Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry.
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 x 50 mm column packed with porous C(18) sorbent with a particle size of 2.5 microm and an average pore diameter of 140 A. 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.
Fountain
Kenneth J
KJ
Waters Corporation, 34 Maple St, Milford, MA 01757, USA.
Gilar
Martin
M
Gebler
John C
JC
D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007477:Ions; D009841:Oligonucleotides / Q000032:analysis* / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D002021:Buffers; D007477:Ions; D009841:Oligonucleotides