Multiple heart-cutting mixed-mode chromatography-reversed-phase 2D-liquid chromatography method for separation and mass spectrometric characterization of synthetic oligonucleotides

• Published in: Journal of Chromatography A Vol. 1625; p. 461338
• Main Authors: Li, Feiyang, Su, Xiaoli, Bäurer, Stefanie, Lämmerhofer, Michael
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 16.08.2020
• Subjects: Mass spectrometry; Mixed-mode chromatography; Multidimensional liquid chromatography; Oligonucleotide; Reversed-phase/weak anion-exchange; Oligonucleotide; Reversed-phase/weak anion-exchange; Mixed-mode chromatography; Mass spectrometry; Multidimensional liquid chromatography
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2020.461338

■Multiple heart-cutting 2D-liquid chromatography method for oligonucleotide analysis.■A mixed-mode RP/WAX column provided high selectivity between similar oligos in 1D.■The 2D with RPLC using a C18 column was used for desalting.■The MS-compatible eluent in 2D devoid of ion-pair allowed sensitive MS detection.■Structurally closely related synthetic oligonucleotides were characterized by TOF-MS. Until today, ion-pair reversed-phase chromatography is still the dominating method for analytical characterization of synthetic oligonucleotides. Its hyphenation with mass spectrometry, however, has some drawbacks such as ion-suppression in electrospray ionization. To overcome this problem, we present in this work a multiple heart-cutting (MHC) two-dimensional liquid chromatography (2D-LC) method with ultra-violet (UV) and electrospray ionization (ESI) mass spectrometry (MS) detection. A reversed-phase/weak anion-exchange (RP/WAX) stationary phase in the first dimension (1D) provides the selectivity for separation of structurally closely related oligonucleotide sequences and deletions (shortmers), respectively, using a mixed pH/triethylammonium phosphate buffer gradient at constant organic modifier content. Heart cuts of the oligonucleotide peaks are transferred to the second dimension (2D) via a multiple heart-cutting valve which is equipped with two loop decks. The 2D RP column is used for desalting via a diverter valve. Active solvent modulation enables to refocus the oligonucleotide peak into a sharp zone by 2D RP entirely free of non-volatile buffer components and ion-pair agents. Oligonucleotides can thus be sensitively detected by ESI-QTOF-MS under MS-compatible conditions.