Multiresponse Optimization of HPLC Method: Simultaneous Estimation of Protease Inhibitors and NNRTI in Human Plasma

Multiresponse optimization approach to develop a simple isocratic, highly sensitive and accurate HPLC method for the simultaneous determination of Efavirenz, Atazanavir, Lopinavir and Ritonavir in human blood plasma along with carvedilol as an internal standard. Optimized the factors (ACN, buffer concentration and flow rate) effecting and interacting with the responses (k1, Rs2,1, Rs3,2 and tR5) applying Central Composite Design a chemometric tool. All the mathematical models as well as response surfaces were defined and derived for the separation using this strategy. Chromatography was performed on Thermo Hypersil C18 column using mobile phase comprising of ACN: 10 mM KH2PO4 (51.2:48.8) with 1 mL min-1 flow rate and detection wavelength was fixed at 210 nm. The analysis time was within 9 min. The method developed was validated by following "Bioanalytical method validation" [USFDA-CDER, 2001]. The developed method can be applied for bioavailability and pharmacokinetic studies.