A Simple and Sensitive RP-HPLC Method for Estimation of Dronedarone in Bulk and Tablet Dosage Forms

 

K. Nageswara Rao¹*, S. Ganapaty² and A. Lakshmana Rao³

¹K. G. R. L. College of Pharmacy, Bhimavaram- 534 202, A.P., India.

²A. U. College of Pharmaceutical Sciences, Visakhapatnam- 530 003, A.P., India.

³V. V. Institute of Pharmaceutical Sciences, Gudlavalleru- 521 356, A.P., India.

 

ABSTRACT:

 

 

INTRODUCTION:

Dronedarone hydrochloride is a benzofuran derivative of amiodarone¹. Chemically Dronedarone is, N-{2-butyl-3-[4-(3-dibutylaminopropoxy)benzoyl]benzofuran-5-yl}methane sulfonamide, hydrochloride² (Fig. 1). Dronedarone is an novel antiarrhythmic agent with multichannel blocking and antiadrenergic properties³. Dronedarone is used to treat certain heart rhythm disorders called atrial fibrillation or atrial flutter⁴. The mechanism of action of Dronedarone is unknown. A few Spectrophotometric⁵, HPLC^6-8 and LC-MS⁹ methods were reported earlier for the estimation of Dronedarone in bulk and pharmaceutical dosage forms. In the present study the authors report a rapid, sensitive, accurate and precise HPLC method for the estimation of Dronedarone in bulk drug and in tablet dosage forms.

 

Fig. 1: Chemical structure of Dronedarone hydrochloride

 

MATERIALS AND METHODS:

Chromatographic conditions:

The analysis of the drug was carried out on a Waters HPLC system equipped with a reverse phase XTerra Symmetry C18 column (100 x 4.6 mm I.D., 5 μm particle size), a 2695 binary pump, a 20 μL injection loop, auto sampler and a 2487 dual absorbance DAD or UV detector and running on Waters Empower software.

 

Chemicals and solvents:

The reference sample of Dronedarone was provided as gift sample from Sumages Pharma Pvt. Ltd., Bhimavaram, India. Dronedarone tablets were purchased from local market. HPLC grade acetonitrile was purchased from E. Merck (India) Ltd., Mumbai, India. Sodium dihydrogen phosphate, triethyl amine and orthophosphoric acid of AR Grade were obtained from S.D. Fine Chemicals Ltd., Mumbai, India. HPLC grade water obtained from Milli Q water purification system was used throughout the study.

 

Preparation of phosphate buffer pH 3.0:

2.5 grams of sodium dihydrogen phosphate was weighed into a 1000 ml beaker, dissolved in HPLC water and 2 ml of triethyl amine was added. Diluted to 1000 ml with HPLC water and pH adjusted to 3.0 with orthophosphoric acid.

 

 

Fig. 2: Calibration curve of Dronedarone

 

Preparation of mobile phase and diluents:

500 ml of the phosphate buffer was mixed with 500 ml of acetonitrile. The solution was degassed in an ultrasonic water bath for 5 minutes and filtered through 0.45 µm filter under vacuum. The same mobile phase was used as diluent.

 

Preparation of standard stock solution:

Accurately weigh and transfer 10 mg of Dronedarone working standard into a 10 mL volumetric flask, add about 7 mL of diluent, sonicate to dissolve it completely and make volume up to the mark with the same solvent. Further pipette 0.4 mL of the above stock solution into a 10 mL volumetric flask and dilute up to the mark with diluent. Mix well and filter through 0.45 µm filter.

 

Preparation of sample solution:

Weigh 20 Dronedarone tablets and calculate the average weight. Accurately weigh and transfer the sample equivalent to 10 mg of Dronedarone into a 10 mL volumetric flask. Add about 7 mL of diluent, sonicate to dissolve it completely and make volume up to the mark with diluent. Mix well and filter through 0.45 µm filter. Further pipette 0.4 mL of the above stock solution into a 10 mL volumetric flask and dilute up to the mark with diluent. Mix well and filter through 0.45 µm filter.

