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. Polythymidylic 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 detections (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.