Validated RP- HPLC Method for the Quantitation of Ritonavir in Bulk and Capsule Dosage forms
C. Jose Gnana Babu*, G. Vijaya Kumar.
Department of Pharmaceutical Analysis, Bharathi College of Pharmacy, Bharathi Nagara, K.M. Doddi, Maddur Taluk, Mandya District, Karnataka, India – 571 422.
*Corresponding Author E-mail: josejino2@yahoo.com, josejino2@gmail.com
ABSTRACT:
A simple, specific, accurate, precise and sensitive reverse phase high performance liquid chromatographic method has been developed for the quantitation of Ritonavir in both pure and capsule dosage forms. An intertsil C- 18, 5 µm column having 250×4.6 mm i.d. in isocratic mode with mobile phase containing acetonitrile: acetate buffer pH 4.0 (70:30). The flow rate was 1.0 ml/min and the effluents were monitored at 248 nm. The retention time was 3.12 min. The linearity was in the range of 10-50 mcg/ml. All the system suitability parameters were found within the range. This method was extensively validated for linearity, precision, specificity, limit of detection, limit of quantitation, accuracy, ruggedness and robustness. The recovery technique was performed to study the accuracy and reproducibility of the proposed method that the recovery of Ritonavir is satisfactory. Thus it can be concluded that the method in the present investigation is simple, sensitive, accurate, rapid and precise. The statistical analysis proves that the method is reproducible and selective. Hence, the above said method can be successfully applied for the routine estimation of Ritonavir in bulk and capsule dosage forms.
KEYWORDS: Ritonavir, RP-HPLC, Validation
Ritonavir is an orally administered antiviral agent1. Ritonavir is a white-to-light-tan powder. It has a bitter metallic taste. It is freely soluble in methanol and ethanol, soluble in isopropanol and practically insoluble in water. Ritonavir is chemically designated as 10-Hydroxy-2-methyl-5-(1-methylethyl)-1-[2-(1-methylethyl)-4thiazolyl]-3,6-dioxo-8,11is(phenylmethyl)-2,4,7,12-tetraazatridecan-13-oic acid, Its molecular formula is C37H48N6O5S2, and the molecular weight is 720.95. It has the melting point of 120-123°C. Extensive literature survey revealed that the few analytical methods have been reported such as spectrophotometric methods2-4 and chromotographic methods5-8. The scope of the work is to develop simple, accurate, precise and economic RP-HPLC method with lesser retention time to estimate the Ritonavir in bulk and capsule dosage forms.
MATERIALS AND METHODS:
A Shimadzu HPLC model containing LC-10 AT pump, variable wavelength programmable UV/VIS detector and Rheodyne injector was employed for the investigation.
All the chemicals used in the investigation were of HPLC grade. The chromatographic analysis was performed on an intertsil C18 column. The mobile phase consisting of acetonitrile and acetate buffer of pH 4.0 in the ratio of 70:30 v/v was selected. The optimized chromatographic conditions are summarized in table 1. The standard solution of Ritonavir was prepared in the concentration of 100 mcg/ml. The ultrasonication were made for the mobile phase and solution and filtered using whatman filter paper No.41. The various dilutions of Ritonavir in the concentration range of 10-50 µg/ml were prepared. The solutions were injected using a 20 µl fixed loop in to the chromatographic system at the flow rate of 1.0 ml/min and the effluents were monitored at 248 nm, chromatograms were recorded.
Table 1: Optimized Chromatographic conditions for the developed method
|
Parameters |
Optimized condition |
|
Column Mobile phase pH Flow rate Injection volume Detection Temperature Retention time Run time |
Intertsil C-18 (5µ) Acetonitrile: Acetate buffer (70:30) 4.0 1.0 ml/min 20 µl 248 nm in uv detector Ambient 3.12 6 min |
The Ritonavir was eluted at 3.12 min as shown in fig.1. The calibration curve was constructed by plotting the average peak area versus concentrations (fig. 2) and regression equation was computed.
Fig. 1: Typical RP-HPLC Chromatogram of Ritonavir by the developed method.
Table 2: System Suitability Test Parameters for the developed method
|
Parameters |
Values |
|
Theoretical plates |
6094 |
|
Asymmetric factor |
1.09 |
|
Tailing factor |
1.11 |
Table 3: Regression analysis of the Calibration curve for the developed method
|
Parameters |
Values |
|
|
Linearity range (µg/ml) Correlation coefficient (r2) Regression equation Slope Intercept |
10-50 0.995 Y= 7491.5 X − 624 7491.5 − 624 |
|
The method was extended for determination of Ritonavir in capsule dosage form. The capsule powder equivalent to 50 mg of Ritonavir was transferred into 50 ml volumetric flask containing 50 ml of mobile phase and flask was kept for ultrasonication for 15 min, then it was diluted up to the mark with mobile phase and the solution was filtered through Whatman filter paper No. 41. The various dilutions were made from the above solution with the mobile phase and analyzed. The amount of Ritonavir present in the sample was computed from the calibration curve.
Table 4: Assay Results of Ritonavir capsules using the developed method
|
Capsules |
Labelled amount (mg) |
bAmount found (mg) |
% Recoveryb |
|
A B |
100 100 |
100.33±0.55 100.45±0.38 |
100.35±0.55 100.39±0.51 |
bMean value ± standard deviation of six determinations
RESULTS AND DISCUSSION:
A suitability test was applied for various parameters. The results obtained were within acceptable limits (Table 2). Thus, the system meets suitable criteria. The calibration curve was obtained for a series of concentration in the range of 10-50 mcg/ml and linearity was found. The data of regression analysis of the calibration curves are shown in Table 3. Selectivity and specificity were studied for the examination of various excipients generally present in the dosage form of Ritonavir. The results indicated that they did not interfere in the assay. The developed method was validated as per the ICH guidelines9 -11. The precision was measured in terms of repeatability, which was determined by sufficient number of aliquots of a homogenous sample. The % RSD was found and lying within the range of ±2. This showed that the precision of the methods are satisfactory. The recovery technique was performed to study the accuracy and reproducibility of the proposed methods. For this, known quantities of the Ritonavir solution were mixed with definite amounts of pre-analyzed formulations and the mixtures were analyzed.
Fig. 2: Calibration curve of Ritonavir by the developed method.
The total amount of Ritonavir was determined by using the proposed method and the amount of added drug was calculated by the difference. The % RSD was less than ± 2.0. This showed that the recovery of Ritonavir by the developed method is satisfactory. Ruggedness and Robustness were determined and the % RSD values were calculated from precision study was less than ± 2.0. Limit of detection (LOD) and Limit of quantitation (LOQ) were determined by the developed method. The results of capsule analysis and recovery studies obtained by the developed method were validated by statistical evaluation (Table 4).
CONCLUSION:
It can be concluded that the method developed in the present investigation are simple, sensitive, accurate, rugged, robust, rapid and precise. Hence, the above said method can be successfully applied for the estimation of Ritonavir in pure and capsule dosage forms.
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Received on 11.06.2012 Modified on 13.07.2012
Accepted on 18.07.2012 © AJRC All right reserved
Asian J. Research Chem. 6(1): January 2013; Page 04-06