Deguchi Kisaburo, Ishikawa Masako, Yokokura Takefumi, Ogata Izumi, Ito Shinya, Mimura Tadao, Ostrander Chad
Naka Customer Center, Hitachi Science Systems Co., 882 Ichige, Hitachinaka 312-8504, Japan.
Rapid Commun Mass Spectrom. 2002;16(22):2133-41. doi: 10.1002/rcm.840.
A new method providing enhanced sensitivity for the analysis of oligonucleotides using an on-line coupled system of reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization ion-trap mass spectrometry (ESI-MS) has been developed. The presented method allows the use of the standard gradient elution of 0.1 M triethylammonium acetate (TEAA) buffer (adjusted to pH 7.0 with acetic acid) and acetonitrile that is typically used for the separation of oligonucleotides in RP-HPLC. An added feature of this method is the ability to combine and mix additional 0.1 M imidazole in acetonitrile after the separation column for improved ESI-MS performance. This is similar to the post-column reaction method in liquid chromatography (LC) and the liquid sheath flow method in LC/ESI-MS, both of which offer the advantage of not compromising the chromatographic separation conditions. The application of this new method is demonstrated to afford improved sensitivity for the analysis of oligonucleotides (20-50 mer) via on-line coupled HPLC/ESI-MS analysis and purification systems.
已开发出一种新方法，该方法使用反相高效液相色谱（RP-HPLC）和电喷雾电离离子阱质谱（ESI-MS）的在线联用系统，提高了对寡核苷酸分析的灵敏度。所提出的方法允许使用0.1 M三乙胺乙酸盐（TEAA）缓冲液（用乙酸调节至pH 7.0）和乙腈的标准梯度洗脱，这是RP-HPLC中通常用于分离寡核苷酸的方法。该方法的一个附加特点是能够在分离柱后将额外的0.1 M咪唑与乙腈混合，以改善ESI-MS性能。这类似于液相色谱（LC）中的柱后反应法和LC/ESI-MS中的液鞘流法，这两种方法都具有不影响色谱分离条件的优点。通过在线联用HPLC/ESI-MS分析和纯化系统，证明了这种新方法在分析寡核苷酸（20 - 50聚体）时具有更高的灵敏度。
