Schappler Julie, Nicoli Raul, Nguyen Dao, Rudaz Serge, Veuthey Jean-Luc, Guillarme Davy
Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland.
Talanta. 2009 Apr 30;78(2):377-87. doi: 10.1016/j.talanta.2008.11.029. Epub 2008 Nov 27.
The coupling of ultra high-pressure liquid chromatography with a single quadrupole mass spectrometer was investigated for the analysis of several cytochromes P450 (CYP450) substrates and respective metabolites. The effect of numerous operating parameters (e.g. mobile phase pH, flow rate, gradient length, MS acquisition mode, dwell time, polarity switching, etc.) on selectivity, sensitivity and acquisition rate was studied. It was demonstrated that basic pH conditions provided the best compromise in terms of sensitivity and chromatographic selectivity with both acidic and basic compounds. The optimal mobile phase flow rate for UHPLC-MS experiments should be comprised between 300 and 600 microL/min for 2.1mm ID columns, while a higher flow rate generated up to 3-fold loss in sensitivity. It was also demonstrated that the fast polarity switching mode represented a valuable tool to improve throughput, maintaining acceptable performance. Finally, limits of detection were included in the range [1-50 ng/mL] in positive ionization mode and [50-250 ng/mL] in negative ionization mode, for investigated compounds.
研究了超高压液相色谱与单四极杆质谱联用技术用于分析几种细胞色素P450（CYP450）底物及其相应代谢产物。研究了众多操作参数（如流动相pH值、流速、梯度长度、质谱采集模式、驻留时间、极性切换等）对选择性、灵敏度和采集速率的影响。结果表明，碱性pH条件在酸性和碱性化合物的灵敏度和色谱选择性方面提供了最佳折衷。对于内径为2.1mm的色谱柱，UHPLC-MS实验的最佳流动相流速应在300至600微升/分钟之间，而较高的流速会导致灵敏度损失高达3倍。还证明了快速极性切换模式是提高通量、保持可接受性能的一种有价值的工具。最后，在所研究的化合物中，正离子模式下的检测限在[1-50纳克/毫升]范围内，负离子模式下的检测限在[50-250纳克/毫升]范围内。
