On-line enhancement technique for the analysis of nucleotides using capillary zone electrophoresis/mass spectrometry.

A new on-line capillary zone electrophoresis/mass spectrometry (CZE/MS), constant pressure-assisted electrokinetic injection (PAEKI), for the analysis of negatively charged nucleotides is reported. PAEKI uses an applied pressure to counterbalance the reverse electroosmotic flow in the capillary column during sample injection, while taking advantage of the field amplification in the sample medium. At balance, the running buffer in the column is stationary, permitting potentially unlimited injection time, and hence unlimited sample enrichment power. The ability of PAEKI to maintain a narrow sample zone over a long injection time seems to be a result of the formation of a high ion concentration band at the boundary of the two media due to rapid deceleration of the migrating ions at the boundary. The injected amount of analytes proved to be linearly proportional to both the field amplification factor, which is expressed as the ratio of resistivities of sample medium to running buffer, and the injection time, which extended up to 1200 s in CZE/MS and 3600 s in CZE/UV. For a 300-s on-line PAEKI injection in CZE/MS, 3 orders of magnitude sample enhancement (5000-fold enrichment) could be observed for the four single nucleotides without compromising separation efficiency and peak shape, and an achievement of detection limits between 0.04 and 0.07 ng/mL. With appropriate sample cleanup, PAEKI can be used in the analysis of single nucleotides in enzyme-digested DNA.