Separation of synthetic cycloalkylated bases, nucleosides and nucleotides by reversed-phase high-performance liquid chromatography.
Reversed-phase high-performance liquid chromatography was used to determine the elution profiles of a series of synthetic cycloalkylated bases, nucleosides, and their corresponding 5'-monophosphates. A 70% aqueous methanol solution proved to be the most efficient solvent system for the separation of a mixture of the bases, all of which were eluted in times ranging from 3.3 to 4.8 min at a flow-rate of 0.8 ml/min. Subsequently, the same percentage of methanol solvent, at 0.8 ml/min, eluted the nucleoside mixture as well, with retention times ranging from 3.3 to 5.0 min. Optimum separation and resolution were achieved with 70% methanol at a flow-rate of 0.6 ml/min for a mixture of the base and nucleoside series. A phosphate buffer, containing acetonitrile-tetrabutylammonium ion, was used to analyze the 5'-monophosphate derivatives. Elution times ranged from 2.6 to 6.1 min at a flow-rate of 1.0 ml/min. Three variables were considered in order to determine optimum conditions for separation and resolution: (a) the percentage of methanol in the solvent; (b) flow-rate of solvent; and (c) the size of the cycloalkylated group of each synthetic analogue. The procedures and conditions described herein have potential use as a monitoring system to detect modified nucleic acid derivative which are prevalent in the body fluids of patients with certain metabolic disorders.
Alick
B
B
Department of Chemistry, Clark College, Atlanta, GA 30314.
Bridges
C
C
Cox
T
T
Earl
V
V
Thedford
R
R
D000478:Alkylation; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D051379:Mice; D009705:Nucleosides / Q000032:analysis*; D009711:Nucleotides / Q000032:analysis*; D012313:RNA / Q000032:analysis; D013056:Spectrophotometry, Ultraviolet; D014407:Tumor Cells, Cultured
D009705:Nucleosides; D009711:Nucleotides; D012313:RNA; D004247:DNA