IMMOBILIZED THYMINE CHROMATOGRAPHY MASS-SPECTROMETRY OF OLIGONUCLEOTIDES

New rapid, sensitive and specific methods are needed to determine the purity of synthetic oligonucleotides and their analogs, such as antise nse and antigene oligonucleotide drugs. Synthetic oligonucleotides are typically separated using HPLC with UV absorbance detection at 260 nm . However, positive identification of oligonucleotides requires more s pecific detection, such as that offered by electrospray mass spectrome try. Size-exclusion, ion-exchange and reversed-phase HPLC columns are usually used for the analysis and purification of oligonucleotides. Am ong these, only reversed-phase ion-pair chromatography is suitable for LC-MS of oligonucleotides, but incomplete resolution of oligonucleoti de mixtures sometimes limits this approach. Therefore, a mixed-mode st ationary phase was developed for electrospray LC-MS of oligonucleotide s incorporating reversed-phase, weak anion-exchange and possibly affin ity properties in order to provide different selectivity than reversed -phase alone. The thymine derivative, 3-(1-thymidyl)propanoic acid, wa s synthesized, purified and then immobilized as a possible affinity li gand on an aminopropyl silica HPLC column using 1-(3-dimethylaminoprop yl)-3-ethylcarbodiimide, for activation. Unreacted amino groups on the column were end-capped by reaction with acetic anhydride. Next, mobil e phase conditions were optimized for the separation of oligonucleotid e mixtures up to 18-mers in length. The parameters investigated includ ed gradients of temperature, pH, ionic strength, reversed-phase, norma l-phase and combinations of these parameters for mixed-mode separation s. Optimum column performance was achieved using gradients that utiliz ed the potential affinity properties of the column. Finally, HPLC-elec trospray mass spectrometric analyses of oligonucleotide mixtures were obtained using the immobilized thymine column and compared to reversed -phase LC-MS analyses. (C) 1998 Elsevier Science B.V.