On-column electroextraction and separation of antisense oligonucleotides in human plasma by capillary gel...

A novel approach is presented for the direct injection, and subsequent separation, of antisense phosphorothioate oligonucleotides in human plasma by capillary gel electrophoresis. The plasma, spiked with the antisense, was simply diluted 1:1 with acidified water and inserted into the sample holder in the capillary electrophoresis instrument. The separation capillary, filled with a dextran solution (replaceable polymer) and a short zone of acidified water at the injection side, was dipped into the plasma sample vial and voltage applied for simultaneous electrokinetic extraction and injection of antisense. The sample vial was then exchanged for the buffer vial, separation voltage applied, and size-sieving separation achieved. Separation time is less than 9 min and total time per analysis cycle 20 min, including rinsing of the capillary, filling with polymer, electroextraction/injection, and separation. This automated method can handle small sample volumes (4 microl) and has a detection limit of 0.5 microgml(-1) for a 16-mer phosphorothioate employing UV-detection. The capillary is stable for about 50 analyses.