ANALYSIS OF ANTISENSE OLIGONUCLEOTIDES BY ON-CAPILLARY ISOTACHOPHORESIS AND CAPILLARY POLYMER SIEVING ELECTROPHORESIS

An attempt was made to evaluate the stability of an antisense oligonuc leotide against nucleases present in HBL 100ras cells. To detect nanom olar concentrations of the oligonucleotide, a sensitive detection syst em was required. A combination of capillary electrophoresis/laser-indu ced fluorescence (CE-LIF) with fluorescence derivatization did not imp rove the sensitivity significantly and also resulted in loss of separa tion of the derivatized sample. On-column isotachophoresis for the pre concentration of oligonucleotide samples in DB-17 coated capillaries f illed with hydroxyethyl cellulose solution could be an alternative. Th e isotachophoresis (ITP) step allows injection of up to 40% of the cap illary volume without loss in peak resolution and peak efficiency. Usi ng ITP-capillary polymer sieving electrophoresis (CPSE), the limit of quantitation at a signal-to-noise ratio of 10 was 73 ng/mL for a 12-me r oligonucleotide. Using these conditions, the gain in sensitivity was 125.