RP-HPLC Method for the Simultaneous Estimation and Validation of Amlodipine Besylate and Atenolol in Bulk and Tablet Dosage Form in Biorelevant Dissolution Medium (Fassif)

 

Palani Shanmugasundaram1*, Kamarapu S. K2

1Director, School of Pharmaceutical Sciences, VISTAS, Vels University, Chennai, Tamilnadu, India.

2Research Scholar, Department of  Pharmaceutical Analysis, School of Pharmaceutical Sciences, VISTAS, Vels University, Chennai, Tamilnadu, India.

*Corresponding Author E-mail: director.sps@velsuniv.ac.in, kamarapu.sudheerkumar@gmail.com

 

ABSTRACT:

Objective: A simple, rapid, and precise reverse phase high performance liquid chromatographic (RP-HPLC) method for simultaneous analysis of Amlodipine Besylate and Atenolol in a tablet dosage form and in Biorelevant media has been developed and validated.

Methods:

The chromatographic separation was achieved using reverse phase C18 column; Kromasil C18 column (250 mm x 4.6 mm x 5μm). The mobile phase used was a mixture of Acetonitrile: Potassium di hydrogen phosphate solution (0.01M, pH 3.0 adjusting with Ortho phosphoric acid): Methanol (15:30:55) at isocratic mode and eluents were monitored at 254 nm using PDA detector.

Results:

By the method Amlodipine Besylate and Atenolol were eluted with retention times of 2.589 and 3.711 min, respectively. The method was continued and validated accordance with ICH guidelines. Validation revealed the method is rapid, specific, accurate, precise, reliable, and reproducible. Calibration curve plots were linear over the concentration ranges 25-125μg/mL for Amlodipine Besylate, 5-25μg/mL for Atenolol. Limits of detection (LOD) were 0.001, and 0.005μg/ml and limits of quantification (LOQ) were 0.004 and 0.015μg/mL for Amlodipine Besylate and Atenolol respectively.

Conclusion:

The statistical analysis was proves the method is suitable for the analysis of Amlodipine Besylate and Atenolol as a bulk and tablet dosage form in biorelevant dissolution media (Fasted State Simulated Intestinal Fluid-FaSSIF) without any interference from the excipients.

 

KEYWORDS:  Amlodipine Besylate and Atenolol,  RP-HPLC, Validation, Biorelevant media (FaSSIF).

 

 


INTRODUCTION:

Amlodipine Besylate is a second generation dihydro pyridine class of calcium channel blockers and is used in the treatment of both hypertension and angina pectoris. Like other calcium channel blockers, Amlodipine Besylate acts by blocking the influx of calcium ions into vascular smooth muscle and cardiac muscle cells during membrane depolarization.

 

This action causes relaxation of vascular and arterial smooth muscle cells, resulting in arterial vasodilation and a decrease in cardiac work and oxygen consumption. Atenolol is a β-blocker seem to be equally effective as an antihypertensive, antianginal and antiarrhymthmic drug widely used as Cardiovascular drug in combination with Amlodipine Besylate. Atenolol competes with sympathomimetic neuro transmitters such as catecholamines for binding at beta (1)-adrenergic receptors in the heart and vascular smooth muscle, inhibiting sympathetic stimulation[1-4]  

Amlodipine Besylate is Chemically 3-ethyl 5-methyl 2-[(2-aminoethoxy) methyl]-4-(2-chlorophenyl)-6-methyl-1, 4-dihydropyridine- 3, 5-dicarboxylate and its molecular formula C20H25ClN2O5 (Fig.1)[5]. It is official in IP-2007[5], USP-2010[6], and BP-2012[7].

 

Fig.1: Chemical structure of Amlodipine Besylate

 

Atenolol is chemically2-(4-{2-hydroxy-3-[(propan-2yl)amino] propoxy} phenyl) acetamide and its molecular formula C14H22N2O3 (Fig.2) [8], It is official in IP-2007 [8] and BP-2012 [9].

