Typical Standard Chromatogram Of Ritonavir And Lopinavir?

Figure 3.1: Typical standard chromatogram of Ritonavir and Lopinavir Figure 3.2: Typical sample chromatogram of Ritonavir and Lopinavir
3.3.2 Method validation The optimized RP-HPLC method for simultaneous assay of Ritonavir and Lopinavir was validated according to ICH guidelines with respect to system suitability, linearity, sensitivity, accuracy, precision and robustness.
3.3.3 System suitability Before sample analysis, the chromatographic parameters used in this analysis must conform the system suitability parameters within the limits. The retention time (Rt), tailing factor (T) and theoretical plate number (N) for the principal peak and its degradation product were evaluated for Ritonavir and Lopinavir. The tailing factors were 0.979
In between the ranges given above, five concentrations were taken and triplicate injections of each concentration were performed. Calibration curves were plotted between analyte concentrations versus that analyte area. Linearity regression analysis of the data gave correlation coefficient value, slope and intercept. For concentrations between 100µg/mL and 1200 µg/mL, the calibration curves were linear. By the values of the correlation coefficients (r2), the linearity of the calibration curves was validated. The correlation coefficients were 0.999 for Ritonavir and 0.999 for Lopinavir. The results of the linearity experiment were listed in Table 3.3 and Table 3.4. Linearity graphs were shown in Figure 3.3 and Figure
The results were summarized in Table 3.5. The sufficient sensitivity of the method was demonstrated by the low values of the limit of detection (LOD) and limit of quantification (LOQ). The chromatograms of Ritonavir and Lopinavir at LOD and LOQ levels were presented in Figures 3.5 and 3.6.
Table 3.5: Sensitivity of the HPLC method for Ritonavir and Lopinavir
Parameters Results Ritonavir Lopinavir
LOD 0.766 2.698
LOQ 2.552 8.996

Figure 3.5: Chromatogram of Ritonavir and Lopinavir at LOD level Figure 3.6: Chromatogram of Ritonavir and Lopinavir at LOQ level
3.3.7 Precision The precision was calculated by injecting six standard solutions of Ritonavir (200 μg/mL) and Lopinavir (800 μg/mL). The peak areas were determined. Relative standard deviation (RSD) of the peak areas of Ritonavir and Lopinavir was then calculated to represent precision. The results were summarized in Table 3.6. The low % RSD values indicated that the method was