Analysis of oligonucleotides by ion-pair reversed-phase liquid chromatography coupled with positive mode electrospray ionization mass spectrometry.

Oligonucleotides are usually analyzed by ion-pair reversed-phase liquid chromatography (IP-RPLC) coupled with negative mode electrospray ionization mass spectrometry (ESI-MS) due to their highly negative charged phosphodiester backbones. Herein, the signal suppression effect of triethylamine (TEA) adducts caused the ion-pair reagent TEA/hexafluoroisopropanol (HFIP) is greatly alleviated after improving the in-source energy in positive mode ESI-MS. This strategy is applied for different RNA sequencing through analyzing their formic acid hydrolysates via IP-RPLC MS. Comparing with negative ion mode, we demonstrate that IP-RPLC MS analysis in positive ion mode is more suitable for RNA sequencing with fewer contaminant interferences. Finally, simultaneous online separation and detection of oligonucleotides and protein digests are achieved in positive ion mode IP-RPLC MS analysis with little interference to each other.