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Analysis of native and chemically modified oligonucleotides by tandem ion‐pair reversed‐phase high‐performance liquid chromatography/electrospray ionization mass spectrometry
Author(s) -
Fountain Kenneth J.,
Gilar Martin,
Gebler John C.
Publication year - 2003
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.959
Subject(s) - chemistry , chromatography , electrospray ionization , mass spectrometry , tandem mass spectrometry , electrospray , oligonucleotide , high performance liquid chromatography , analytical chemistry (journal) , top down proteomics , selected reaction monitoring , dna , biochemistry
Ion‐pair reversed‐phase high‐performance liquid chromatography (IP‐RP‐HPLC) was utilized in tandem with negative‐ion electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOFMS) for the analysis of native and chemically modified oligonucleotides. Separation was performed on a 1.0 × 50 mm column packed with porous C 18 sorbent with a particle size of 2.5 μm and an average pore diameter of 140 Å. A method was developed which maximizes both chromatographic separation and mass spectrometric sensitivity using an optimized buffer system containing triethylamine and 1,1,1,3,3,3‐hexafluoro‐2‐propanol with a methanol gradient. The ESI‐TOFMS tuning parameters were also optimized in order to minimize in‐source fragmentation and achieve the best sensitivity. Analyses of native, phosphorothioate, and guanine‐rich oligonucleotides were performed by LC/MS. Detection limits were at sub‐picomole levels with an average mass accuracy of 125 ppm. The described method allowed for the LC/MS analysis of oligonucleotides up to 110mer in length with little alkali cation adduction. Since sensitive detection of oligonucleotides was achieved with ultraviolet (UV) detection, we utilized a combination of UV‐MS for quantitation (UV) and characterization (MS) of oligonucleotides and their failure sequence fragments/metabolites. Copyright © 2003 John Wiley & Sons, Ltd.