Liquid chromatography–tandem mass spectrometric determination of tenofovir-diphosphate in human peripheral blood mononuclear cells

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• 作者：Tracy King ; Lane Bushman ; Jennifer Kiser ; Peter L. Anderson ; Michelle Ray ; Thomas Delahunty and Courtney V. Fletcher
• 关键词：Tenofovir-diphosphate ; LC– ; MS– ; MS ; Peripheral blood mononuclear cells
• 刊名：Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences
• 出版年：2006
• 出版时间：7 November 2006
• 年：2006
• 卷：843
• 期：2
• 页码：147-156
• 全文大小：652 K

文摘

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.