 

Fig.2: Chemical structure of Atenolol

 

In the scientific Literature survey reveals that various analytical methods have been reported for the assay of Amlodipine Besylate and Atenolol in pure form and in pharmaceutical formulations. While British Pharmacopoeia described liquid chromatography method for the assay of Amlodipine Besylate[7]. Non-aqueous titration method is specified in Indian Pharmacopoeia for the assay of Atenolol[8]. Many methods have been reported in the literature for the estimation of Amlodipine Besylate and Atenolol individually[10-12] and in other combination [13-32].

 

The present investigation was aimed at developing a fully validated RP-HPLC method for the simultaneous estimation of Amlodipine Besylate and Atenolol in bulk and pharmaceutical combined dosage form in biorelevant dissolution medium (FaSSIF)  that is more economical, simple, precise and accurate than the previous methods.

 

MATERIALS AND METHODS:

1. Experimental:

1.1. Materials and Methods:

Pharmaceutical grade working standards Amlodipine Besylate and Atenolol were obtained from Hetero Labs, Jedcharla, India. All chemicals and reagents were HPLC grade and were purchased from Merck Chemicals, Mumbai, India.

 

1.2. Instrumentation:

The analysis was performed using SHIMADZU(UFLC-LC20) High Performance liquid chromatography, analytical balance 0.1mg Sensitivity (SHIMADZU), PDA Detector (Standard cell) and data handling system (LC-20), pH meter (lab India), Sonicator. The column used is Thermo Hypersil Keystone ODS C18 column (250x2.5mm packed with 5μm size Stationary phase) with the flow rate 1.0ml/min (isocratic).

 

1.3. Preparation of blank Fasted State Simulated Intestinal Fluid (FaSSIF):

Accurately weighed 1.74g of Sodium hydroxide pellets, 19.77g of Sodium dihydrogen orthophosphate, and 30.93g of Sodium chloride dissolve in 5 L of purified water and adjust the pH 6.5 exactly by using 1N Hydrochloric acid [32].

 

1.4. Preparation of FaSSIF33:

Accurately weighed 3.3g of sodium taurocholate dissolve in 500 mL blank FaSSIF solution, add 11.8 mL of a solution to 100mg/mL lecithin in methylene chloride, and forming an emulsion. The methylene chloride was eliminated under vacuum at 40°C. Then draw a vacuum for 15 minutes at 250mbar and also followed by 15 minutes at 100mbar. These results gave in a clear, micellar solution, having no perceptible odor for methylene chloride. After that, it was cool to room temperature and adjusts the volume upto 2L with blank FaSSIF [32].

 

1.5. Preparation of Standard Stock solution:

Accurately weighed 10 mg of Amlodipine Besylate and Atenolol working standard and separately transferred into a 10ml clean dry volumetric flasks, add about 7mL of biorelevant media ( FaSSIF ) to each volumetric flask and sonicate to dissolve it completely and make volume up to the mark with the same solvent. Calibration standards at five levels were prepared by appropriately mixed and further diluted stock standard solutions in the concentration ranges from 25-125μg/mL for Amlodipine Besylate and 5-25μg/mL for Atenolol. Samples in triple injections were made for each prepared concentration. Peak areas were plotted against the corresponding concentration to obtain the linearity graphs.

 

1.6. Preparation of Standard solution:

The above standard stock solution was containing 1000μg/mL of each Amlodipine Besylate and Atenolol in separate volumetric flasks. Then transferred the 1ml of Amlodipine Besylate and 0.1ml of Atenolol of prepared standard stock solution into a clean 10ml volumetric flask and made upto the mark with diluent. And finally the standard solution concentrations were 100μg/mL and 10μg/mL of Amlodipine Besylate and Atenolol respectively.

 

1.7. Preparation of Test solution:

For the analysis of a tablet dosage form, 20 tablets were weighed individually and their average mass was determined. Then, the tablets were crushed to a fine powder. The powder equivalent to 50mg of Amlodipine Besylate and 5mg of Atenolol were transferred to a 100mL volumetric flask and dissolved in 100mL of biorelevant media (FaSSIF), sonication was done for 15 min with swirling. After sonication, the solution was filtered through a membrane filter paper (#0.45μ). From the above stock solution 2mL was transferred in to 10mL volumetric flask and made volume upto the mark with diluent, the final concentrations were 100μg/mL and 10μg/mL of Amlodipine Besylate and Atenolol respectively, then injected into the chromatographic system, and analyzed quantitatively. The analysis was repeated six times and the possibility of excipient interference with the analysis was examined.

