For the precision study of the present method, 6.0 µg/mL of Tenofovir AF, 480.0 µg/mL of Darunavir, 120.0 µg/mL of Emtricitabine, and 90.0 µg/mL of Cobicistat spiked solution was prepared from the primary standard stock solution. The prepared solution was injected for six times and measured the area for all six injections using the HPLC. The %RSD for the area of six replicate injections was found to be within the specified limits. The % RSD for the area of six standard injections results should not be more than 2%.
For this study the spiked solution of all four APIs was prepared like precision study and was injected on the other day for six times and measured the area for all six injections in HPLC. The % RSD for the area of six standard injections results should not be more than 2%.
Accuracy of the present developed method was studied by recovery study. The fixed dose combination tablet powder was kept constant and standard solutions of Tenofovir AF, Darunavir, Emtricitabine and Cobicistat were spiked at the levels of 50%, 100% and 150%. The total amount obtain was calculated for each concentration level. Percentage recovery was calculated for each level and overall recovery were calculated for the all studied drugs. The % Recovery for each level should be between 98.0 and 102.0%.
It was performed to validate whether an analytical system working properly. It was evaluated by injecting the working standard drugs of Tenofovir AF (6.0 µg/mL), Darunavir (480.0 µg/mL) and Emtricitabine (120.0 µg/mL) and cobicistat (90.0 µg/mL) six times. Several parameters like theoretical plate, retention time, asymmetric factor has been considered for the calculation of percentage relative standard.
For the study of linearity of the present developed method, different levels of the Tenofovir AF, Darunavir, Emtricitabine and Cobicistat has been prepared from the standard solution with suitable dilution. For Tenofovir AF 2.0–10.0 µg/mL, for Darunavir 160.0–180.0 µg/mL, for Emtricitabine 40.0–200.0 µg/mL, and 30.0–150.0 µg/mL solutions were prepared at different levels and injected for the linearity study. Calibration curve was plotted for all the drugs at different levels. Concentration and peak area were considered to construct the linearity graph and obtained data was subjected to regression analysis.
Limit of detection
For the study of detection limit of the individual component of the spiked solution, the specified sample has been prepared from the standard stock solution which contains Tenofovir AF (6 µg/mL), Darunavir (480 µg/mL) and Emtricitabine (120 µg/mL) and cobicistat (90 µg/mL). For the Tenofovir AF 2 mL was pipetted in to the 10 mL flask, filled with diluent and further 1.19 mL was transferred to 10 mL volumetric flask and volume was filled with the diluent to achieve 0.14 µg/mL of tenofovir. Similarly, for darunavir 2 mL of standard solution was diluted to 10 mL, and further 0.45 mL was diluted to 10 mL with diluent to obtained 2.14 µg/mL. For Emtricitabine, 1 mL of the standard solution was diluted to 10 mL and further 0.5 mL was diluted to 10 mL to achieve 0.6 µg/mL. Cobicistat LOD solution was prepared by diluting 6 mL of the standard solution to 10 mL, then 1.36 mL from the prepared solution was transferred and diluted to 10 mL with the diluent to obtain 7.32 µg/mL of Cobicistat. Signal to noise ratio was calculated from the baseline and signal noise and should be 3.
Limit of quantitation
For the study of quantitation limit, sample solutions were prepared from the working standard.
Solution. 0.47 µg/mL of Tenofovir AF, 7.12 µg/mL of Darunavir, 2.10 µg/mL of Emtricitabine, 24.42 µg/mL of Cobicistat diluted solutions were prepared from the standard solution by diluting suitable volumes with the diluent in 10 mL of volumetric flask. The prepared solutions were injected into the chromatographic system for the analysis. Signal to noise ratio was calculated from the baseline noise and signal and quantitation limit ratio should be 10.
In the robustness study of the present developed method, several optimised parameters like, flow rate of the mobile phase, Organic composition of the mobile phase, detection wavelength of the optimised conditions were deliberately changes in minor level. The flow rate was changes at the level of ± 0.1, The organic composition was changes to ± 10%, and the detection wavelength was changes to ± 2 nm. The peak areas in all such conditions for all the studied APIs were considered to observed the robustness. The percentage relative standard deviation was calculated for the parameters and should not be more than 2.
Force degradation study
Force degradation study of the present sample solution  was conducted using ICH prescribed stress condition such as acidic, alkaline, oxidative, thermal and photolytic stress conditions. All the types of degradation studies have been performed in triplicate and mean peak area has been considered for the calculation.
Degradation under acidic condition
Pipette 2 mL of above solution into a 10 mL volumetric flask and 3 mL of 0.1N HCl was added. Then, the volumetric flask was kept at 60ºC for 6 h and then neutralized with 0.1N NaOH and make up to 10 mL with diluent. Filter the solution with 0.22 microns syringe filters and place in vials.
Degradation under alkaline condition
Pipette 2 mL of above solution into a 10 mL volumetric flask and 3 mL of 0.1N NaOH was added in 10 mL of volumetric flask. Then, the volumetric flask was kept at 60ºC for 6 h and then neutralized with 0.1N HCl and make up to 10 mL with diluent. Filter the solution with 0.22 microns syringe filters and place in vials.
Thermal induced degradation
Tenofovir, Darunavir, Emtricitabine and Cobicistat sample was taken in petridish and kept in Hot air oven at 1100 C for 24 h. Then the sample was taken and diluted with diluents and injected into HPLC and analyzed.
Pipette 2 mL of above stock solution into a 10 mL volumetric flask and 1 mL of 3% w/v of hydrogen peroxide added in 10 mL of volumetric flask and the volume was made up to the mark with diluent. The volumetric flask was then kept at room temperature for 15 min. Filter the solution with 0.45 microns syringe filters and place in vials.
The photolytic degradation was done by exposing of drug content under the UV light for 15 min to 7 days. The drug degradation observed in the above specific photolytic degradation condition.
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