Analysis of Oligonucleotides by HPLC-Electrospray Ionization Mass Spectrometry
A Apffel1, J A Chakel, S Fischer
1Biomeasurements Group, Hewlett-Packard Laboratories, Palo Alto, California 94304, and California Analytical Division, Hewlett-Packard Company, Palo Alto, California 94304.
Abstract:
A new interface procedure has been developed that allows, for the first 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, single-base resolution can be obtained with low levels of cation adduct formation 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 the HPLC mobile phase, adjusted to pH 7.0 with triethylamine. This novel additive results in both good HPLC separation and efficient electrospray ionization. The broad potential of this new method is demonstrated for synthetic homopolymers of thymidine (PolyT), fragments based on the pBR322 plasmid sequence, and phosphorothioate ester antisense oligonucleotides. This approach will be of particular utility for the characterization of DNA probes and PCR primers and quality control of antisense compounds such as phosphorothioates and their metabolites, as well as of materials used in clinical trials.
Related Concept Videos
Mass Spectrometry: Complex Analysis
GC–MS is a powerful hyphenated method commonly used in forensics and environmental...
Electrospray Ionization (ESI) Mass Spectrometry
ESI utilizes electrical energy to transfer ions from the liquid phase of the sample into the...
Peptide Identification Using Tandem Mass Spectrometry
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
Ion-Exchange Chromatography
Mass Spectrum: Interpretation
Mass Spectrometry: Overview


