Rousu Timo, Hokkanen Juho, Pelkonen Olavi R, Tolonen Ari
Novamass Ltd, Medipolis Center Oulu, Finland.
Front Pharmacol. 2010 Aug 6;1:10. doi: 10.3389/fphar.2010.00010. eCollection 2010.
Liquid chromatography-mass spectrometry (LC-MS) with generic gradient elution for a large number of chemically different compounds is a common approach in drug development, used to acquire a large amount of data in a short time frame for drug candidates. The analysis with non-optimized parameters however may lead to a poor method performance for many compounds, and contains a risk of losing important information. Here, generic electrospray time of flight (ESI-TOF) MS methods in various pH conditions were tested for 55 chemically diverse compounds (10 acids, 25 bases, 17 neutrals, and 3 amphoterics), aiming to find best analytical conditions for each compound, for studies of in vitro metabolic properties in liver preparations. The effect of eluent pH and elution gradient strength on chromatographic performance and electrospray MS ionization efficiency were examined for each compound. The data are evaluated how well the best generic approach could cover the analysis of test compounds and how many compounds would still need completely different analytical conditions after that. Aqueous mobile phase consisting of 0.05% acetic acid and 5 mM ammonium acetate (pH 4.4) showed the best general suitability for the analyses, showing adequate performance for metabolite profiling for 41 out of 55 compounds either in positive or negative ion mode. In positive ion mode, the main limitation of performance in various pH conditions was generally not the lack of ionization, but rather the poor chromatographic performance (inadequate retention or poor peak shape), suggesting that more emphasis should be put in finding conditions providing best chromatographic performance, rather than highest ionization properties. However, a single generic approach for a large number of different compounds is not likely to produce good results for all compounds. Preferably, at least two or three different conditions are needed for the coverage of a larger number of structurally diverse compounds.
液相色谱 - 质谱联用（LC-MS）采用通用梯度洗脱来分析大量化学性质不同的化合物，这是药物研发中的常用方法，用于在短时间内获取大量候选药物的数据。然而，使用未优化参数进行分析可能导致许多化合物的方法性能不佳，并存在丢失重要信息的风险。在此，针对55种化学性质各异的化合物（10种酸、25种碱、17种中性化合物和3种两性化合物）测试了不同pH条件下的通用电喷雾飞行时间（ESI-TOF）质谱方法，旨在为每种化合物找到最佳分析条件，用于肝脏制剂体外代谢特性的研究。考察了洗脱液pH和洗脱梯度强度对每种化合物色谱性能和电喷雾质谱电离效率的影响。评估了最佳通用方法对测试化合物分析的覆盖程度，以及在此之后仍有多少化合物需要完全不同的分析条件。由0.05%乙酸和5 mM乙酸铵（pH 4.4）组成的水性流动相对分析显示出最佳的总体适用性，在正离子或负离子模式下，对55种化合物中的41种进行代谢物谱分析时表现出足够的性能。在正离子模式下，不同pH条件下性能的主要限制通常不是缺乏电离，而是色谱性能不佳（保留不足或峰形不佳），这表明应更加注重寻找提供最佳色谱性能而非最高电离特性的条件。然而，针对大量不同化合物的单一通用方法不太可能对所有化合物都产生良好结果。最好需要至少两种或三种不同条件来覆盖更多结构多样的化合物。
