Rapid quantitative determination of L-FMAU-TP from human peripheral-blood mononuclear cells of hepatitis B virus-infected patients treated with L-FMAU by ion-pairing, reverse-phase, liquid chromatography/electrospray tandem mass spectrometry.

The purpose of this study was to develop an analytical method for the determination of 2'-fluoro-5-methyl-beta-l-arabinofuranosyl uracil triphosphate (L-FMAU-TP) in human peripheral blood mononuclear cells (PBMCs), and its application in the determination of cellular levels of L-FMAU-TP in PBMCs isolated from patients treated with 2'-fluoro-5-methyl-beta-l-arabinofuranosyl uracil (L-FMAU). An ion-pairing liquid chromatography (IPC) method, coupled with negative ion electrospray ionization tandem mass spectrometry (ESI-MS/MS), was developed for the accurate and repeatable detection of L-FMAU-TP, with a limit of detection of 1.6 pmol/10 cells. The calibration curve for L-FMAU-TP was linear over the concentration range 1.6 to 80 pmol/10(6) cells. The intra- and inter-day precision was lower than 11.2%, and the accuracy was between 97.1 and 106.9%. When applied to the determination of L-FMAU-TP in PBMCs isolated from HBV-infected patients undergoing L-FMAU treatment, the levels reached a steady state concentration 4 weeks after daily single oral administration of 20 mg L-FMAU, and these levels were maintained for up to 12 weeks, but then decreased 12 weeks after drug cessation. The terminal half-life of L-FMAU-TP in PBMCs after drug cessation was estimated to be 15.6 days.