Forced Degradation and Stability Indicating Method Development and Validation of Ratinovir by RP-HPLC In Bulk and Pharmaceutical Dosage Form

A stable, simple, accurate, precise, robust and highly selective Reverse Phase High Performance Liquid Chromatographic (RP-HPLC) method was developed and validated using ritonavir. Chromatographic separation was achieved using cyber labs, LC 100 separation module, Agilent C18 column at temperature 30°C. Flow rate selected was 1ml/min. Both drugs are identified with UV detector at 256nm. Mobile phase employed was Methanol: Water (50:50), which resulted best sensitivity. Developed method was validated in terms of linearity, range (25-150μg/ml), precession (correlation coefficient is less than 0.999), robustness, accuracy(recovery was 101.96%) and under stress conditions drug degradation was less than 10%.The validation of proposed stability indicating method was verified by recovery studies and can be applicable in routine pharmaceutical analysis.


INTRODUCTION
Ritonavir is an HIV protease inhibitor that interferes with the reproductive cycle of HIV. Although it was initially developed as an independent antiviral agent, it has been shown to possess advantageous properties in combination regimens with low-dose ritonavir and other protease inhibitors. It is now more commonly used as a booster of other protease inhibitors and is available in both liquid formulation and as capsules.

Figure 1: Structure of Ritonavir
A Very few analytical methods have been reported for the assay of ritonavir individually and various methods have been reported for combination with other drugs in formulations. Literature survey reveals that no analytical method was there for determination of ritonavir individually in dosage forms. So it was felt worthwhile to develop a stable, simple, rapid, accurate, precise and more economical high performance liquid chromatographic method for estimation of ritonavir in bulk and its dosage form. (2)(3)(4)(5)(6)

Instruments
Reference standards of Ritonavir was obtained as gift samples from Aurobindo Pharmaceuticals, Hyderabad. Market formulation of this norvir was procured from the local market. HPLC grade water and methanol were obtained from Merck (India). The LC system cyber labs with UV detector. The output signal was monitored and integrated using LC solutions Software.

Preparation of Mobile Phase:
Mix a mixture of Methanol 500 ml (50%) and 500 ml of Water HPLC (50%) and degas in ultrasonic water bath for 5 minutes.

RESULTS AND DISCUSSION
The purpose of the present study was to develop a stable, rapid and sensitive RP-HPLC method for the estimation of ritonavir in bulk and pharmaceutical dosage form.

Method Optimization:
After conducting several trials one method was optimized. The optimized parameters were given in table no.1 and chromatogram was shown in figure.2  The described method has been validated for the estimation of ritonavir using following parameters as per ICH guidelines. (7) Accuracy Accuracy of the method was demonstrated at three different concentration levels (50-150%) by spiking a known quantity of standard drugs into analyzed sample in triplicate. The percentage recovery was found to be 101.9%. The results of accuracy (

Precision:
For the precision of the method, three replicate were injected into the system on same day and %RSD was calculated, i.e 0.17. Results of precision are given in Table 3, which indicated that the method is precise.

Intermediate Precision:
For the precision of the method, three replicate were injected into the system on two different non consecutive days, in each case %RSD was 0.53. Results of precision are given in Table 4, which indicated that the method is precise.

Linearity:
To establish linearity of the proposed method, five different sets of drug solution was prepared and analyzed. Standard curves were constructed in the concentration range of 25-150μg/ml of ritonavir ( Figure 3). The correlation coefficient was determined as 0.9965 and the results were shown in Table 5.   Experiment was conducted by changing the parameters like flow rate and Temperature. The system suitability parameters were within the limit. Results were shown in table 7.

FORCED DEGRADATION STUDIES:
Samples were subjected to stress conditions like acid, alkaline, oxidative, thermal and photolytic and percentage of drug degradation was calculated. Impurity peaks were observed in stress conditions. Drug degradation was less than 10% in all stress conditions. The results were shown in  ACKNOWLEDGEMENT: We are thankful to Pulla Reddy Institute of Pharmacy and to the Department of Pharmaceutical Analysis Quality Assurance for providing the facilities such as lab, equipment and chemicals to carry out the work. We express our sincere gratitude to Aurobindo Laboratories limited, for supply of Ritonavir drug sample.