPLC method for the simultaneous determination of amlodipine besylate and azilsartan medoxom in tablet dosage form Indian Drugs, 2016. 53(06): p. 51-61.

11.  Jigna Zankat, M.B., Jigisha Patel Development and validation of analytical method for simultaneous estimation of Azilsartan medoxomil and Amlodipine besylate in synthetic mixture The Pharmaceutical and Chemical Journal, 2015(2(2)): p. 22-29.

12.  G. Kumara Swamy. A novel stability indicating validated RP-HPLC method for simultaneous determination of azilsartan and amlodipine besylate hydrochloride in bulk and tablet dosage form European Journal of Biomedical and Pharmaceutical Sciences 2015. 2(3): p. 316-332.

 

 

 

Received on 27.05.2017          Accepted on 28.07.2017        

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