 

1.8. Optimization of HPLC Method:

The HPLC method was optimized and developed with a simultaneous method for Amlodipine Besylate and Atenolol. The mixed standard solution (100μg/mL of Amlodipine Besylate and 10μg/mL of Atenolol) injected in HPLC by the followed chromatographic conditions. The chromatographic separation was achieved on a Thermo Hypersil Keystone ODS C18 column (250x2.5mm, 5μm). The isocratic mobile phase consisting of Acetonitrile: Potassium di hydrogen phosphate solution (0.01M, pH 3.0 adjusting with Ortho phosphoric acid): Methanol (15:30:55) was used throughout the analysis. The flow rate of the mobile phase was 1.0ml/min. Detection was monitored at wavelength of 254nm. The column temperature was kept at ambient and injection volume was 20μl.

 

RESULTS AND DISCUSSION:

The simultaneous estimation of Amlodipine Besylate and Atenolol was done by RP-HPLC and in the optimized method the mobile phase consists of Acetonitrile: Potassium di hydrogen phosphate solution (0.01M, pH 3.0 adjusting with Ortho phosphoric acid): Methanol (15:30:55). Then finally filtered using 0.45μ membrane filter paper and degassed in sonicator for 15 minutes. The detection is carried out using PDA detector at 254nm. The solutions are following at the constant flow rate of 1.0 ml/min. The retention time for Amlodipine Besylate and Atenolol was 2.589 and 3.711minutes respectively. Linearity ranges for Amlodipine Besylate and Atenolol were 25-125μg/mL and 5-25μg/mL respectively and the results were found for in the acceptable as (R2) = 0.999 and 0.9983 for Amlodipine Besylate and Atenolol respectively. LOD were 0.001 and 0.005μg/ml and LOQ were 0.004 and 0.015μg/mL for Amlodipine Besylate and Atenolol respectively. The all parameters value of RSD is less than 2.0% indicating the accuracy and precision of the method. The percentage recoveries were found 99.8% and 100.2% Amlodipine Besylate and Atenolol respectively.

 

1. Method Development and Optimization:

The HPLC procedure was optimized with a view to develop a suitable LC method for the analysis of Amlodipine Besylate and Atenolol in fixed dose for bulk and combined dosage form. It was found that Acetonitrile: Potassium di hydrogen phosphate solution (0.01M, pH 3.0 adjusting with Ortho phosphoric acid): Methanol (15:30:55) gave acceptable retention time (2.589 and 3.711min), theoretical plates, and good resolution for Amlodipine Besylate and Atenolol at the flow rate of 1.0ml/min (Table. 1; Fig. 1 & 2).

 

Table No. 1: Optimized Chromatographic Conditions

Parameters

Method

Stationary phase (column)

Thermo Hypersil Keystone ODS C18 column

(250x2.5mm, 5μm)

Mobile Phase

15:30:55 v/v/v (Acetonitrile: Potassium di hydrogen 

 phosphate solution : Methanol)

pH

2.8 ± 0.02

Flow rate (ml/min)

1.0

Run time (minutes)

6.0

Column temperature (°C)

Ambient

Volume of injection loop (μl)

20

Detection wavelength (nm)

254                                                                                                                           

Drugs Rt (min)

2.589 & 3.711

 

Fig.1: Blank Chromatogram

Fig.2: Chromatogram of Mixed Standared Amlodipine Besylate and Atenolol at 254nm from bulk drug

 

2. Assay:

Three batches of compound tablets were analyzed using the developed method. Satisfactory results were obtained that the mean percentage found for Amlodipine Besylate and Atenolol were in good agreement with the label claimed. The mean percentage found (Table-2) indicated that the proposed method could be adopted for the determination Amlodipine Besylate and Atenolol in combined tablet dosage form as shown in Fig. 3.

