A method for quantitating the intracellular metabolism of AZT amino acid phosphoramidate pronucleotides by capillary high-performance liquid chromatography-electrospray ionization mass spectrometry.
A methodology has been developed for the analysis of the intracellular metabolism of 3'-azido-3'-deoxythymidine (AZT) amino acid phosphoramidates utilizing reverse-phase high-performance liquid chromatography interfaced with negative ion electrospray ionization mass spectrometry (LC/ESI(-) -MS). The presented work demonstrates the potential of capillary LC/MS and LC/MS/MS to identify and quantitate the cellular uptake and metabolism of nucleoside phosphoramidate. Significant intracellular amounts of D- and L-phenylalanine methyl ester or D- and L-tryptophan methyl ester AZT phosphoramidates were observed for human T-lymphoblastoid leukemia (CEM) cells incubated for 2 and 4 h with the prodrugs. AZT-MP was the primary metabolite observed for human T-lymphoblastoid leukemia (CEM) cells. In this paper, the details of using LC/MS to analyze AZT amino acid phosphoramidates in biological samples are discussed. LC/MS is an efficient method for analyzing multiple samples containing several analytes in a short period of time. The method also provides high selectivity and sensitivity, and requires minimal sample preparation. This approach should be broadly applicable for the analysis of the intracellular metabolism of nucleoside prodrugs and pronucleotides.
Kim
Jisook
J
Department of Medicinal Chemistry, College of Pharmacy, and the Cancer Research Center, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Park
Soobong
S
Tretyakova
Natalia Y
NY
Wagner
Carston R
CR
D000577:Amides / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D019380:Anti-HIV Agents / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D054306:Dideoxynucleotides; D006801:Humans; D015458:Leukemia, T-Cell / Q000378:metabolism*; D010756:Phosphoric Acids / Q000378:metabolism*; D011355:Prodrugs / Q000378:metabolism*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D013942:Thymine Nucleotides / Q000008:administration & dosage / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D015215:Zidovudine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000378:metabolism*
D000577:Amides; D000596:Amino Acids; D019380:Anti-HIV Agents; D054306:Dideoxynucleotides; D010756:Phosphoric Acids; D011355:Prodrugs; D013942:Thymine Nucleotides; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D015215:Zidovudine; C011067:phosphoramidic acid