Quantitative determination of short single-stranded oligonucleotides from blood plasma using capillary...

The quantitative determination of short (< 20 bases) single-standed (ss-) oligonucleotides (oligos) from blood plasma using capillary gel electrophoresis with laser-induced fluorescence is reported. Oligos were derivatized on column after equilibration of the column with a 1:150 dilution of OliGreen dye. The resulting fluorescent complex was detected and measured with an argon ion laser detector using excitation/emission wavelengths of 488/520 nm, respectively. The method involves precipitation of plasma proteins with phenol-chloroform followed by dilution and drop analysis in nanopure water for 30 min on a 0.025 microns cellulose acetate membrane. This treatment lowers the ionic strength of the plasma sample resulting in a significant improvement of the electrokinetic loading (5 kV, 10 s) of the analyte. Optimal electrophoretic separation was achieved at 13 kV using 4 M urea in a 10% polyacrylamide gel filled capillary, 100 mM Tris borate as the running buffer, and a temperature of 30 degrees C. Oligos were determined in the presence of p(dT)20/40 as internal standard. The observed migration times were 6.35 and 6.60 min for the oligo and internal standard, respectively. The migration times and fluorescent yield of the complex were temperature dependent. Increasing the separation temperature (20 to 60 degrees C) resulted in a decrease in the migration time and fluorescent yield of the oligonucleotide-dye complex. A linear response over a broad concentration range (0.02-1.5 micrograms/mL, R2 = 0.997) was obtained. The limit of quantitation was set at 20 ng/mL (CV% = 11.3%). The intraday variability was 9.44, 5.28, and 9.2% for 190, 760, and 1520 ng/mL plasma samples, respectively. Data are presented to illustrate the practicality of the method for the pharmacokinetic evaluation of GS522 and potential metabolites in plasma after intravenous administration to rats.