title: Liquid chromatography-tandem mass spectrometric determination of tenofovir-diphosphate in human peripheral blood mononuclear cells.
abstract: To facilitate the evaluation of drug safety, virologic activity, and pharmacokinetics, an anion exchange isolation of tenofovir-diphosphate (TFV-DP) from human peripheral blood mononuclear cells (hPBMCs), coupled with dephosphorylation, desaltation, and detection by LC-MS-MS was validated. hPBMCs were harvested from whole blood, lysed, and a suspension of intracellular tenofovir moieties was produced. TFV-DP was isolated from TFV-monophosphate (TFV-MP) and tenofovir (TFV), dephosphorylated with acid phosphatase to form TFV and then desalted and concentrated, making it possible for tandem mass spectral detection. An LC-MS-MS methodology was developed and validated for the determination of TFV concentrations, which directly correspond with the intra-hPBMC TFV-DP concentration. The assay was linear in the range of 50-10,000 fmol per sample. The lower limit of quantitation (LLOQ) of the method is 10 fmol per million cells with 5 million hPBMCs used. This paper outlines the development and validation of the determination of TFV-DP concentrations in femtomoles per million hPBMCs.
authors.affiliation: Antiviral Pharmacology Laboratory, Department of Clinical Pharmacy, School of Pharmacy, University of Colorado Health Sciences Center, Box C238, 4200 East 9th Avenue, Denver, CO 80262, USA.
authors.forename: Tracy
authors.initials: T
authors.lastname: King
authors.forename: Lane
authors.initials: L
authors.lastname: Bushman
authors.forename: Jennifer
authors.initials: J
authors.lastname: Kiser
authors.forename: Peter L
authors.initials: PL
authors.lastname: Anderson
authors.forename: Michelle
authors.initials: M
authors.lastname: Ray
authors.forename: Thomas
authors.initials: T
authors.lastname: Delahunty
authors.forename: Courtney V
authors.initials: CV
authors.lastname: Fletcher
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000097:blood; D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D063065:Organophosphonates / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000068698:Tenofovir
chemical_list: D063065:Organophosphonates; D000068698:Tenofovir; D000225:Adenine