Hachey D L, Patterson B W, Reeds P J, Elsas L J
USDA/ARS Childrens Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030.
Anal Chem. 1991 May 1;63(9):919-23. doi: 10.1021/ac00009a017.
A rapid, single-step procedure for the extraction and derivatization of organic alpha-keto acids from microliter quantities of human plasma has been developed. The keto acids were analyzed as the pentafluorobenzyl (PFB) ester by methane negative chemical ionization gas chromatography/mass spectrometry. The PFB esters possess excellent chromatographic properties and required no further derivatization to block the keto group. They fragment to produce intense carboxylate anions, often as the sole ion in the spectrum, and offer detection limits below 1 pmol. This derivative is suitable for isotopic analysis of organic keto acids because it does not introduce any additional isotopic complexity into the target molecule. Normal human plasma 4-methyl-2-oxopentanoic acid levels were 34.9 +/- 5.3 mumol.L-1 and could be determined with 1.1% precision by isotope dilution GC/MS. We have used this procedure to study leucine and 4-methyl-2-oxopentanoic acid metabolism by using stable isotopically labeled tracers in a variety of normal and abnormal conditions.
已开发出一种从微升量人血浆中提取和衍生有机α-酮酸的快速单步程序。通过甲烷负离子化学电离气相色谱/质谱法将酮酸分析为五氟苄基（PFB）酯。PFB酯具有出色的色谱特性，无需进一步衍生化来封闭酮基。它们裂解产生强烈的羧酸根阴离子，通常是光谱中的唯一离子，检测限低于1 pmol。这种衍生物适用于有机酮酸的同位素分析，因为它不会给目标分子引入任何额外的同位素复杂性。正常人血浆中4-甲基-2-氧代戊酸水平为34.9±5.3μmol·L-1，通过同位素稀释气相色谱/质谱法可精确测定1.1%。我们已使用该程序，通过在各种正常和异常条件下使用稳定同位素标记的示踪剂来研究亮氨酸和4-甲基-2-氧代戊酸的代谢。