 

Table.2: Assay data for Atenolol

Tablet

(AMTAS-AT)

Label

Claim

(mg)

Amount

Estimated*

(mg)

%

Amount

Estimated

Acceptance

Range

Amlodipine Besylate

5

4.99

99.8

 

98%-102%

Atenolol

50

50.10

100.2

* Mean of 3 determinations

 

 

Fig.3: Chromatogram of Amlodipine Besylate and Atenolol at 254nm from pharmaceutical dosage form (AMTAS-AT)

 

3. Validation of Developed method34-36:

The proposed method was validated with the aspect of system suitability test, specificity, linearity and range, accuracy, precision, LOD, LOQ, stability and robustness according to the ICH guidelines.

 

3.1. System suitability:

A Standard solution of Amlodipine Besylate and Atenolol working standard was prepared as per procedure and was injected six times into the HPLC system. The system suitability parameters were evaluated from standard Chromatograms obtained by calculating the % RSD of retention times, tailing factor, theoretical plates and peak areas from five replicate injections as shown in Table 3-4.

 

Table-3: Results of System suitability Test for ATENOLOL

Injection

Retention time(Rt)

Peak Area

 Plate count

Tailing factor

1

3.711

1185786

 

6389

1.3

 

2

3.702

1184759

6455

1.3

3

3.698

1187496

6234

1.6

4

3.708

1190478

6478

1.3

5

3.715

1183897

6502

1.3

6

3.714

1184759

6384

1.2

Mean

-

1186196

-

-

SD

-

2433.47

-

-

% RSD

-

0.20

-

-

 

Table-4: Results of System suitability Test for AMLODIPINE

Injection

Retention time(Rt)

Peak Area

 Plate count

 Tailing

Factor

1

2.589

 

2008408

5752

 

1.4

 

2

2.570

2008412

5758

1.3

3

2.572

2008357

5672

1.2

4

2.578

2007478

5674

1.4

5

2.582

2008475

5749

1.3

6

2.584

2008364

5843

1.4

Mean

-

2008249

-

-

SD

-

380.0

-

-

% RSD

-

0.01

-

-

 

3.2. Linearity:

Linearity was evaluated by analysis of working standard solutions of Amlodipine Besylate and Atenolol of five different concentrations. The range of linearity ranges from 25-125μg/ml for Amlodipine Besylate and 5-25μg/ml for Atenolol (Table. 5). The result of correlation coefficients of Amlodipine Besylate and Atenolol (R2) = 0.999 & 0.9983 respectively (Fig. 4-5). There was an excellent correlation between peak areas and concentrations of each drug.

 

Fig.4: Linearity graph for Amlodipine Besylate

 

Fig.5: Linearity graph for Amlodipine Atenolol

 

Table No. 5: Data for Linearity

Analyte

Conc. range

(μg/mL)

Correlation

Coefficient

(R2)

Slope

Intercept

Amlodipine Besylate

25-125

0.999

15628x

821206

Atenolol

5-25

0.9983

46684x

506834

 

3.3. Accuracy:

The accuracy of the method was determined by calculating the recovery studies at three levels (50%, 100% and 150%) by standard addition method. Known amounts of standard Amlodipine Besylate and Atenolol were added to the pre quantified samples and they were subjected to proposed HPLC method. The recoveries results of Amlodipine Besylate and Atenolol in pharmaceutical preparation are shown in the Table-6-7.


Table.6: Accuracy Study of Amlodipine Besylate

Sample Id

Conc found (µg/ml)

Concn Obtained (µg/ml)

% Recovery 

Mean recovery

Statistical Analysis

50%

5

5.01

100.2

 

 

 

%RSD= 0.505

50%

5

4.96

99.2

99.73

50%

5

4.99

99.8

 

100%

10

9.95

99.5

 

 

 

%RSD=0.66

100%

10

9.87

98.7

98.8

100%

10

9.82

98.2

 

150%

15

14.64

97.6

 

 

 

%RSD=1.45

150%

15

14.76

98.4

98.8

150%

15

15.06

100.4

 

 

Table.7: Accuracy Study of Atenolol

Sample Id

Conc (µg/ml)

Concn Obtained (µg/ml)

% Recovery of  drug

Mean accuracy

% RSD

50%

5

4.92

98.0

 

 

99.2

 

 

1.2

50%

5

4.96

99.2

50%

5

5.02

100.4

100%

10

9.95

99.5

 

 

99.5

 

 

0.2

100%

10

9.94

99.4

100%

10

9.98

99.8

150%

15

14.78

98.6

 

99.0

0.530

150%

15

14.94

99.6

150%

15

14.83

98.8

 

 


3.4. Precision:

Precision study was performed to find out intraday and interday variations. The intraday and interday precision study of Amlodipine Besylate and Atenolol was carried out by estimating the correspondence response 3 times on the same day and on 3 different days for 3 different concentrations of Amlodipine Besylate and Atenolol and the results were reported in terms of % relative standard deviation (% RSD). All results fall within acceptance limits (RSD < 2), as shown in Table-8.

