The Estimation of Maraviroc in Tablet dosage form by RP-HPLC.

 

L. Satyanarayana1*, Prof. S.V. Naidu2, Prof. M. Narasimha Rao2, Prof. C. Ayyanna2 and Alok Kumar1.

1Department of Pharmaceutical Chemistry, Omega College of Pharmacy, Edulabad, Ghatkesar, Ranga Reddy Dist., Hyderabad-500 034.

2Centre for Biotechnology, Department of Chemical Engineering, College of Engineering (A), Andhra University, Vishakapatnam - 530003

 

ABSTRACT:

A simple, precise, rapid and accurate reverse phase HPLC method was developed for the estimation of Maraviroc in tablet dosage form. A RP Inertsil ODS-3V C-18, 250x4.6 mm, 5μm partical size, with mobile phase consisting of 0.02M Dipotasium hydrogen orthophosphate in water pH 2.5 with orthophosphoric acid and Acetonitrile in the ratio of 60:40 v/v was used. The flow rate was 0.8 ml/min and the effluents were monitored at 210 nm. The retention time was 4.330 min. The detector response was linear in the concentration of 80-240µg/ml. The respective linear regression equation being Y= 31018.059X + 44222.4. The limit of detection and limit of quantification was 0.2µg and 0.6µg/ml respectively. The percentage assay of Maraviroc was 99.42%. The method was validated by determining its accuracy, precision and system suitability.

The results of the study showed that the proposed RP-HPLC method is simple, rapid, precise and accurate, which is useful for the routine determination of Maraviroc in bulk drug and in its pharmaceutical dosage form.

 

KEYWORDS: Maraviroc, RP-HPLC, Estimation, and Tablets.

 

 

INTRODUCTION:

Maraviroc (Selzentry®-300mg) is an antiretroviral drug in the CCR5 receptor antagonist class used in the treatment of HIV infection1,2. Chemically it is 4,4-difluoro-N-[(1S)-3-[(3-exo)-3-[3-methyl-5-(1-methylethyl)-4H-1,2,4-triazol-4-yl]-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl]-cyclohexanecarboxamide. Its molecular weight is 513.66 and molecular formula is C29H41F2N5O. Literature survey reveals no chromatographic methods for the estimation of Maraviroc from pharmaceutical dosage forms. The availability of an HPLC method with high sensitivity and selectivity will be very useful for the determination of Maraviroc in pharmaceutical formulations. The aim of the study was to develop a simple, precise and accurate reversed-phase HPLC method for the estimation of Maraviroc in bulk drug samples and in pharmaceutical dosage form.

 

Structure of Maraviroc

EXPERIMENTAL:

Materials and Methods:

Maraviroc was obtained as a gift sample from Hetero Drugs Ltd, Hyderabad. Acetonitrile and water used were of HPLC grade (Qualigens). Commercially available Maraviroc tablets (Selzentry® 300mg, Pfizer) were procured from local market.

 

Instrument:

Quantitative HPLC was performed on liquid Chromatograph, Waters separation 2996, PDA detector module equipped with automatic injector with injection volume 20µl, and 2693 pump. A RP Inertsil ODS-3V C-18 column (250x4.6 mm i.d; particle size 5μm) was used. The HPLC system was equipped with Empower Software.

 

HPLC Conditions:

The contents of the mobile phase were 0.02M Dipotassium hydrogen orthophosphate in water pH-2.5 with orthophosphoric acid and Acetonitrile in the ratio of 60:40 v/v. They were filtered before use through a 0.45μm membrane filter, and pumped from the respective solvent reservoirs to the column at a flow rate of 0.8 ml/min. The run time was set at 10.0 min and the column temperature was ambient. Prior to the injection of the drug solution, the column was equilibrated for at least 30 min with the mobile phase flowing through the system. The eluents were monitored at 210 nm.

 

Preparation of Standard Stock solution: A standard stock solution of the drug was prepared by dissolving 200 mg of Maraviroc in 100 ml volumetric flask containing 30 ml of water, sonicated for about 15 min and then made up to 100 ml with water to get 2000µg/ml standard stock solution.

 

Working Standard solution: 5ml of the above stock solution was taken in 50 ml volumetric flask and thereafter made up to 50 ml with mobile phase to get a concentration of 200µg/ml.

 

Preparation of Sample solution: Twenty tablets (Selzentry® 300mg, Pfizer) were weighed, and then powdered. A sample of the powdered tablets, equivalent to 200mg of the active ingredient, was mixed with 30 ml of water in 100 ml volumetric flask. The mixture was allowed to stand for 1 hr with intermittent sonication to ensure complete solubility of the drug, and then filtered through a 0.45 μm membrane filter, followed by adding water up 100 ml to obtain a stock solution of 2000µg/ml. Transfer for 5ml of this   solution to a 50 ml of volumetric flask and made upto sufficient volume with mobile phase to give an concentration of 200µg/ml.

