Metabolomic analysis of key central carbon metabolism carboxylic acids as their 3-nitrophenylhydrazones by UPLC/ESI-MS.
Metabolomic analysis of key central carbon metabolism carboxylic acids as their 3-nitrophenylhydrazones by UPLC/ESI-MS.
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DOI：
10.1002/elps.201200601
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发表时间：
2013-10
期刊：
影响因子：
2.9
通讯作者：
Borchers, Christoph H.
中科院分区：
文献类型：
--
作者：
Han, Jun;Gagnon, Susannah;Eckle, Tobias;Borchers, Christoph H.
关键词：
多种羟基、酮、二和三羧酸属于中心碳代谢 (CCM) 的细胞代谢物。对这些羧酸盐进行灵敏且可靠的分析对于许多生物和细胞工程研究非常重要。在这项工作中，我们研究了 3-硝基苯肼作为衍生剂，并优化了使用 LC/MS 和电喷雾电离测量 10 种 CCM 相关羧酸化合物的反应条件，包括乙醇酸盐、乳酸盐、苹果酸盐、富马酸盐、琥珀酸盐、柠檬酸盐、异柠檬酸盐、丙酮酸盐、草酰乙酸和 α-酮戊二酸盐作为其 3-硝基苯腙。利用我们开发的衍生化方案，并在三重四极杆仪器上使用负离子多重反应监测，所有的羧酸盐在约 100 动态范围内均表现出良好的线性。 200 至 2000 以上。检测和定量的柱上限从高飞摩尔到低皮摩尔。十种分析物中有八种的分析准确度确定在 89.5% 至 114.8% 之间（CV≤7.4%，n=6）。使用四极杆飞行时间仪器，通过 UPLC/MS 和全质量检测成功测定了从 13C 标记的小鼠心脏中提取的这些羧酸盐的同位素分布模式，表明该分析方法可用于代谢通量分析。总之，这项工作展示了一种有效的化学衍生化 LC/MS 方法，用于生物基质中这些关键 CCM 中间体的代谢组学分析。
Multiple hydroxy-, keto-, di-, and tri-carboxylic acids are among the cellular metabolites of central carbon metabolism (CCM). Sensitive and reliable analysis of these carboxylates is important for many biological and cell engineering studies. In this work, we examined 3-nitrophenylhydrazine as a derivatizing reagent and optimized the reaction conditions for the measurement of ten CCM related carboxylic compounds, including glycolate, lactate, malate, fumarate, succinate, citrate, isocitrate, pyruvate, oxaloacetate, and α-ketoglutarate as their 3-nitrophenylhydrazones using LC/MS with electrospray ionization. With the derivatization protocol which we have developed, and using negative-ion multiple reaction monitoring on a triple-quadrupole instrument, all of the carboxylates showed good linearity within a dynamic range of ca. 200 to more than 2000. The on-column limits of detection and quantitation were from high femtomoles to low picomoles. The analytical accuracies for eight of the ten analytes were determined to be between 89.5 to 114.8% (CV≤7.4%, n=6). Using a quadrupole time-of-flight instrument, the isotopic distribution patterns of these carboxylates, extracted from a 13C-labeled mouse heart, were successfully determined by UPLC/MS with full-mass detection, indicating the possible utility of this analytical method for metabolic flux analysis. In summary, this work demonstrates an efficient chemical derivatization LC/MS method for metabolomic analysis of these key CCM intermediates in a biological matrix.
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影响因子：
82.9
作者：
通讯作者：
--
DOI：
10.1046/j.1432-1033.2003.03798.x
发表时间：
2003-11-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
David, H;Åkesson, M;Nielsen, J
通讯作者：
Nielsen, J
影响因子：
4.1
作者：
Kloos, D.;Derks, R. J. E.;Giera, M.
通讯作者：
Giera, M.
影响因子：
4.1
作者：
Peters, R;Hellenbrand, J;Van der Wal, S
通讯作者：
Van der Wal, S
影响因子：
6.1
作者：
Banos, Clara-Eugenia;Silva, Manuel
通讯作者：
Silva, Manuel