 

Calibration plot:

About 10 mg of Dronedarone was weighed accurately, transferred into a 10 mL volumetric flask and dissolved in 7 mL of a 50:50 v/v mixture of phosphate buffer and acetonitrile. The solution was sonicated for 15 min and the volume made up to the mark with a further quantity of the diluent to get a 1000 µg/mL solution. From this, a working standard solution of the drug (40 µg/mL) was prepared by diluting with the above solution to 10 mL in a volumetric flask. Further dilutions ranging from 10-60 µg/mL were prepared from the solution in 10 mL volumetric flasks using the above diluent. 20 µL of each dilution was injected six times into the column at a flow rate of 0.8 mL/min and the corresponding chromatograms were obtained. From these chromatograms, the average area under the peak of each dilution was computed. The calibration graph constructed by plotting concentration of the drug against peak area (Fig. 2) was found to be linear in the concentration range of 10-60 µg/mL of the drug. The relevant data are furnished in Table 1. The regression equation of this curve was computed. This regression equation was later used to estimate the amount of Dronedarone in tablet dosage forms.

 

Table 1. Calibration data of the method

Concentration (mg/mL)	Mean peak area (n=6)
10	1963417
20	3514290
30	5425095
40	6985736
50	8692380
60	10896246

 

Procedure:

A mixture of phosphate buffer and acetonitrile in the ratio of 50:50 v/v was found to be the most suitable mobile phase for ideal separation of Dronedarone. The solvent mixture was filtered through 0.45 μm membrane filter and sonicated before use. It was pumped through the column at a flow rate of 0.8 mL/min. The column was maintained at ambient temperature. The pump pressure was set at 800 psi. The column was equilibrated by pumping the mobile phase through the column for at least 30 min prior to the injection of the drug solution. Inject 20 mL of the standard, sample solutions into the chromatographic system and measure the area for the Dronedarone peak. The detection of the drug was monitored at 290 nm. The run time was set at 6 min. Under these optimized chromatographic conditions the retention time obtained for the drug Dronedarone was 2.578 min. A typical chromatogram showing the separation of the drug is given in Fig. 3.

 

Fig. 3:  Typical   chromatogram of Dronedarone

 

Validation of the proposed method:

The specificity, linearity, precision, accuracy, limit of detection, limit of quantification, robustness and system suitability parameters were studied systematically to validate the proposed HPLC method as per the ICH guidelines for the estimation of Dronedarone¹⁰. Solution containing 40 µg/mL solution of Dronedarone was subjected to the proposed HPLC analysis to check method precision and intermediate precision of the method and the results are furnished in Table 2. The accuracy of the HPLC method was assessed by analyzing solutions of Dronedarone at 50, 100 and 150 % concentration levels by the proposed method. The results are furnished in Table 3. The system suitability parameters are given in Table 4.

 

Estimation of Dronedarone in tablet dosage forms:

Commercial formulations of Dronedarone tablets were chosen for testing the suitability of the proposed method to estimate Dronedarone in tablet formulations. Twenty tablets were weighed and powdered. An accurately weighed portion of this powder equivalent to 10 mg of Dronedarone was transferred into a 10 mL volumetric flask and dissolved in 5 mL of a 50:50 v/v mixture of phosphate buffer and acetonitrile. The contents of the flask were sonicated for 15 min and a further 3 mL of the diluent was added, the flask was shaken continuously for 15 min to ensure complete solubility of the drug. The volume was made up with the diluent and the solution was filtered through a 0.45 µm membrane filter. This solution containing 40 µg/mL of Dronedarone was injected into the column six times. The average peak area of the drug was computed from the chromatograms and the amount of the drug present in the tablet dosage form was calculated by using the regression equation obtained for the pure drug. The relevant results are furnished in Table-5.

