INTRODUCTION:

Atazanavir and Ritonavir are protease inhibitors and both are widely used in combination as Antiviral agents. Ritonavir inhibits the CYP3-A mediated metabolism of Atazanavir, thereby increases plasma level of Atazanavir.¹ Various methods have been reported for analysis of RTV and ATV in pharmaceutical formulations and in human plasma individually or in combination with other antiviral drugs using liquid chromatography (LC) ^2-7, LC/MS ^8-9 and UV spectroscopy.¹¹ Aim of the present study to develop and validate accurate method and determine both drugs concurrently by simple, rapid, and selective HPLC method that could be used for quality control and routine analysis. The proposed method was validated in accordance with International Conference on Harmonization (ICH) guideline.¹²

MATERIAL AND METHODS:

Material:

Atazanavir and Ritonavir were procured from Cipla Ltd., Mumbai, India. Acetonitrile (S. D. fine chemicals Ltd., Mumbai, India) and phosphoric acid (RFCL Ltd., India) were of HPLC grade. Triple distilled water was used. Potassium dihydrogen phosphate was of analytical grade (S. D. fine chemicals Ltd., Mumbai, India). VIRATAZ-R Tablets was manufactured by Hetero Drugs Ltd. Each tablets claimed to contain 100 mg RTV and 300 mg ATV.

Instruments:

Agilent 1100 series HPLC system (Agilent, USA) comprising QUAT Agilent solvent delivery module, Agilent ALS auto injector with 20 µL loop, degasser and VWD UV detector were used for analysis. A reversed-phase Thermo Hypersil C18 (250 × 4.6 mm, 5 µ) column (Phenomenex, Germany) was used for separation. The chromatographic data were processed using Chem Station software (Agilent).

Chromatographic Conditions:

The optimized mobile phase was consisted of acetonitrile and 0.05M KH₂PO₄ buffer (pH 3 ± 0.5) in the ratio of 55:45 (v/v). The mobile phase was degassed by ultrasonication for 20 min. The mobile phase was pumped from the solvent reservoir to the column at a flow rate of 1.0 ml/min. The run time was set at 15 min and the column temperature was maintained at 25 ± 2° C. The volume of injection loop was 20 µl. Mobile phase was used as diluents throughout experiment. The column was equilibrated for at least 30 min with the mobile phase flowing through the system, prior to injection of the solutions. Eluent was monitored at 245 nm and data were acquired, stored and analyzed using Chem Station software

Figure 1 Chromatogram of Atazanavir and Ritonavir

Preparation of Standard Stock Solution:

Standard stock solution of the drug was prepared by dissolving atazanavir (300 mg) and ritonavir (100 mg) in a 100 ml volumetric flask containing 75 ml of the diluents. It was sonicated for about 20 min and then made up to volume with the diluent.

Preparation of test solution:

Twenty tablets were weighed to obtain the average tablet weight, which were then powdered. Sample of the powdered tablets, claimed to contain 300 mg of Atazanavir and 100 mg of Ritonavir, were mixed with 75 ml diluent in a 100 ml volumetric flask. This mixture was sonicated for 20 minute to ensure complete solubility of the drug. The volume was made up to mark with the diluent. This mixture was shaken well and then filtered. The filtrate (5.0 ml) was transferred to a volumetric flask (50 ml) and made up to sufficient volume with the diluent.

Method Validation:

The develop method was validated as per ICH guideline for linearity, range, accuracy, precision, specificity, limit of detection, limit of quantification and robustness parameter.

Specificity:

To perform the specificity of proposed method, blank sample was injected in chromatograph and chromatogram was recorded.

Linearity:

The linearity of the method was determined at the five concentration levels ranging from 150-360 μg/ml for Atazanavir and 50-120 μg/ml for Ritonavir. Working standard solutions of Atazanavir and Ritonavir were prepared by suitable dilution of the standard stock solution with the diluent to obtain concentration of Atazanavir in the range of 150-360 µg/ml and Ritonavir 50-120 µg/ml. Each of these drug solutions (20 µl) were injected into the injector and the peak areas were recorded. The calibration graph was constructed by plotting amount injected (µg/ml) versus mean peak area (m AU).