 

3.5. LOD and LOQ:

The LOD and LOQ for Amlodipine Besylate and Atenolol were separately determined by based on calculating the signal-to-noise ratio. Detection limit=3.3 σ/s; quantification limit=10 σ/s; where σ is the standard deviation of y-intercept of regression line and‘s’ is the slope of the calibration curve. Results were shown in the Table-9.

 

Table-8: Intra-day and Inter-day precision of Amlodipine Besylate and Atenolol Standard solutions

Drug

Concentration* (μg/ml)

Intra-day

Inter-day

Peak Area

% RSD

Peak Area

% RSD

Amlodipine Besylate

5

2005053

0.15

2010800

0.19

10

2007362

0.26

2002956

0.24

15

2007473

0.26

2012800

0.26

Atenolol

5

1183951

0.22

1184689

0.20

10

1184689

0.14

1188199

0.18

15

1186232

0.16

1195842

0.15

* Mean of each 3 determinations

 

 

 

Table-9: LOD and LOQ for Amlodipine Besylate and Atenolol

Parameter

Amlodipine Besylate

Atenolol

LOD

0.001

0.005

LOQ

0.004

0.015

 

3.6. Robustness:

The robustness study was done by making small changes in the optimized method parameters like ±1% change in flow rate and composition of mobile phase. There was no significant impact on the retention time and tailing factor. Results were sown in Table-10 and Table-11.


 

Table.10: Robustness data for Amlodipine

Std. Replicate

Variation in flow rate

Variation in Mobile phase composition

Flow Rate 0.8ml/min

Flow Rate1.2ml/min

Acetonitrile: Buffer: Methanol (15:35:50)

Acetonitrile: Buffer: Methanol (10:30:60)

1

2492492

1676589

1951632

1979168

2

2495874

1675428

1954783

1967452

Mean

2494183

1676009

1953208.0

1973310

SD

2391.4

820.9

2228.0

8284.46

%RSD

0.09

0.04

0.11

0.4

Retention time

3.150

2.168

2.618

2.572

Tailing factor

1.4

1.3

1.3

1.3

Theoretical plates

5752

4207

4577

4476

 

Table.11: Robustness data for Atenolol

Parameter

Variation in flow rate

Variation in Mobile phase composition

Standard

 

Flow Rate

0.8ml/min

Flow Rate

1.2ml/min

Acetonitrile:Buffer: Methanol

(15:35:50)

Acetonitrile:Buffer: Methanol

(10:30:60)

1

1500192

100524

1196996

1153397

2

1500426

100468

1198547

1154782

Mean

1500309

100496

1197772

1154090

SD

165.5

39.59

1096.2

979.34

%RSD

0.01

0.03

0.09

0.08

Retention time

4.674

3.121

4.394

3.331

Tailing factor

1.2

1.2

1.2

1.2

Theoretical plates

7187

5412

6498

6471

 


CONCLUSION:

The RP-HPLC method has been developed and validated for the simultaneous estimation of Amlodipine Besylate and Atenolol in tablet dosage form by using Biorelevent Dissolution Media (FaSSIF). The results show that the method is accurate, precise, linear, robust, simple and rapid. Acceptable regression values, %RSD and standard deviations which make it is versatile and valuable for simultaneous estimation of two drugs in bulk and pharmaceutical dosage forms. The run time is relatively short. The results of this developed RP-HPLC method could be conveniently adopted for quality control analysis of Amlodipine Besylate and Atenolol simultaneously from tablet dosage form in Biorelevent dissolution media.

 

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Received on 11.03.2017          Modified on 14.04.2017

Accepted on 02.08.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2017; 10(10):3379-3385.

DOI: 10.5958/0974-360X.2017.00601.1