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.
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/10(6)cells). The assay was able to determine ZDV-TP concentrations accurately and precisely within the range of 5-640 fmol/10(6)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.
King
Tracy
T
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.
Bushman
Lane
L
Anderson
Peter L
PL
Delahunty
Thomas
T
Ray
Michelle
M
Fletcher
Courtney V
CV
D005591:Chemical Fractionation / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D054306:Dideoxynucleotides; D004355:Drug Stability; D004797:Enzyme-Linked Immunosorbent Assay; D015658:HIV Infections / Q000097:blood / Q000188:drug therapy; D006801:Humans; D007474:Ion Exchange; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D013942:Thymine Nucleotides / Q000097:blood*; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000097:blood / Q000627:therapeutic use
D054306:Dideoxynucleotides; D013942:Thymine Nucleotides; D015215:Zidovudine; C052873:zidovudine triphosphate