Analysis of antisense oligonucleotides by on-capillary isotachophoresis and capillary polymer sieving electrophoresis.
An attempt was made to evaluate the stability of an antisense oligonucleotide against nucleases present in HBL 100ras cells. To detect nanomolar concentrations of the oligonucleotide, a sensitive detection system was required. A combination of capillary electrophoresis/laser-induced fluorescence (CE-LIF) with fluorescence derivatization did not improve the sensitivity significantly and also resulted in loss of separation of the derivatized sample. On-column isotachophoresis for the preconcentration of oligonucleotide samples in DB-17 coated capillaries filled with hydroxyethyl cellulose solution could be an alternative. The isotachophoresis (ITP) step allows injection of up to 40% of the capillary volume without loss in peak resolution and peak efficiency. Using 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-mer oligonucleotide. Using these conditions, the gain in sensitivity was 125.
Laboratorium voor Farmaceutische Chemie en Analyse van Geneesmiddelen, Faculteit Farmaceutische Wetenschappen, K.U. Leuven, Belgium. kamran=khan%fca%far@cc3.kuleuven.ac.be
K
K
Khan
A
A
Van Schepdael
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T
Saison-Behmoaras
A
A
Van Aerschot
J
J
Hoogmartens
D001940:Breast; D002461:Cell Line, Transformed; D002482:Cellulose / Q000031:analogs & derivatives; D004355:Drug Stability; D004586:Electrophoresis; D019075:Electrophoresis, Capillary / Q000379:methods*; D004720:Endonucleases / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism; D005453:Fluorescence; D011905:Genes, ras; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007834:Lasers; D016376:Oligonucleotides, Antisense / Q000032:analysis* / Q000378:metabolism; D011108:Polymers; D012680:Sensitivity and Specificity; D012996:Solutions; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms
D016376:Oligonucleotides, Antisense; D011108:Polymers; D012996:Solutions; D002482:Cellulose; C002283:hydroxyethylcellulose; D004720:Endonucleases; D005092:Exonucleases