Separation and identification of oligonucleotides by hydrophilic interaction liquid chromatography (HILIC)—inductively coupled plasma mass spectrometry (ICPMS)

Abstract

A method for the separation and detection of oligonucleotides utilizing hydrophilic interaction liquid chromatography (HILIC) with inductively coupled plasma mass spectrometry (ICPMS) is described. Polythymidilic acids of various lengths (10, 15, 20 and 30 nucleotides) were separated under gradient HILIC conditions. Selective detection of oligonucleotides was possible through monitoring m/z 47, corresponding to 31P16O+, using ICPMS. Oxygen was used as a reaction gas in the collision/reaction cell to produce PO+ by reacting with phosphorus in the gas phase, thereby effectively eliminating the interferences for phosphorus normally seen at m/z 31. Limits of detection (LODs) were determined to be 1.69 pmol, 1.21 pmol, 1.0 pmol and 0.55 pmol loaded on column for the 10, 15, 20 and 30-mer, respectively.

Comprehensive hydrophilic interaction and ion-pair reversed-phase liquid chromatography for analysis of di- to deca-oligonucleotides

“…Only a few publications describing the analysis of ONs by HILIC are available. Easter et al described the use of HILIC on a diol column and on a TSK-gel Amide-80 column [40]. The use of methacrylate-based monoliths has been described for the separation of three ONs by HILIC [41].…”

Chromatographic methods for the determination of therapeutic oligonucleotides

“…ICP-MS provides elemental and isotopic information with a wide linear dynamic range that may exceed six orders of magnitude. Elemental phosphorus from phosphodiester linkages or both elemental phosphorus and sulfur from phosphorothioate linkages can be measured using ICP-MS with good sensitivity [68]. However, since ICP is a destructive ionization mode, it cannot provide additional information about the structure of the analyte.…”

“…A recent paper by Easter et al introduced oligo separations on a hydrophilic interaction liquid chromatography (HILIC) column (2 × 150 mm) with detection by inductively coupled plasma mass spectrometry [68]. HILIC columns use water as the strong eluent and allow for separations without the use of IP agents.…”

A high-throughput flow injection inductively coupled plasma mass spectrometry method for quantification of oligonucleotides

“…A recent development in phosphorus determination by a quadrupole-based ICP-MS is to determine the oxide ion 31 P 16 O + (47 u), thus avoiding the isobaric interferences at 31 u, as long as any other spectral interference at mass 47 u (e.g., 47 Ti + ) is negligible. This is normally realized with a reaction/collision cell using oxygen as a reaction gas [17,22,23]. However, under optimized conditions the oxide ion can be formed without the use of oxygen as reaction gas [24].…”