Comparison of mobile‐phase systems commonly applied in liquid chromatography‐mass spectrometry of nucleic acids

Abstract

LC-MS represents an important technology for the qualitative and quantitative analysis of nucleic acids. For MS, ESI in negative ion mode is used. The chromatographic method of choice is ion-pair (IP) RP chromatography. Chromatographic separations are usually accomplished by gradients of an organic modifier in aqueous solutions of IP reagents. Commonly applied IP reagents are 2.3 mM triethylamine/400 mM 1,1,1,3,3,3-hexafluoro-2-propanol (TEA/HFIP, pH 7.0) and 10-25 mM cyclohexyldimethylammonium acetate (CycHDMAA, pH 8.4). Direct comparison of mass spectrometric performance of the two solvent systems revealed that the TEA/HFIP system offers better detection sensitivity than the CycHDMAA system. This is mainly attributable to the depletion of HFIP during droplet formation and solvent evaporation. Removal of the anionic counterion facilitates oligonucleotide ionization, and the oligonucleotides are desorbed as highly charged ions into the gas phase. TEA/HFIP-based mobile phases are recommended for developing quantitative assays targeting defined oligonucleotides. The CycHDMAA system allows the formation of cyclohexyldimethylammonium adducts. These adducts are cleaved in the gas phase, and this decomposition gives rise to charge state reduction. Ammonium adduct formation is of particular importance in preventing adducting with metal ions. Thus, adducts with metal ions are efficiently suppressed with CycHDMAA. For the TEA/HFIP system, however, such adducting represents a severe problem particularly if large oligonucleotides are analyzed. Thus, CycHDMAA-based mobile phases are recommended for qualitative assays such as LC-MS-based genotyping.

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

“…A PS-DVB capillary monolith column 21,[24][25][26][27][28][29][30][31][32][33] , a C 18 reverse-phase particulate silica column 23,40,41 and a hydrophobic interaction chromatography (HILIC) column 42 have been used for the LC/ESI-MS analysis of oligonucleotides. Among them, the capillary monolith column is superior to the others in terms of separation capacity, however, the capillary monolith column used in past studies was made in-house and operated at a low flow rate (2 µl/min), which requires instrumentation dedicated to micro LC.…”

“…LC provides an on-line separation of the analytes from coexisting substances and does not require a laborious sample preparation prior to ionization 21,22 . Application of LC/ESI-MS genotyping for differentiation of pathogen species, single-nucleotide polymorphisms and short tandem repeats has been reported using a capillary polymer-monolith column that can separate longer oligonucleotides 1,21,[24][25][26][27][28][29][30][31][32][33] .…”

“…Because the present method employs a heated ESI probe, which nebulizes the eluate with hot nitrogen gas at 350 °C, this effect may be over-emphasized. Instead of HFIP-TEA buffer, Erb and Oberacher recommended cyclohexyldimethylammonium acetate (CycHDMAA; pH 8.4) for genotyping analysis because of a reduction in adduct formation with trace metal ions 33 . The authors deduced that CycHDMAA itself suppressed the formation of the metal adduct.…”

Assessing the Interplay between the Physicochemical Parameters of Ion-Pairing Reagents and the Analyte Sequence on the Electrospray Desorption Process for Oligonucleotides

“…Erb and Oberacher [40] compared triethylamine (TEA) with dimethylcyclohexylamine (DMCHA) and concluded that TEA generates a stronger signal for a 27-mer DNA. Sharma et al [41] used six different alkylamines (including DMCHA, TPA and TEA) with a 17-mer DNA strand and observed the best MS sensitivity with dimethylbutylamine (DMBA).…”

Corona Discharge Suppression in Negative Ion Mode Nanoelectrospray Ionization via Trifluoroethanol Addition

“…39,41,42 Fluorosolvent addition has also been utilized extensively as a method to retain and improve ionization of hydrophilic small molecules and oligonucleotides through ion-pairing on reverse-phase resins and improved ESI droplet desolvation. 43–46 Furthermore, small needle-tip geometry has been reported to decrease minimum spray voltage below ambient atmosphere dielectric limits. 47 While these approaches have been effective, instrument modification, dilution of precious sample, and analyte solubility are still of concern, particularly with regards to online separations and “native” mass spectrometry experiments.…”