 

Linearity: Aliquots of standard Maraviroc stock solution were taken in different 10 ml volumetric flasks and diluted up to the mark with the mobile phase such that the final concentrations of Maraviroc are in the range of 80-240μg/ml. Each of these drug solutions (20µl) was injected three times into the column, and the peak areas and retention times were recorded. Evaluation was performed with PDA detector at 210 nm and a Calibration graph was obtained by plotting peak area versus concentration of Maraviroc (Fig 2).

 

The plot of peak area of each sample against respective concentration of Maraviroc was found to be linear in the range of 80–240µg/ml with correlation coefficient of 0.999. Linear regression least square fit data obtained from the measurements are given in table I.  The respective linear regression equation being Y= 31018.059X + 44222.4 The regression characteristics, such as slope, intercept, and %RSD were calculated for this method and given in Table I.

 

Table I: Linear Regression Data for Calibration curves.

Parameters

Results of proposed HPLC Method

Concentration range (µg/ml)

Slope (m)

Intercept (c)

Correlation coefficient

% RSD

Standard error of estimate

80-240

31018.059

44222.4

0.999

0.8

96595.78

 

Assay: 20µl of sample solution was injected into the injector of liquid chromatograph. The retention time was found to be 4.330 minutes. The amount of drug present per tablet was calculated by comparing the peak area of the sample solution with that of the standard solution. The data are presented in Table II.

 

Table II: Results of HPLC Assay and Recovery studies

Sample

Amount claim

(mg/tablet)

%  found by the proposed method

% Recovery*

1.

2.

3.

200

200

200

99.66

99.24

99.38

109.97

109.65

109.822

*Average of three different concentration levels.

 

Recovery Studies:

Accuracy was determined by recovery studies of Maraviroc, known amount of standard was added to the preanalysed sample and subjected to the proposed HPLC analysis. Results of recovery study are shown in Table II. The study was done at three different concentration levels.

 

RESULTS AND DISCUSSION:

The system suitability tests were carried out on freshly prepared standard stock solution of Maraviroc. Parameters that were studied to evaluate the suitability of the system are given in Table III.

 

Table III   Validation Summary

Validation Parameter

Results

System Suitability

Theoretical Plates (N)

Tailing factor

Retention time in minutes

Resolution

% Area

 

6034.07

1.35

4.330

3.50

99.61

LOD (µg/ml)

LOQ (µg/ml)

0.2

0.6

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The limit of detection (LOD) and limit of quantification (LOQ) for Maraviroc were found to be 0.2µg/ml and 0.6µg/ml respectively. The signal to noise ratio is 3 for LOD and 10 for LOQ.

 

Fig 1:  Typical Chromatogram of Maraviroc by HPLC

 

From the typical chromatogram of Maraviroc as shown in fig 1, it was found that the retention time was 4.330 min. A mixture of 0.02M Dipotassium hydrogen orthophosphate in water pH-2.5 with orthophosphoric acid and Acetonitrile in the ratio of 60:40 v/v was found to be most suitable to obtain a peak well defined and free from tailing. In the present developed HPLC method, the standard and sample preparation required less time and no tedious extraction were involved. A good linear relationship (r=0.999) was observed between the concentration range of 80-240µg/ml. Low values of standard deviation are indicative of the high precision of the method. The assay of Maraviroc tablets was found to be 99.42%. From the recovery studies it was found that about 109.81% of Maraviroc was recovered which indicates high accuracy of the method. The absence of additional peaks in the chromatogram indicates non-interference of the common excipients used in the tablets. This demonstrates that the developed HPLC method is simple, linear, accurate, sensitive and reproducible.

 

Fig-2: Calibration curve of the Maraviroc by RP-HPLC.

 

Thus, the developed method can be easily used for the routine quality control of bulk and tablet dosage forms of Maraviroc within a short analysis time.

ACKNOWLEDGEMENTS:

The authors are grateful to M/s Hetero Drugs, Hyderabad for the supply of as a gift sample Maraviroc and to the Management, Omega College of Pharmacy, Hyderabad, for providing the necessary facilities to carry out the research work.

 

REFERENCES:

1.        Biswas P, Tambussi G, Lazzarin A ;  The status of co-receptor inhibition to counter HIV entry". Expert Opinion on Pharmacotherapy, 8 (7): 923–33, (2007).

2.        Stefania Notari, Chiara Tommasi, Emanuele Nicastri, Rita Bellagamba, Massimo Tempestilli, Leopoldo Paolo Pucillo, Pasquale Narciso and Paolo Ascenzi; Simultaneous Determination of Maraviroc and Raltegravir in Human Plasma by HPLC-UV; IUBMB Life, 61(4): 470–475,(2009).

 

 

Received on 19.08.2011

Accepted on 11.09.2011        

© A&V Publication all right reserved

Research Journal of Pharmaceutical Dosage Forms and Technology. 3(5): Sept.-Oct. 2011, 230-232