Analysis Of Oligonucleotides By Ion-pair Hydrophilic Interaction Liquid Chromatography/electrospray Ionization Mass Spectrometry (IP-HILIC/ESIMS) | 89408
Journal of Chromatography & Separation Techniques
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Synthetic oligonucleotides are widely used in the polymerase chain reaction (PCR) as DNA primers or in molecular biology as probes to screen for diseases, viral infections, and to identify genes. Sensitive and selective methods have always been demanded for the characterization of oligonucleotides, especially, when the oligonucleotides are applied as therapeutics. Ionpair reversed-phase (IP-RP) liquid chromatography has been commonly used for the analysis of oligonucleotides, but ion suppression is a major problem when coupling with electrospray mass spectrometry (ESI-MS). Although the introduction of hexafluoroisopropanol (HFIP) in the mobile phase has improved MS detection sensitivity of oligonucleotides, this mobile phase system results in a severe problem with adducts formation particularly if large oligonucleotides are analysed. An alternative chromatographic approach, hydrophilic interaction liquid chromatography (HILIC), was recently employed for the analysis of oligonucleotides. It provided enhanced MS sensitivity with fewer adducts but lacked chromatographic resolution for some oligonucleotides. Here we improve chromatographic resolution whilst maintaining MS sensitivity by adding an ion-pairing reagent, triethylammonium acetate (TEAA), into the HILIC mobile phase. The IP-HILIC approach produces lower retention capacity and has the added benefit of providing simpler MS spectra, with fewer charge states, when comparing with HILIC.
Lingzhi Gong is research interests include mass spectrometry based analysis of small molecules and biomolecular molecules (nucleic acids, peptides, proteins) through hyphenated to a chromatographic method (mainly liquid chromatography), and research into chromatographic retention mechanisms. His current work focuses on characterizing single- and double-stranded DNA/RNA, and protein/peptide - DNA crosslinking complex using hyphenated liquid chromatography and electrospray mass spectrometry (LC-ESIMS). Lingzhi currently runs Core Mass Spectrometry Research Facility at Queen Mary University of London.