Limit of detection (LOD) and Limit of Quantification (LOQ):

The LOD and LOQ values were determined by measuring signal to noise (S/N) ratio. As per ICH guideline, generally concentration of substance that will give signal-to-noise ratio 2:1 or 3:1 is considered as LOD, while signal-to-noise ratio 10:1 is considered as LOQ. To demonstrate LOD and LOQ level experimentally, the LOD and LOQ solutions of Atazanavir and Ritonavir were prepared and injected into the liquid chromatograph to find out S/N ratio.

Accuracy:

To examine the accuracy of the developed method, recovery studies were carried out by standard addition method at three different concentration levels in triplicate by spiking standard Atazanavir and Ritonavir solution in previously analyzed tablet solution.

Precision:

Intra-day and inter-day precision were determined by analyzing samples of atazanavir and Ritonavir at three different concentration level in triplicate within range. Intra-day and inter-day precision were performed by injecting solutions containing Atazanavir (150 µg/ml, 300 µg/ml and 360 µg/ml) and Ritonavir (50 µg/ml, 100 µg/ml and 120 µg/ml) in triplicate and calculate percentage relative standard deviation (% RSD). Repeatability was performed by injecting solution containing Atazanavir (300 µg/ml) and Ritonavir (100 µg/ml) six times in chromatograph and calculate % RSD.

Robustness:

Robustness of the method was studied by using mobile phase (acetonitrile- 0.05 M KH₂PO₄ buffer solution (pH 3)) in the proportion of 57:43 and 53:47 (v/v) and the flow rate (0.9 ml/min and 1.1 ml/min).

RESULTS AND DISCUSSION:

The aim of the present work was to develop a rapid, precise, accurate and cost effective HPLC method for simultaneous estimation of atazanavir and ritonavir in its pharmaceutical tablet formulation, using the reverse phase (RP) C₁₈ column with UV detection and validate develop method as per US FDA guideline and ICH guideline. The validation parameters were summarized in Table 1. As per US FDA reviewer guidance document for validation of chromatographic methods; chromatographic parameters- capacity factor >2, tailing factor <2, theoretical plate number >2000 and resolution >2 are required. To achieve these chromatographic parameters in the proposed LC method, the mixture of acetonitrile and buffer solution in different proportion at various flow rates were tried. The mobile phase consisted of acetonitrile- KH₂PO₄ buffer (pH 3) in proportion of 55:45 (v/v) at 1.0 ml/min flow rate, proved to be better than other proportion used. Atazanavir and Ritonavir showed equal absorbance at 245 nm in mobile phase, hence 245 nm was selected as the wavelength of detection. In the proposed method; capacity factor, tailing factor and theoretical plate number obtained were 3.79, 1.20, and >10373 for Atazanavir and 4.62, 1.17, and >10200 for Ritonavir respectively. The retention time of Atazanavir and Ritonavir was found to be 6.8 ± 0.5 and 8.5 ± 0.5 min respectively with resolution 6.02. A typical chromatogram obtained by the proposed HPLC method is shown in Fig. 2.

The method was found to be specific. There was no peak found in blank sample chromatogram at Atazanavir and Ritonavir peak retention time.

Atazanavir and Ritonavir were found to be linear over the concentration range from 150-360 µg/ml and 50-120 µg/ml, respectively. The regression equations for the linearity curve of Atazanavir was obtained y=20.62x-99.121 with the correlation coefficient(r) 0.9999, while y=12.842x-9.303 with the correlation coefficient (r) 0.9998 for Ritonavir, where y=mean peak area and x=concentration (µg/ml). The value of correlation coefficient >0.99 indicate excellent linearity of the developed HPLC method. The LOD and LOQ values were found to be 0.9 µg/ml and 3 µg/ml for Atazanavir and 3µg/ml and 10µg/ml for Ritonavir, respectively.

