Development and validation of a cost-effective and sensitive bioanalytical HPLC-UV method for determination of lopinavir in rat and human plasma.

Qin, Chaolong; Feng, Wanshan; Chu, YenJu; Lee, Jong Bong; Berton, Mattia; Bettonte, Sara; Teo, Yeong Yeu; Stocks, Michael J; Fischer, Peter M; Gershkovich, Pavel

A simple, sensitive and cost-effective HPLC-UV bioanalytical method for determination of lopinavir (LPV) in rat and human plasma was developed and validated. The plasma sample preparation procedure includes a combination of protein precipitation using cold acetonitrile and liquid-liquid extraction with n-hexane-ethyl acetate (7:3, v/v). A good chromatographic separation was achieved with a Phenomenex Gemini (C18, 150 mm × 2.0 mm, 5 μm) column at 40°C with gradient elution, at 211 nm. Calibration curves were linear in the range 10-10,000 ng/mL, with a lower limit of quantification (LLOQ) of 10 ng/mL using 100 μL of plasma. The accuracy and precision in all validation experiments were within the criteria range set by the guidelines of the Food and Drug Administration (FDA). This method was successfully applied to a preliminary pharmacokinetic study in rats following intravenous bolus administration of LPV. Moreover, the method was subsequently fully validated for human plasma allowing its use in therapeutic drug monitoring. In conclusion, this novel, simple and cost-efficient bioanalytical method for determination of LPV is useful for pharmacokinetic and drug delivery studies in rats, as well as therapeutic drug monitoring in human patients.