INTRODUCTION:

Darunavir (Prezista®300mg) is a drug used to treat HIV infection. It is in the protease inhibitor class. Prezista® is an OARAC recommended treatment option for treatment-naïve and treatment-experienced adults and adolescents^[^1]. Chemically it is [(1R,5S,6R)-2,8-dioxabicyclo[3.3.0]oct-6-yl] N-[(2S,3R)-4-[(4-aminophenyl)sulfonyl-(2-methylpropyl)amino]-3-hydroxy-1-phenyl- butan-2-yl] carbamate. Its molecular weight is 547.6 and molecular formula is C₂₇H₃₇N₃O₇S. Literature survey reveals no chromatographic methods for the estimation of Darunavir from pharmaceutical dosage forms. The availability of an HPLC^[^2-3] method with high sensitivity and selectivity will be very useful for the determination of Darunavir in pharmaceutical formulations. The aim of the study was to develop a simple, precise and accurate reversed-phase HPLC method for the estimation of Darunavir in bulk drug samples and in pharmaceutical dosage form.

Structure of Darunavir

EXPERIMENTAL:

Materials and Methods:

Darunavir was obtained as a gift sample from Hetero Drugs Ltd, Hyderabad. Acetonitrile and water used were of HPLC grade (Qualigens). Commercially available Darunavir tablets (Prezista 300 mg, Tibotec Company)) 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 5 μm) was used. The HPLC system was equipped with Empower Software.

HPLC Conditions:

The contents of the mobile phase were 0.02M Dipotassium hydrogen orthophosphate +0.02M Potassium Dihydrogen orthophosphatein in water and Acetonitrile in the ratio of 40:60 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 1.0 ml/min. The run time was set at 15.0 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 265 nm.

Preparation of Standard Stock solution: A standard stock solution of the drug was prepared by dissolving 200 mg of Darunavir 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 (Prezista 300 mg, Tibotec Company)) 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 0.45μm 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 Darunavir 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 Darunavir 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 265 nm and a Calibration graph was obtained by plotting peak area versus concentration of Darunavir (Fig 2).

The plot of peak area of each sample against respective concentration of Darunavir was found to be linear in the range of 80–240µg/ml with correlation coefficient of 0.998.Linear regression least square fit data obtained from the measurements are given in table I. The respective linear regression equation being Y= 44076.08X + 726603. 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

44076.08

726603

0.998

0.3

189092

Assay: 20µl of sample solution was injected into the injector of liquid chromatograph. The retention time was found to be 5.859 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*

200

98.75

98.50

98.37

109.47

109.65

109.22

*Average of three different concentration levels.

Recovery Studies:

Accuracy was determined by recovery studies of Darunavir, 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 Darunavir. 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

7280.76

1.20

5.859

2.28

99.96

LOD (µg/ml)

LOQ (µg/ml)

0.1

0.3

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

The limit of detection (LOD) and limit of quantification (LOQ) for Darunavir were found to be 0.1µg/ml and 0.3µg/ml respectively. The signal to noise ratio is 3 for LOD and 10 for LOQ.

From the typical chromatogram of Darunavir as shown in fig 1, it was found that the retention time was 5.859 min. A mixture of 0.02M Dipotassium hydrogen orthophosphate +0.02M Potassium Dihydrogen orthophosphatein in water and Acetonitrile in the ratio of 40:60 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=0.998) 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 Darunavir tablets was found to be 98.58%. From the recovery studies it was found that about 109.49% of Darunavir 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 1: Typical Chromatogram of Darunavir by HPLC

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

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

ACKNOWLEDGEMENTS:

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

REFERENCES:

1. Ghosh AK, Dawson ZL, Mitsuya H. "Darunavir, a conceptually new HIV-1 protease inhibitor for the treatment of drug-resistant HIV". Bioorg. Med. Chem.15 (24): 7576–80, (2007).

2. B, Ramprasad A.Lanka, Srinivasu Pamidi, Jayachandra R. Peddareddigari, JVLNS Rao New RP-HPLC Method for the Determination of Darunavir in Tablet Dosage Form; Asian J. Pharm. Res, Vol 1, Issue 1, 10-14, (2011)

3. Clotet, Nicholas Bellos, Jean-Michel Molina, David Cooper, Jean-Christophe Goffard, Adriano Lazzarin, Andrej Wöhrmann, Christine Katlama, Timothy Wilkin, Richard Haubrich, Efficacy and safety of darunavir-ritonavir at week 48 in treatment-experienced patients with HIV-1 infection in POWER 1 and 2: a pooled subgroup analysis of data from two randomised trials, The Lancet, Volume 369, Issue 9568, 1169-1178 (2007).

Received on 19.08.2011 Accepted on 22.08.2011

Asian J. Res. Pharm. Sci. 1(3): July-Sept. 2011; Page 74-76