WeO-09
ANALYSIS OF OLIGONUCLEOTIDES BY HPLC, ELECTROSPRAY IONIZATION MASS SPECTROMETRY (ESI-MS), HPLC/ESI-MS, AND MALDI-TOF MS
Alex Apffel, John Chakel and William Hancock
Biomeasurements Group, Hewlett-Packard Laboratories, 3500 Deer Creek Road, Palo Alto, CA 94304, USA
A comparison of analysis of oligonucleotides and small nucleic acids (<500mers), using a number of techniques, including HPLC, ESI-MS, HPLC/ESI-MS and MALDI-TOF has been made to characterize the strengths and weakness of each approach in terms of resolution, sensitivity and speed of analysis. The use of combinations triethylamine, piperidine and imidazole containing solvents for ESI-MS results in strong multiply charged spectra with significant reduction in sodium adducts for oligonuceotides up to 100mers. However, for mixtures of >5-10 components, the multiply charged spectra become difficult to deconvolute. The combination of HPLC and ESI-MS could result in mass specificity with reasonable resolution. Reversed Phase HPLC yields optimal separations with mobile phase consisting of 10-20% acetonitrile in 100mM TEAA buffer, pH 7.0. Unfortunately, optimal HPLC conditions yield poor ESI-MS sensitivity, and vice versa. Compromise solutions can be used with mixed results. Size Exclusion Chromatography (SEC) on TSK PW.sub(xl). can be used with near optimal ESI-MS conditions and acceptable chromatographic resolution. MALDI-TOMS can be used for similar application to yield extremely sensitive (fmol) detection, with fair resolution for oligonucleotides up to 500mers, but usually requires manual desalting of the samples to reduce sodium adducts. These techniques are shown for analysis of synthetic oligonucleotides, anti-sense phosphorothioate oligodeoxynucleotides and small fragments produced by endonuclease digests of pBR322 plasmids.