Huber C G, Krajete A
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University, Innrain 52a, A-6020 Innsbruck, Austria.
Anal Chem. 1999 Sep 1;71(17):3730-9. doi: 10.1021/ac990378j.
Ion-pair reversed-phase high-performance liquid chromatography was successfully coupled to negative-ion electrospray ionization mass spectrometry by using 60 × 0.20 mm i.d. capillary columns packed with 2.3-μm micropellicular, octadecylated poly(styrene/divinylbenzene) particles as stationary phase and gradients of acetonitrile in 50 mM aqueous triethylammonium bicarbonate as mobile phase. Systematic variation of the eluent composition, such as concentration of ion-pair reagent, anion in the ion-pair reagent, solution pH, and acetonitrile concentration led to the conclusion that most parameters have opposite effects on chromatographic and mass spectrometric performances. The use of acetonitrile as sheath liquid enabled the rapid and highly efficient separation and detection of phosphorylated and nonphosphorylated oligonucleotides ranging in size from 8 to 40 nucleotides. High-quality full-scan mass spectra showing little cation adduction were acquired from which the molecular masses of the separated oligonucleotides were calculated with an accuracy of 0.011%. With calibration curves being linear over at least 2 orders of magnitude, the lower limits of detection for a oligodeoxythymidine 16-mer were 104 fmol with full scan and 710 amol with selected-ion-monitoring data acquisition. The potential of ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry was demonstrated for mixed-sequence oligomers by the characterization of a reaction mixture from solid-phase synthesis of a 40-mer oligonucleotide.
采用内径为60×0.20 mm的毛细管柱，以填充2.3-μm微球型十八烷基化聚（苯乙烯/二乙烯基苯）颗粒为固定相，50 mM碳酸氢三乙铵水溶液中乙腈梯度为流动相，成功地将离子对反相高效液相色谱与负离子电喷雾电离质谱联用。洗脱液组成的系统变化，如离子对试剂浓度、离子对试剂中的阴离子、溶液pH值和乙腈浓度，得出大多数参数对色谱和质谱性能有相反影响的结论。使用乙腈作为鞘液能够快速、高效地分离和检测大小在8至40个核苷酸范围内的磷酸化和非磷酸化寡核苷酸。获得了高质量的全扫描质谱，几乎没有阳离子加合，由此计算出分离寡核苷酸的分子量，准确度为0.011%。校准曲线在至少2个数量级上呈线性，16聚体寡脱氧胸苷的全扫描检测下限为104 fmol，选择离子监测数据采集的检测下限为710 amol。通过对40聚体寡核苷酸固相合成反应混合物的表征，证明了离子对反相高效液相色谱-电喷雾电离质谱对混合序列寡聚物的分析潜力。
