Analysis of oligonucleotides by hydrophilic interaction liquid chromatography coupled to negative ion electrospray ionization mass spectrometry

• Published in: Journal of Chromatography A Vol. 1218; no. 32; pp. 5480 - 5486
• Main Authors: Gong, Lingzhi, McCullagh, James S.O.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 12.08.2011; Elsevier
• Subjects: Acetonitrile; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, High Pressure Liquid - instrumentation; Chromatography, High Pressure Liquid - methods; Concentration (composition); Eluents; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; HILIC chromatography; Ionization; LC/MS; Liquid chromatography; Mass spectrometry; MS friendly buffer; Nucleic acids; Oligonucleotides; Oligonucleotides - analysis; Separation; Spectrometry, Mass, Electrospray Ionization - methods; Tandem Mass Spectrometry - methods; LC/MS; HILIC chromatography; MS friendly buffer; Oligonucleotides; Chemical analysis; Coupled method; Hydrophilic Interaction Liquid chromatography; HPLC chromatography; Electrospray; Analysis method; Time of flight method; Negative ion; Oligonucleotide; Mass spectrometry
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2011.06.044

Hydrophilic interaction liquid chromatography (HILIC) is here successfully coupled to negative-ion electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) for the analysis of synthetic and chemically modified oligonucleotides. Separation was performed on a 2.1 mm × 100 mm PEEK ZIC ® HILIC column packed with hydrophilic stationary phase with a permanent zwitterionic functional group and a particle size of 3.5 μm with an average pore diameter of 200 Å. A method was developed to separate homogeneous and heterogeneous oligonucleotides as well as methylated oligonucleotides using a quaternary pumping system containing ammonium acetate and water with an acetonitrile gradient. Analyses of oligonucleotides were performed by LC/MS with a detection limit of 2.5 picomole (20 mer) with signal to noise ratio (S/N) of 4.12. The influence of the eluent composition, type of buffer and its concentration, and organic modifier were also evaluated. The HILIC LC/MS method presented in this paper used common, ‘MS friendly’, mobile phases achieving sensitive and selective oligonucleotide analysis.