pharmaceutical
Applications
note
RestekCorporation • (800)356-1688 • (814)353-1300 •
#59141
pharmaceutical
Analyze Nucleotides, Nucleosides, Purine, and Pyrimidine Bases
Simultaneously with the Ultra IBD Column
Mixtures of nucleotides, nucleosides, and their respective
purine or pyrimidine bases are difficult to analyze by reversed
phase/high performance liquid chromatography (RP/HPLC).
These compounds cover a wide range of polarities and
functionalities, from the acidic nucleotides to the basic
purines and pyrimidines, making it very difficult to retain and
resolve all of them with conventional alkyl stationary phases.
Traditional HPLC analysis of these compounds often uses a
combination of reversed phase-ion pairing (RP-IP) and/or ion
exchange (IEX) mode. Nucleotides often are analyzed by
anion exchange, while nucleosides sometimes are analyzed
by cation exchange. These methods are not compatible with
all the solutes in the mixtures and they lack ruggedness.
This Applications Note demonstrates that all three classes of
compounds (nucleotides, nucleosides, and bases) can be
analyzed by RP/HPLC using one column and the same,
simple isocratic mobile phase. This provides greater
convenience, reproducibility, and ruggedness in developing
methods for these mixtures. By using a unique, intrinsically
base-deactivated stationary phase (i.e., the Ultra IBD
column), simple RP/HPLC conditions were identified that
can resolve any common purine or pyrimidine base from its
related ribonucleoside and mono-, di-, and triphosphate
nucleotides.
Nucleotides and nucleosides are derived from the nitrog-
enous bases shown in Figure 1. These nitrogenous bases are
either purines (adenine and guanine) or pyrimidines
(cytosine, uracil, and thymine). A nucleoside consists of a
purine or pyrimidine base linked to a five-carbon sugar
(pentose). A nucleotide is composed of a nucleoside plus
one or more phosphate groups. Figure 2 shows the structures
of a nucleoside and three nucleotides derived from adenine.
Figure1
Nitrogenous purine and pyrimidine bases form nucleotides and nucleosides
Figure2
A nucleoside and three nucleotides derived from adenine
adenine
guanine
cytosine
uracil
thymine
adenosine
5'-AMP
5'-ADP
5'-ATP
