nsc-614846 and carbovir-triphosphate

Our previous negative ESI-LC-MS/MS method developed for nucleoside reverse transcriptase inhibitor (NRTI) triphosphate (-TP) measurements in human peripheral blood mononuclear cells (PBMC) encountered some specificity problems for several NRTI-TP and simultaneous endogenous nucleotide triphosphates analysis. As LC-MS/MS offers several possibilities to circumvent such problems, we have investigated the contribution of the positive electrospray ionization mode in enhancing the specificity of the intracellular analyses of triphosphate metabolites of lamivudine, abacavir, and tenofovir. For intracellular NRTI-TP analysis, after disruption of PBMCs, concentrated supernatants were directly injected into the LC-MS/MS system, dimethylhexylamine being used as ion-pairing agent to resolve NRTI-TP. MS/MS detection was performed after positive electrospray ionization. Total run time was 12 min instead of 26 min for NRTI-TP analysis. The validation parameters of the method met the international requirements, and endogenous chromatographic interferences were eliminated. The use of positive ESI, offering a better specificity and a slightly better sensitivity than the negative ESI mode for these compounds, resulted in specificity enhancement and more robust assay methods.

Topics: Adenine; Anti-HIV Agents; Cytidine Triphosphate; Deoxyguanine Nucleotides; Dideoxynucleosides; Dideoxynucleotides; HIV Infections; Humans; Lamivudine; Leukocytes, Mononuclear; Organophosphonates; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Tenofovir

Two enantiomers of carbovir, a carbocyclic analog of 2',3'-dideoxyguanosine, were compared with respect to their phosphorylation and the phosphorylation of their nucleotides by mammalian enzymes. 5'-Nucleotidase catalyzed the phosphorylation of (-)-carbovir, which is active against HIV (human immunodeficiency virus), but did not phosphorylate (+)-carbovir. (-)-Carbovir monophosphate was 7,000 times more efficient as a substrate for GMP kinase than was (+)-carbovir monophosphate. Pyruvate kinase, phosphoglycerate kinase, and creatine kinase phosphorylated both enantiomers of carbovir diphosphate at similar rates. Nucleoside-diphosphate kinase preferentially phosphorylated the (-)-enantiomer. Both enantiomers of carbovir triphosphate were substrates and alternative substrate inhibitors of HIV reverse transcriptase. Thus, the contrasting HIV-inhibitory activities of carbovir enantiomers were due to differential phosphorylation by cellular enzymes and not due to enantioselectivity of HIV reverse transcriptase.

Topics: Antiviral Agents; Creatine Kinase; Deoxyguanine Nucleotides; Dideoxynucleosides; Guanylate Kinases; HIV; Models, Molecular; Molecular Conformation; Molecular Structure; Nucleoside-Phosphate Kinase; Phosphoenolpyruvate Carboxykinase (GTP); Phosphoglycerate Kinase; Phosphorylation; Phosphotransferases; Pyruvate Kinase; Reverse Transcriptase Inhibitors; Stereoisomerism; Templates, Genetic