Table 1: Summary of validation parameter:

Parameters	Atazanavir	Ritonavir
Linearity range	150-360 µg/ml	50-120 µg/ml
Correlation Co-efficient	0.9999	0.9998
Precision (% RSD) Intra dayInter day	0.22-0.32% 0.53-0.63%	0.51-0.57% 0.69-1.10%
% Mean Recovery	99.5-101.4%	99.7-101.6%
Limit of detection	0.9 µg/ml	3 µg/ml
Limit of quantification	3 µg/ml	10 µg/ml

The mean absolute recovery determined by spiking known amounts of Atazanavir (150, 300, 360 µg/ml) and Ritonavir (50, 100 120 µg/ml) solutions to the previously analyzed tablet solution in triplicate. The results were shown in Table 2. The average percent recoveries obtained as 99.5-101.4 % for Atazanavir and 99.7-101.6% for Ritonavir indicates good accuracy of the method.

Table 2: Results of recovery study:

Amount of Atazanavir spiked (mg/ml)	Amount of Atazanavir found (mg/ml) Mean ± S.D. (n =3)	% Recovery Mean ± S.D. (n =3)
150	149.25±1.33	99.5±0.89
300	304.08±2.30	101.4±0.77
360	361.59±3.01	100.4±0.84

Amount of Ritonavir spiked (mg/ml)	Amount of Ritonavir found (mg/ml) Mean ± S.D. (n =3)	% Recovery Mean ± S.D. (n =3)
50	49.86±0.44	99.7±0.88
100	101.56±0.56	101.6±0.56
120	121.40±0.84	101.2±0.70

This method was validated for intra-day and inter-day precision. The RSD values for intra-day and inter-day precision obtained were 0.22-0.32% and 0.53-0.63 % for atazanavir and 0.51-0.57% and 0.69-1.10% for ritonavir respectively, indicating good precision. Repeatability was found to be 0.47% for atazanavir and 0.51% for ritonavir. The results of precision were shown in Table 3.

The proposed method was found to be robust because change in flow rate and mobile phase composition did not change system suitability criteria such as symmetry of peak and number of theoretical plate. In order to demonstrate the stability of both standard and test solutions during analysis, both the solutions were analyzed over a period of 12 hr at room temperature. The results showed that for the solutions, retention time and peak area of atazanavir and ritonavir almost unchanged and no significant degradation was observed within the indicated period.

The proposed method was successfully applied to the analysis of one marketed tablet formulation containing atazanavir (300 mg/tablet) and ritonavir (100 mg/tablet). The results obtained are given in Table 4. The average drug content was found to be 101.3 % and 101.6 % respectively.

Table 3: Results of intra-day and inter-day precision study:

Concentration of Atazanavir (µg/ml)	Concentration of Atazanavir (µg/ml)	Atazanavir % RSD		Ritonavir % RSD
Concentration of Atazanavir (µg/ml)	Concentration of Atazanavir (µg/ml)	Intra day	Inter day	Intra day	Inter day
150	50	0.32	0.63	0.57	1.10
300	100	0.22	0.53	0.51	0.69
360	120	0.30	0.56	0.54	0.83

Table 4: Estimation of atazanavir and ritonavir in combined tablet dosage form by the proposed HPLC method:

Drug	Labeled claim Mg/tablet	Amount found Mg/tablet Mean ± S.D. (n = 3)	% Assay Mean ± S.D. ( n= 3)
Atazanavir	300	303.92±0.28	101.3±0.1
Ritonavir	100	101.57±0.36	101.6±0.4

CONCLUSION:

The results of the study showed that the proposed HPLC method is simple, rapid, precise, accurate, specific and sensitive for determination of atazanavir and ritonavir, without any interference from the excipients. Hence, this method is suitable for routine analysis of atazanavir and ritonavir in their combined tablet dosage form.

ACKNOWLEDGEMENT:

The authors are thankful to M/s Cipla Ltd., Mumbai, India for providing gift samples of Atazanavir and Ritonavir.

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Received on 22.12.2012 Modified on 30.12.2012

Research J. Pharm. and Tech. 6(2): Feb. 2013; Page 200-203