Analysis of intracellular didanosine triphosphate at sub-ppb level using LC-MS/MS.
An analytical procedure has been developed for the analysis of intracellular didanosine triphosphate (ddATP). An electrospray ionization tandem mass spectrometer (ESI-MS) was interfaced to liquid chromatography (LC) using a mobile phase CH3OH/H2O (25/75) containing 1% formic acid for the analysis of the 5'-triphosphate metabolite of the antiviral didanosine. In this procedure, ddATP was extracted from CEM-T4 cells, isolated using an exchange anion solid phase extraction procedure, enzymatically dephosphorylated and then analyzed by LC-MS/MS within a 1 min run time. The influence of several parameters (electrospray ionization interface, acidic modifiers of the mobile phase) has been studied. A calibration curve was generated and the linear regression analysis yielded a regression coefficient (r(2)) greater than 0.999. Using LC-MS/MS detection in single reaction monitoring mode (SRM), the limit of quantitation of ddA in CEM-T4 cells was 0.02 ng ml(-1). Furthermore, this procedure could be used to perform simultaneous detection of five nucleoside reverse transcriptase inhibitors, such as AZT, 3TC, ddA, ddC and d4T and make LC-MS/MS a method of choice for Therapeutic Drug Monitoring (TDM) in a clinical environment.
Cahours
X
X
Institut de Chimie Organique et Analytique, Associé au CNRS, Université d'Orléans, BP 6759 45067, Orleans, France.
Tran
T T
TT
Mesplet
N
N
Kieda
C
C
Morin
P
P
Agrofoglio
L A
LA
D002478:Cells, Cultured; D002853:Chromatography, Liquid; D003838:Deoxyadenine Nucleotides / Q000032:analysis*; D016049:Didanosine / Q000032:analysis; D054306:Dideoxynucleotides; D006801:Humans; D011863:Radioimmunoassay; D015203:Reproducibility of Results; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization
D003838:Deoxyadenine Nucleotides; D054306:Dideoxynucleotides; D012996:Solutions; C037773:2',3'-dideoxyadenosine triphosphate; D016049:Didanosine