Yang Wen-Chu, Regnier Fred E, Adamec Jiri
Bindley Bioscience Center, Purdue University, West Lafayette, IN, USA.
Electrophoresis. 2008 Nov;29(22):4549-60. doi: 10.1002/elps.200800156.
A new approach to the selective comparative metabolite profiling of carboxylic acids in rat urine was established using CE-MS and a method for positively pre-charged and (2)H-coded derivatization. Novel derivatizing reagents, N-alkyl-4-aminomethyl-pyridinum iodide (alkyl = butyl, butyl-d9 or hexyl), containing quaternary amine and stable-isotope atoms (deuterium), were introduced for the derivatization of carboxylic acids. CE separation in positive polarity showed high reproducibility (0.99-1.32% RSD of migration time) and eliminated problems with capillary coating known in CE-MS anion analyses. Essentially complete ionization and increased hydrophobicity after the derivatization also enhanced MS detection sensitivity (e.g. formic acid was detected at 0.5 pg). Simultaneous derivatization of one sample using two structurally similar reagents, N-butyl-4-aminomethyl-pyridinum iodide (BAMP) and N-hexyl-4-aminomethyl-pyridinum iodide, provided additional information for recognizing a carboxylic acid in an unknown sample. Moreover, characteristic fragmentation acquired by online CE-MS/MS allowed for identification and categorization of carboxylic acids. Applying this method on rat urine, we found 59 ions matching the characteristic patterns of carboxylic acids. From these 59, 32 ions were positively identified and confirmed with standards. For comparative analysis, 24 standard carboxylic acids were derivatized by chemically identical but isotopically distinct BAMP and N-butyl-d9-4-aminomethyl-pyridinium iodide, and their derivatization limits and linearity ranges were determined. Comparative analysis was also performed on two individual urine samples derivatized with BAMP and N-butyl-d9-4-aminomethyl-pyridinium iodide. The metabolite profiling variation between these two samples was clearly visualized.
采用毛细管电泳-质谱联用（CE-MS）以及正电荷预充和（2）H编码衍生化方法，建立了一种用于大鼠尿液中羧酸选择性比较代谢物谱分析的新方法。引入了新型衍生化试剂N-烷基-4-氨甲基吡啶碘化物（烷基=丁基、丁基-d9或己基），其含有季铵和稳定同位素原子（氘），用于羧酸的衍生化。正极性的CE分离显示出高重现性（迁移时间的相对标准偏差为0.99 - 1.32%），并消除了CE-MS阴离子分析中已知的毛细管涂层问题。衍生化后基本完全电离和增加的疏水性也提高了质谱检测灵敏度（例如，甲酸在0.5 pg时被检测到）。使用两种结构相似的试剂N-丁基-4-氨甲基吡啶碘化物（BAMP）和N-己基-4-氨甲基吡啶碘化物对一个样品进行同时衍生化，为识别未知样品中的羧酸提供了额外信息。此外，通过在线CE-MS/MS获得的特征性碎片有助于羧酸的鉴定和分类。将该方法应用于大鼠尿液，我们发现59个离子与羧酸的特征模式匹配。其中32个离子通过标准品得到了阳性鉴定和确认。为了进行比较分析，24种标准羧酸用化学性质相同但同位素不同的BAMP和N-丁基-d9-4-氨甲基吡啶碘化物进行衍生化，并测定了它们的衍生化限度和线性范围。还用BAMP和N-丁基-d9-4-氨甲基吡啶碘化物对两个个体尿液样品进行了衍生化，并进行了比较分析。这两个样品之间的代谢物谱变化清晰可见。
