Separation of near-infrared fluorescent conjugates of dATP and related compounds by capillary electrophoresis

Capillary electrophoresis was examined as a means for the separation and quantitation of deoxyadenosine triphosphate (dATP) and other nucleotides that were labeled with the near-infrared fluorescent dye IRD700 or related tags. Under the final optimized conditions the labeled dATP was separated from several possible impurities, including the unconjugated forms of IRD700 and dATP, as well as dADP, dAMP and their corresponding IRD700 conjugates. The assay was performed under two sets of conditions. First, the sample was injected onto a 50 cm×75 μm I.D. fused-silica capillary at 25 kV in the presence of a pH 9.5, 140 mM borate running buffer. The resulting peaks were monitored at both 254 and 680 nm, where the latter wavelength was used to identify any species that contained the IRD700 label. A second injection was then performed under the same conditions but with a fixed concentration of dTTP now being added to the running buffer; this resulted in the formation of a complex between the dTTP and any dATP, dADP or dAMP-containing components, which changed their rates of migration and allowed them to be differentiated from unconjugated IRD700 or dye contaminants. Only 6 nl of a 1:10 diluted sample were required per analysis. The limit of detection at this injection volume was approximately 1.0 μM (or 6·10−15 mol for a 6-nl injection) for each monitored component. The linear range extended up to at least 80 μM. The analysis time was 20 min per injection and the day-to-day precision was ±2–3%. The same method was also found to be useful in examining related conjugates, such as those based on the dye IRD40.