Quantitation of zidovudine triphosphate concentrations from human peripheral blood mononuclear cells by anion exchange solid phase extraction and liquid chromatography-tandem mass spectroscopy; an indirect quantitation methodology

Tracy King, Lane Bushman, Peter L. Anderson, Thomas Delahunty, Michelle Ray, Courtney V. Fletcher

Research output: Contribution to journalArticlepeer-review

41 Scopus citations

Abstract

To facilitate the assessment of drug safety and determination of phamacokinetics, an anion exchange isolation of zidovudine triphosphate (ZDV-TP) from human peripheral blood mononuclear cells (hPBMC), coupled with dephosphorylation, desaltation, and detection by liquid chromatography-tandem mass spectroscopy (LC-MS-MS) was validated. hPBMCs were harvested from whole blood, lysed, and a suspension of intracellular ZDV-TP was produced. ZDV-TP was isolated from ZDV, ZDV-monophosphate (ZDV-MP), and ZDV-diphosphate (ZDV-DP), which were all present in the cell lysate, by performing a salt gradient anion exchange SPE. Isolated ZDV-TP was dephosphorylated with acid phosphatase to its parent drug form, ZDV. ZDV was then desalted and concentrated for tandem mass spectral detection. An LC-MS-MS methodology was developed and validated for the determination of molar ZDV directly corresponding to the intra-hPBMC molar ZDV-TP concentration. ZDV-TP concentrations were determined in femtomoles per million hPBMCs (fmol/106 cells). The assay was able to determine ZDV-TP concentrations accurately and precisely within the range of 5-640 fmol/106 cells with 10 million cells per sample analyzed. Inter- and intra-day accuracy and precision data for back calculated standards and quality controls fell within 15% of nominal. The assay correlated well with a previous ELISA method developed and validated in our laboratory, and has been successfully used to quantitate ZDV-TP concentrations in patients being routinely monitored and treated with ZDV.

Original languageEnglish (US)
Pages (from-to)248-257
Number of pages10
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume831
Issue number1-2
DOIs
StatePublished - Feb 2 2006

Keywords

  • LC-MS-MS
  • Zidovudine-triphosphate
  • hPBMC

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology

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