 

Table 2. Precision data of the proposed HPLC method

Concentration of Dronedarone (40 mg/mL)	Peak area
	Method precision	Intermediate precision
Injection-1	6867436	6883073
Injection-2	6883073	6946738
Injection-3	6990756	6990756
Injection-4	6990756	6992673
Injection-5	6973102	6973102
Average	6941024	6957268
Standard Deviation	75599.5	45391.2
% RSD	1.09	0.65

 

 

Table 3. Accuracy studies

 

 

Table 4. System suitability parameters

 

Table 5. Assay studies

 

RESULTS AND DISCUSSION:

In the proposed method, the retention time of Dronedarone was found to be 2.578 min. Quantification was linear in the concentration range of 10-60 µg/mL. The regression equation of the linearity plot of concentration of Dronedarone over its peak area was found to be y=70288.4+176454.446x (r²=0.999), where x is the concentration of Dronedarone (µg/mL) and y is the corresponding peak area. The number of theoretical plates calculated was 2972.3, which indicates efficient performance of the column. The limit of detection and limit of quantification were found to be 0.008 μg/mL and 0.028 μg/mL respectively, which indicate the sensitivity of the method. The use of phosphate buffer and acetonitrile in the ratio of 50:50 v/v resulted in peak with good shape and resolution. The high percentage of recovery indicates that the proposed method is highly accurate. No interfering peaks were found in the chromatogram of the formulation within the run time indicating that excipients used in tablet formulations did not interfere with the estimation of the drug Dronedarone by the proposed HPLC method.  

 

CONCLUSION:

The proposed HPLC method is rapid, sensitive, accurate and precise for the determination of Dronedarone and can be reliably adopted for routine quality control analysis of Dronedarone in its tablet dosage forms.

 

ACKNOWLEDGEMENTS:

The authors are thankful to M/s Sumages Pharma Pvt. Ltd., Bhimavaram, India, for providing a reference sample of Dronedarone.

 

REFERENCES:

1.       G. Manakshe and B. Hygriv Rao. Dronedarone-current status in management of atrial fibrillation. Indian Heart Journal, 2012, 64(2), 181-186.

2.       S. Mohanarangam, B. Satyanarayana, R.E. Chandrashekar, B. Vijayabhaskar and P. Pratap Reddy. A novel and efficient synthesis of Dronedarone hydrochloride, an antiarrhythmic drug substance. Journal of the Chinese Chemical Society, 2011, 58, 841-845.

3.       M.J. Tafreshi and J. Rowles. A review of the investigational antiarrhythmic agent Dronedarone. Journal of Cardiovascular Pharmacology and Therapeutics, 2007, 12, 15-26.

4.       P. Touboul, J. Brugada, A. Capucci, H.J. Crijns, N. Edvardsson and S.H. Hohnloser. Dronedarone for prevention of atrial fibrillation: A dose-ranging study. European Heart Journal, 2003, 24, 1481-1487.

5.       A. Patel, J. Akhtar and C. Sharma. Spectrophotmetric estimation of Dronedarone in pure drug and pharmaceutical formulation. American Journal of Biochemical and Pharmaceutical Research, 2012, 1(2), 266-271. 

6.       A. Patel and J. Akhtar. RP-HPLC method development and validation of Dronedarone HCl in its pure form and tablet dosage form. Journal of Chemical and Pharmaceutical Research, 2012, 4(4), 2173-2179.

7.       T. Naresh, S.S. Shakil, K.V. Surendranath, K. Ravi Kiran and Suresh Kumar. A stability indicating HPLC method for Dronedarone in bulk drugs and pharmaceutical dosage forms. American Journal of Analytical Chemistry, 2012, 3, 544-551.  

8.       R.W. Bolderman, J.J. Hermans and J.G. Maessen. Determination of the class III antiarrhythmic drugs Dronedarone and Amiodarone, and their principal metabolites in plasma and myocardium by high-performance liquid chromatography and UV-detection. Journal of Chromatography B, 2009, 877(18-19), 1727-1731.

9.       C. Xie, S. Yang, D. Zhong, X. Dai and X. Chen. Simultaneous determination of Dronedarone and its active metabolite Debutyldronedarone in human plasma by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study, Journal of Chromatography B, 2011, 879(28), 3071-3075.

10.     ICH Harmonised Tripartite Guideline, Q2 (R1), Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonisation, Geneva. 2005, 1-13.

 

 

Received on 28.10.2012        Modified on 09.11.2012

Accepted on 11.11.2012        © AJRC All right reserved