Anderson L W, Zaharevitz D W, Strong J M
Division of Cancer Treatment, National Institutes of Health, Bethesda, Maryland 20892.
Anal Biochem. 1987 Jun;163(2):358-68. doi: 10.1016/0003-2697(87)90236-3.
A method is described for simultaneous quantification of glutamine and glutamate plasma levels and isotopic enrichments in these compounds. Glutamine and glutamate are analyzed intact as their tertiary-butyldimethylsilyl derivatives. Deuterated glutamine and glutamate are used as internal standards for quantification by reverse isotope dilution. Preparation of plasma samples is accomplished by adding ammonium formate as an ion-pairing agent followed by extraction of the amino acids into 4.3:1 methanol:water. Negligible amounts of glutamine to glutamate conversion are observed during the sample preparation and GC/MS analysis. Since glutamine is analyzed intact, both single and double [15N]glutamine labels can be quantified. [15N]Glutamine at 0.2 to 11 mol% excess was measured in plasma with an average relative standard error of 3.8%, and [15N]glutamate over a range of 0.4 to 9 mol% excess was measured with a mean relative standard error of 12%. At glutamate levels above 1 mol% excess 15N, the mean relative standard error was 6%. Finally, automated sample injection into the GC/MS and automated data reduction are used for the analysis of samples by GC/MS.
本文描述了一种同时定量测定血浆中谷氨酰胺和谷氨酸水平以及这些化合物中同位素丰度的方法。谷氨酰胺和谷氨酸作为叔丁基二甲基甲硅烷基衍生物被完整分析。氘代谷氨酰胺和谷氨酸用作内标，通过反向同位素稀释进行定量。血浆样品的制备是通过加入甲酸铵作为离子对试剂，然后将氨基酸萃取到4.3:1的甲醇:水中。在样品制备和气相色谱/质谱分析过程中，观察到谷氨酰胺向谷氨酸的转化量可忽略不计。由于谷氨酰胺是完整分析的，单重和双重[15N]谷氨酰胺标记都可以定量。血浆中过量0.2至11 mol%的[15N]谷氨酰胺的测量平均相对标准误差为3.8%，过量0.4至9 mol%的[15N]谷氨酸的测量平均相对标准误差为12%。在15N过量的谷氨酸水平高于1 mol%时，平均相对标准误差为6%。最后，使用自动进样到气相色谱/质谱仪和自动数据处理来通过气相色谱/质谱仪分析样品。
