The combination of dynamic pH junction, sweeping
(using borate complexation), and large volume sample
stacking (LVSS) is investigated as three consecutive steps
for on-line focusing in the sensitive quantitation of urinary
nucleosides by CE-UVD. A low conductivity aqueous sample
matrix free from borate and a high conductivity BGE (containing
borate, pH 9.25) are needed to fulfill the required
conditions for dynamic pH junction, LVSS, and sweeping.
Parameters affecting the separation and the enrichment efficiency
are studied such as buffer concentration, separation
voltage, capillary temperature, sample composition, and sample
injection volume. Prerequisite for the developed strategy is
the extraction of the nucleosides from urine using a
phenylboronate affinity gel, which is described to be a unique
means for the selective enrichment of cis-diol metabolites
under alkaline conditions. The impact of ionic constituents
remaining in the eluate after extraction on focusing efficiency
and resolution is investigated. The developed method is applied
to the analysis of blank and spiked urine samples.
Fundamental aspects underlying the proposed enrichment
procedure are discussed. A detection limit as low as
10 ng mL−1 is achieved. To the best of our knowledge, this
LOD represents the lowest LOD reported so far for the analysis
of nucleosides using CE with UV detection and provides
a comparable sensitivity to CE/MS. Because of the high
sensitivity, the proposed method shows a great potential for the analysis of nucleosides in human urine and other types of
biological fluids.