High sensitivity measurement of amino acid isotope enrichment using liquid chromatography-mass spectrometry
High sensitivity measurement of amino acid isotope enrichment using liquid chromatography-mass spectrometry
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DOI：
10.1016/j.jchromb.2012.07.036
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发表时间：
2012-09-15
影响因子：
3
通讯作者：
Poeze, Martijn
中科院分区：
文献类型：
--
作者：
van Eijk, Hans M. H.;Wijnands, Karolina A. P.;Poeze, Martijn
测量体内注入的稳定同位素富集代谢物如氨基酸的掺入或转化在代谢研究中起着关键作用。在基因敲除小鼠模型中经常探测特定途径，限制了可用的样品量。虽然与燃烧同位素比质谱法（C-IRMS）相比不太精确，但气相色谱-质谱法（GC-MS）或液相色谱-质谱法（LC-MS）技术因此通常是测量目标代谢物的同位素富集的选择方法。然而，在代谢消耗的条件下，这些系统的精度变得有限。在本文中，进行了研究，以提高灵敏度和精度的同位素富集LC-MS测量。离子统计和分辨率被确定为关键因素时，使用线性阱质谱仪的应用。结合自动化柱前衍生和精心选择的溶剂混合物，使我们能够在血浆浓度低至5 μ mol/l时测量同位素富集至0.005%，与替代方法相比，提高了100倍。所得到的方法现在允许测量氨基酸精氨酸转化为瓜氨酸的体内转化，瓜氨酸作为精氨酸和瓜氨酸血浆水平耗尽的体内鼠内毒素血症模型中一氧化氮产生的标志物。(C)2012爱思唯尔有限公司版权所有。
Measurement of the incorporation or conversion of infused stable isotope enriched metabolites in vivo such as amino acids plays a key role in metabolic research. Specific routes are frequently probed in knockout mouse models limiting the available amount of sample. Although less precise as compared to combustion-isotope ratio mass spectrometry (C-IRMS), gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) techniques are therefore often the method of choice to measure isotopic enrichment of target metabolites. However, under conditions of metabolic depletion, the precision of these systems becomes limiting. In this paper, studies were performed to enhance the sensitivity and precision of isotope enrichment measurements using LC-MS. Ion-statistics and resolution were identified as critical factors for this application when using a linear trap mass spectrometer. The combination with an automated pre-column derivatization and a carefully selected solvent mix allowed us to measure isotopic enrichments down to 0.005% at plasma concentrations as low as 5 mu mol/l, an improvement by a factor of 100 compared to alternative methods. The resulting method now allowed measurement of the in vivo conversion of the amino acid arginine into citrulline as a marker for the production of nitric oxide in an in vivo murine endotoxemia model with depleted plasma levels of arginine and citrulline. (C) 2012 Elsevier B.V. All rights reserved.