REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ESTIMATION OF ATAZANAVIR AND RITONAVIR IN PHARMACEUTICAL DOSAGE FORM

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ABOUT AUTHORS:
Venkatesh. J , M. Singaiah Chowdary*, Haritha. D, Anuroop , Dr. V.V.L.N Prasad, Anjani Prasad Reddy. V.
Department of Pharmaceutical Analysis and Quality assurance,
School of pharmacy, Anurag Group of Institutions, Venkatapur (V).
Ghatkesar (M), Rangareddy (D)
*chowdary.mannam1@gmail.com

ABSTRACT:
A new simple, rapid, specific, accurate, precise and novel reverse phase High Performance Liquid Chromatography (RP-HPLC) method has been developed for the simultaneous estimation of atazanavir and ritonavir in the combined pharmaceutical dosage form. The chromatographic separation for Atazanavir and Ritonavir were achieved with mobile phase containing mixed phosphate buffer (pH 4.0) and acetonitrile (45:55 % v/v), Symmetry C18 (4.6 x 100mm, 3.5mm, Make: ACE)at 5
°C and UV detection at 237 nm. The compounds were eluted in the isocratic mode at a flow rate of 0.9 ml min-1. The retention times of atazavir 4.29 ± 0.09 min and ritonavir at 5018 ± 0.09 min. The above method was validated in terms of linearity, accuracy, precision, LOD, LOQ etc. in accordance with ICH guidelines.

Reference ID: PHARMATUTOR-ART-1957

INTRODUCTION
Atazanavir, chemically methyl N-[(1S)-1-{N'-[(2S,3S)-2-hydroxy-3-[(2S)-2-[(methoxycarbonyl)amino]-3,3-dimethylbutanamido]-4-phenylbutyl]-N'-{[4-(pyridin-2-yl)phenyl]methyl}hydrazinecarbonyl}-2,2-dimethylpropyl]carbamate [1] (Fig.1), Atazanavir selectively inhibits the virus-specific processing of viral Gag and Gag-Pol polyproteins in HIV-1 infected cells by binding to the active site of HIV-1 protease, thus preventing the formation of mature virions. Atazanavir is not active against HIV-2. Ritonavir, chemically, (1,3-thiazol-5-ylmethyl N-[(2S,3S,5S)-3-hydroxy-5-[(2S)-3-methyl-2-{[methyl({[2-(propan-2-yl)-1,3-thiazol-4-yl]methyl})carbamoyl]amino}butanamido]-1,6-diphenylhexan-2-yl]carbamate  [2] (Fig.2). Ritonavir inhibits the HIV viral proteinase enzyme which prevents cleavage of the gag-pol polyprotein, resulting in noninfectious, immature viral particles.

Literature survey reveals that there are several analytical methods for the estimation of atazanavir and ritonavir individually or in combination with other drugs [3-8]. Although the combination use of atazanavir and ritonavir is continuously increasing, there is no simple and economical RP-HPLC method for the determination of these drugs in the combined pharmaceutical dosage form. The purpose of present study is to investigate a simple, precise, accurate and economic RP-HPLC method in simultaneous determination of atazanavir and ritonavir in the combined pharmaceutical dosage form.

MATERIAL AND METHODS
Atazavir and ritonavir gift samples were obtained from Sura pharma labs. Acetonitrile (HPLC grade) and dipotassium hydrogen phosphate (AR grade) were purchased from Merck Ltd, India. Water for HPLC was obtained from Qualigen fine chemicals, Mumbai, India. Analytical reagent potassium dihydrogen orthophosphate (AR grade) was obtained from Rankem Pvt. Ltd, Mumbai.Chromatographic separation was performed on Waters® HPLC system equipped with  Waters 2489 UV/Visible detector and Empower software. Symmetry C18 (4.6 x 100mm, 3.5mm, Make: ACE) and constant flow pump and Auto injector with 20 μL loop were used. The composition of the mobile phase was in the ratio of mixed phosphate buffer (pH 4.0) and acetonitrile (45:55 % v/v) and was delivered at a flow rate of 0.9 ml min-1. The mobile phase was filtered through a 0.45 μ membrane filter and sonicated for 15 min. Analysis was performed at 5
°Ctemperature.

Preparation of standard solution
Accurately weigh and transfer 10 mg of Atazanavir and 10mg of Ritonavir working standard into a 10ml clean dry volumetric flask add about 7ml of Diluent and sonic ate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution).

Further pipette 0.6ml of Atazanavir & Ritonavir the above stock solution into a 10ml volumetric flask and dilute up to the mark with diluent. A typical chromatogram obtained from the analysis of drugs using the developed method was shown in Fig.3.


Fig.3. A typical chromatogram of standard atazanavir and ritonavir measured at 237 nm.

Preparation of sample solution:
Accurately weigh and transfer 568.2 mg of Atazanavir and Ritonavir Tablet powder  into a 100ml clean dry volumetric flask add about 70ml of Diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution).

Further pipette 0.1ml of atazanavir and ritonavirof the above stock solution into a 10ml volumetric flask and dilute up to the mark with diluent.

RESULTS AND DISCUSSION
Symmetry C18 (44.6 x 100mm, 3.5mm, Make: ACE), column maintained at ambient temperature 5
°C was used for the separation and the method was validated for the estimation of atazanavir and ritonavir in tablets. The composition, pH and the flow rate of the mobile phase were optimized. A mobile phase consisting of phosphate buffer (pH 4.0): acetonitrile (45:55 % v/v) set at a flow rate of 0.9 ml min-1 was selected for use of further studies after several preliminary investigatory chromatographic runs. Under the described experimental conditions, all peaks were well defined and free from tailing. The effects of small deliberate changes in the mobile phase composition, and flow rate were evaluated as a part of testing for method robustness.

Method validation:
The proposed method was validated as per International Conference on Harmonization (ICH) guidelines.

Linearity and Range
Linearity was established by least squares linear regression analysis of the calibration curve. The calibration curves were linear over the concentration range of 40-80 μg mL-1   foratazanavir,  40-80 μg mL-1 for ritonavir. Peak areas were plotted versus respective concentrations and linear regression analysis was performed on the resultant curves. Correlation coefficient values were found to be 0.999 and 0.999 for ramipril and Amlodipine besylate respectively, (Figs. 4, 5). The results are given in Table 1.

Fig.4. Calibration curve of Atazanavir


Fig.5. Calibration curve of Ritonavir

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