ANALYSIS OF OLIGONUCLEOTIDES BY HPLC-ELECTROSPRAY IONIZATION MASS-SPECTROMETRY

A new interface procedure has been developed that allows, for the firs t time, the high-efficiency analysis of synthetic oligonucleotides up to 75 bases by reversed-phase HPLC and on-line electrospray ionization mass spectrometry, For oligonucleotides up to 30 bases in length, sin gle-base resolution can be obtained with low levels of cation adduct f ormation in the negative ion electrospray mass spectra, A key part of the method uses 1,1,1,3,3,3-hexafluoro-2-propanol as an additive to th e HPLC mobile phase, adjusted to pH 7.0 with triethylamine. This novel additive results in both good HPLC separation and efficient electrosp ray ionization, The broad potential of this new method is demonstrated for synthetic homopolymers of thymidine (PolyT), fragments based on t he pBR322 plasmid sequence, and phosphorothioate ester antisense oligo nucleotides. This approach will be of particular utility for the chara cterization of DNA probes and PCR primers and quality control of antis ense compounds such as phosphorothioates and their metabolites, as wel l as of materials used in clinical trials.