Simultaneous quantification of emtricitabine and tenofovir nucleotides in peripheral blood mononuclear cells using weak anion-exchange liquid chromatography coupled with tandem mass spectrometry.
Emtricitabine (FTC) and tenofovir (TFV) are widely used antiviral agents that require intracellular phosphorylation to become active. This article describes the development and validation of an assay for the simultaneous quantification of FTC mono-, di- and triphosphate (FTC-MP, -DP and -TP), TFV and TFV mono- and diphosphate (TFV-MP and -DP) in peripheral blood mononuclear cells. Reference compounds and internal standards were obtained by thermal degradation of FTC-TP, TFV-DP, stable isotope-labeled TFV-DP and stable isotope-labeled cytosine triphosphate. Cells were lysed in methanol:water (70:30, v/v) and the extracted nucleotides were analyzed using weak anion-exchange chromatography coupled with tandem mass spectrometry. Calibration ranges in PBMC lysate from 0.727 to 36.4, 1.33 to 66.4 and 1.29 to 64.6 nM for FTC-MP, FTC-DP and FTC-TP and from 1.51 to 75.6, 1.54 to 77.2 and 2.54 to 127 nM for TFV, TFV-MP and TFV-DP, respectively, were validated. Accuracies were within -10.3 and 16.7% deviation at the lower limit of quantification at which the coefficients of variation were less than 18.2%. At the other tested levels accuracies were within -14.3 and 9.81% deviation and the coefficients of variation lower than 14.7%. The stability of the compounds was assessed under various analytically relevant conditions. The method was successfully applied to clinical samples.
Jansen
Robert S
RS
Department of Pharmacy & Pharmacology, Slotervaart Hospital/The Netherlands Cancer Institute, Louwesweg 6, 1066 EC Amsterdam, The Netherlands. Robert.jansen@slz.nl
Rosing
Hilde
H
Kromdijk
Wiete
W
ter Heine
Rob
R
Schellens
Jan Hm
JH
Beijnen
Jos H
JH
D000225:Adenine / Q000031:analogs & derivatives* / Q000097:blood; D000227:Adenine Nucleotides / Q000097:blood; D000838:Anions / Q000737:chemistry; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000097:blood; D004355:Drug Stability; D000068679:Emtricitabine; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D016014:Linear Models; D009711:Nucleotides / Q000097:blood*; D063065:Organophosphonates / Q000097:blood*; D010766:Phosphorylation; D011742:Pyrimidine Nucleotides / Q000097:blood; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000068698:Tenofovir
D000227:Adenine Nucleotides; D000838:Anions; D009711:Nucleotides; D063065:Organophosphonates; D011742:Pyrimidine Nucleotides; D003841:Deoxycytidine; D000068698:Tenofovir; D000068679:Emtricitabine; D000225:Adenine