Modified DNAs and RNAs often exist in trace amounts, which makes detection challenging. Adding ionizable groups to nucleobases (NBs) can enhance sensitivity in liquid chromatography-tandem mass spectrometry (LC-MS2). Traditional derivatization methods for modified nucleosides require complex preparation under harsh conditions and cover only limited modifications. We present an ultrafast (microseconds) derivatization technique using ionizable imidazole, facilitated by microdroplets through supersonic electrospray-induced aldehyde condensation for direct LC-MS2 analysis. This technique universally tags the exocyclic amine (−NH2) or pyrrolic nitrogen (−NH) of five NBs without base damage, particularly guanosine, which is prone to oxidation. Sensitive modifications located on ring carbon, like oxidative lesions and epigenetic marks, or nontagging sites of ring nitrogen, like N-alkylation on guanosine, and depurinating adducts, remain intact. The rapid derivatization avoids artifact generation and allows online postcolumn in situ detection, maintaining chromatographic separation. This method achieves over 90% derivatization ratio and improves quantification sensitivity by more than 10-fold. It also enables a quick test for tagging site-blocked modifications. We demonstrate this method by locating stable adduction sites of 4-hydroxy-estradiol (4OHE2) in chromatin DNA and quantifying 4OHE2-induced oxidation and released adducts in MCF-7 cells, achieving 13–43-fold higher sensitivity with significantly reduced sample amounts. Key parameters influencing detection efficiency were systematically explored for routine application.
Do et al. (Thu,) studied this question.