Department of Surgery, Maastricht University Medical Center, Maastricht, The Netherlands.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Sep 15;905:31-6. doi: 10.1016/j.jchromb.2012.07.036. Epub 2012 Aug 18.
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 μmol/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.
在体测量输注的稳定同位素标记代谢物（如氨基酸）的掺入或转化对于代谢研究至关重要。在敲除小鼠模型中，通常会探测特定的途径，从而限制了可用样本的数量。尽管与燃烧同位素比质谱（C-IRMS）相比，气相色谱-质谱（GC-MS）或液相色谱-质谱（LC-MS）技术的精度较低，但这些技术通常是测量目标代谢物同位素丰度的首选方法。然而，在代谢耗竭的情况下，这些系统的精度会受到限制。本文研究了使用 LC-MS 提高同位素丰度测量的灵敏度和精度。在使用线性阱质谱仪时，离子统计学和分辨率被确定为该应用的关键因素。与自动柱前衍生化和精心选择的溶剂混合物相结合，使我们能够在血浆浓度低至 5 μmol/l 时测量低至 0.005%的同位素丰度，与替代方法相比提高了 100 倍。由此产生的方法现在可以测量体内氨基酸精氨酸转化为瓜氨酸，作为体内内毒素血症模型中一氧化氮产生的标志物，该模型中血浆精氨酸和瓜氨酸水平耗竭。
