Bioanalytical method development of nevirapine, fosamprenavir calcium and its metabolite amprenavir by RP-HPLC in rat plasma.

The current analytical work focuses on the development of a bioanalytical method for anti-HIV drugs, nevirapine, fosamprenavir calcium, and its metabolite amprenavir. Fosamprenavir is a prodrug of amprenavir, digested by cellular phosphatases in vivo. Rat plasma was isolated from the collected rat blood by centrifugation and used for preparing stock solutions. A reverse-phase column (ODS C-18) was used for the development of the method and validation. The bioanalytical method was developed using the isocratic mode. The solvent system used is Methanol: Water: ACN (800 mL: 200 mL: 50 mL). The method was run at a flow rate of 0.5 mL/min, and 258 nm was the detection wavelength. The developed method showed retention at 8.705 min for nevirapine, 11.923 min for fosamprenavir calcium, and 14.391 min for amprenavir. The calibration curve was linear with a correlation coefficient (r) of 0.9916 for nevirapine, 0.9909 for fosamprenavir calcium, and 0.9879 min for amprenavir. The RSD of the accuracy, precision, and stability study of the method was found to be less than 2 % and was found to be acceptable. The method is reliable and does not show any kind of interference due to the plasma sample. Thus, the results support that a reliable, reproducible, and efficient method was developed, and validation was carried out for the estimation of the drug nevirapine, fosamprenavir, and its metabolite amprenavir in rat plasma samples.