School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet 1, 1211 Geneva 4, Switzerland.
Pfizer, Inc., Worldwide Medicinal Chemistry, La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121, USA.
J Chromatogr A. 2018 Aug 10;1562:96-107. doi: 10.1016/j.chroma.2018.05.055. Epub 2018 May 28.
The aim of this study was to evaluate the suitability of SFC-MS for the analysis of a wide range of compounds including lipophilic and highly hydrophilic substances (log P values comprised between -6 and 11), for its potential application toward human metabolomics. For this purpose, a generic unified chromatography gradient from 2 to 100% organic modifier in CO was systematically applied. In terms of chemistry, the best stationary phases for this application were found to be the Agilent Poroshell HILIC (bare silica) and Macherey-Nagel Nucleoshell HILIC (silica bonded with a zwitterionic ligand). To avoid system overpressure at very high organic modifier proportion, columns of 100 × 3 mm I.D. packed with sub-3 μm superficially porous particles were selected. In terms of organic modifier, a mixture of 95% MeOH and 5% water was selected, with 50 mM ammonium formate and 1 mM ammonium fluoride, to afford good solubility of analytes in the mobile phase, limited retention for the most hydrophilic metabolites and suitable peak shapes of ionizable species. A sample diluent containing 50%ACN/50% water was employed as injection solvent. These conditions were applied to a representative set of metabolites belonging to nucleosides, nucleotides, small organic acids, small bases, sulfated/sulfonated metabolites, poly-alcohols, lipid related substances, quaternary ammonium metabolites, phosphate-based substances, carbohydrates and amino acids. Among all these metabolites, 65% of the compounds were adequately analyzed with excellent peak shape, 23% provided distorted peak shapes, while only 12% were not detected (mostly metabolites having several phosphate or several carboxylic acid groups).
本研究旨在评估 SFC-MS 分析范围广泛的化合物的适用性，包括亲脂性和高亲水性物质（log P 值在-6 到 11 之间），以将其潜在应用于人类代谢组学。为此，系统地应用了从 2%到 100%有机改性剂的通用统一色谱梯度。从化学角度来看，发现最适合这种应用的固定相是 Agilent Poroshell HILIC（裸露硅胶）和 Macherey-Nagel Nucleoshell HILIC（硅胶键合两性离子配体）。为避免在高有机改性剂比例下系统超压，选择了 100×3mm ID 的填充有亚 3μm 表面多孔颗粒的色谱柱。从有机改性剂的角度来看，选择了 95%甲醇和 5%水的混合物，加入 50mM 甲酸铵和 1mM 氟化铵，以在流动相中提供良好的分析物溶解度、对最亲水代谢物的有限保留和可电离物质的合适峰形。使用含有 50%ACN/50%水的样品稀释剂作为进样溶剂。这些条件应用于一组具有代表性的代谢物，包括核苷、核苷酸、小分子酸、小碱基、硫酸化/磺化代谢物、多醇、脂质相关物质、季铵代谢物、基于磷酸盐的物质、碳水化合物和氨基酸。在所有这些代谢物中，65%的化合物得到了充分的分析，具有极好的峰形，23%的化合物提供了变形的峰形，而只有 12%的化合物未被检测到（主要是具有几个磷酸盐或几个羧酸基团的代谢物）。
