二异丙基乙胺/六氟异丙醇介导的离子对超高效液相色谱/质谱法用于磷酸盐和羧酸盐代谢物分析：在细胞代谢研究中的应用
Diisopropylethylamine/hexafluoroisopropanol-mediated ion-pairing ultra-high-performance liquid chromatography/mass spectrometry for phosphate and carboxylate metabolite analysis: utility for studying cellular metabolism.
作者信息
Guo Lili, Worth Andrew J, Mesaros Clementina, Snyder Nathaniel W, Glickson Jerry D, Blair Ian A
机构信息
Penn SRP and Center for Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA, 19104, USA.
A.J. Drexel Autism Institute, Drexel University, Philadelphia, PA, 19104, USA.
出版信息
Rapid Commun Mass Spectrom. 2016 Aug 30;30(16):1835-45. doi: 10.1002/rcm.7667.
RATIONALE
Mass spectrometric (MS) analysis of low molecular weight polar metabolites can be challenging because of poor chromatographic resolution of isomers and insufficient ionization efficiency. These metabolites include intermediates in key metabolic pathways, such as glycolysis, the pentose phosphate pathway, and the Krebs cycle. Therefore, sensitive, specific, and comprehensive quantitative analysis of these metabolites in biological fluids or cell culture models can provide insight into multiple disease states where perturbed metabolism plays a role.
METHODS
An ion-pairing reversed-phase ultra-high-performance liquid chromatography (IP-RP-UHPLC)/MS approach to separate and analyze biochemically relevant phosphate- and carboxylic acid-containing metabolites was developed. Diisopropylethylamine (DIPEA) was used as an IP reagent in combination with reversed-phase liquid chromatography (RP-LC) and a triple quadrupole mass spectrometer using selected reaction monitoring (SRM) and negative electrospray ionization (NESI). An additional reagent, hexafluoroisopropanol (HFIP), which has been previously used to improve sensitivity of nucleotide analysis by UHPLC/MS, was used to enhance sensitivity.
RESULTS
HFIP versus acetic acid, when added with the IP base, increased the sensitivity of IP-RP-UHPLC/NESI-MS up to 10-fold for certain analytes including fructose-1,6-bisphosphate, phosphoenolpyruvate, and 6-phosphogluconate. It also improved the retention of the metabolites on a C18 reversed-phase column, and allowed the chromatographic separation of important isomeric metabolites. This methodology was amenable to quantification of key metabolites in cell culture experiments. The applicability of the method was demonstrated by monitoring the metabolic adaptations resulting from rapamycin treatment of DB-1 human melanoma cells.
CONCLUSIONS
A rapid, sensitive, and specific IP-RP-UHPLC/NESI-MS method was used to quantify metabolites from several biochemical pathways. IP with DIPEA and HFIP increased the sensitivity and improved chromatographic separation when used with reversed-phase UHPLC.
原理
由于异构体的色谱分辨率较差以及电离效率不足，对低分子量极性代谢物进行质谱（MS）分析具有挑战性。这些代谢物包括关键代谢途径中的中间体，如糖酵解、磷酸戊糖途径和三羧酸循环。因此，对生物体液或细胞培养模型中的这些代谢物进行灵敏、特异且全面的定量分析，可为多种代谢紊乱起作用的疾病状态提供深入了解。
方法
开发了一种离子对反相超高效液相色谱（IP-RP-UHPLC）/MS方法，用于分离和分析具有生化相关性的含磷酸盐和羧酸的代谢物。二异丙基乙胺（DIPEA）用作离子对试剂，与反相液相色谱（RP-LC）以及使用选择反应监测（SRM）和负电喷雾电离（NESI）的三重四极杆质谱仪联用。另外一种试剂六氟异丙醇（HFIP），此前已被用于提高UHPLC/MS分析核苷酸的灵敏度，此次用于增强灵敏度。
结果
与乙酸相比，当与离子对碱一起添加时，HFIP使IP-RP-UHPLC/NESI-MS对某些分析物（包括果糖-1,6-二磷酸、磷酸烯醇丙酮酸和6-磷酸葡萄糖酸）的灵敏度提高了10倍。它还改善了代谢物在C18反相柱上的保留，并实现了重要异构体代谢物的色谱分离。该方法适用于细胞培养实验中关键代谢物的定量分析。通过监测雷帕霉素处理DB-1人黑素瘤细胞引起的代谢适应性，证明了该方法的适用性。
结论
采用快速、灵敏且特异的IP-RP-UHPLC/NESI-MS方法对几种生化途径中的代谢物进行定量分析。与反相UHPLC联用时，DIPEA和HFIP形成的离子对提高了灵敏度并改善了色谱分离效果。
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