Determination of flavopiridol (L86 8275; NSC 649890) in human plasma by reversed-phase liquid chromatography with electrochemical detection

Abstract

Purpose: Flavopiridol is a flavone which inhibits several cyclin-dependent kinases, and exhibits potent growth-inhibitory activity against a number of human tumor cell lines both in vitro, and when grown as xenografts in mice. It is currently being evaluated in a phase I clinical trial at the National Cancer Institute. The objective of this project was to develop and validate an analytical method for the assay of flavopiridol in human plasma, with sufficient sensitivity to permit the plasma pharmacokinetics of flavopiridol to be studied during clinical trials. Methods: Flavopiridol was isolated from human plasma samples by extraction with t-butylmethyl ether following alkalinization with borate buffer (pH 8.0). The extract was evaporated, the residue was dissolved in mobile phase, and analyzed by reversed-phase high-pressure liquid chromatography. Chromatography was accomplished with a polymer-based C₁₈ column eluted with a mobile phase consisting of methanol-phosphate buffer, pH 11.0 (53:47 v/v). Electrochemical detection (ECD) was employed. Results: Flavopiridol was recovered from human plasma with an efficiency of 85–87%. Calibration curves were linear over the concentration range 10–500 nM (4.4–219 ng/ml). Plasma standard concentrations were measured with an accuracy and precision ranging from 3.2% to 10%. Regression analysis of flavopiridol concentrations of 15 clinical trial plasma samples ranging in concentration from approximately 50 to 4000 μM quantitated by both ECD and mass spectrometry showed close agreement. The equation of the regression line was y = 1.02x + 8 with a correlation coefficient of 0.969. Continuous infusion of flavopiridol in four patients for 72 h at a rate of 50 mg/m² per day, resulted in mean steady-state plasma concentrations of from 200 to 300 nM. Levels declined in a biexponential manner following termination of the infusion, falling to approximately 10 nM after 48 h. Conclusions: An analytical method for the assay of flavopiridol in human plasma was developed with sensitivity to at least 10 nM. The assay is accurate, precise and specific, and is suitable for determination of plasma flavopiridol concentrations for pharmacokinetic studies during clinical trials.