High-Performance Isotope Labeling for Profiling Carboxylic Acid-Containing Metabolites in Biofluids by Mass Spectrometry

We have developed a new isotope labeling method, based on the use of isotope-coded p-dimethylaminophenacyl (DmPA) bromide as a reagent, combined with liquid chromatography−mass spectrometry (LC−MS) for high-performance metabolome analysis with a focus on profiling carboxylic acid-containing metabolites. Derivatization is simple, fast (1 h plus 30 min for quenching the reaction), and applicable to a wide range of carboxylic acids with a high yield and little or no side reaction products. This labeling method is demonstrated to be not only effective in introducing an isotope tag for accurate metabolite quantification but also improving the chromatographic retention of the metabolites in reversed-phase (RP) LC, enhancing ESI efficiency by 2−4 orders of magnitude, and facilitating the identification of metabolite peaks in LC−MS. In triplicate experiments of a 1:1 ratio of 13C-/12C-DmPA labeled human urine, we were able to detect 2671, 2546, and 2820 ion pairs from metabolites containing one or more carboxylic acid groups.

本研究开发了一种新型同位素标记法，以同位素编码的对二甲氨基苯甲酰溴（p-dimethylaminophenacyl (DmPA) bromide）为标记试剂，结合液相色谱-质谱联用法（liquid chromatography−mass spectrometry (LC−MS)），用于以含羧酸类代谢物表征分析为核心的高性能代谢组学分析。该衍生化反应操作简便、耗时短（仅需1小时，外加30分钟终止反应），可适用于绝大多数羧酸类底物，且衍生化产率高，副反应产物极少甚至无副产物生成。实验证明，该标记法不仅可有效引入同位素标签以实现代谢物的准确定量，还能改善代谢物在反相液相色谱（reversed-phase (RP) LC）中的色谱保留行为，将电喷雾电离（ESI）效率提升2~4个数量级，同时助力LC-MS中代谢物峰的鉴定工作。在以1:1比例混合碳13（13C）与碳12（12C）标记的DmPA的人体尿液样本开展的三次重复实验中，我们成功从含有一个或多个羧酸基团的代谢物中检测到2671、2546及2820个离子对。