Highly Selective Fluorometric Determination of Dicarboxylic Acids Based on Intramolecular Excimer-Formfng Derivatization with a Pyrene-Labeling Reagent.

基于使用芘标记试剂进行分子内准分子形成衍生化的二羧酸的高选择性荧光测定。

• 批准号: 12672101
• 负责人: YAMAGUCHI Masatoshi
• 金额: $ 2.05万
• 依托单位: Fukuoka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12672101/
• 关键词: blood; urine; dicarboxylic acids; excimer fluorescence; derivatization; HPLC; グルタル酸血症; 診断

A highly selective and sensitive method was developed to determine dicarboxylic acids, based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(l-pyrene)butanoic acid hydrazide (PBH) in the presence of l-ethyl-3-(3-dimrthylaminopropyDcarbodiimide, followed by reversed phase high performance liquid chromatography (HPLC). Dicarboxylic acids, having two carboxylic acid moieties in a molecule, were converted to the corresponding dipyrene-labeled derivatives by reaction (40ーC, 60 min) with PBH. The derivatives afforded intramolecular excimer fluorescence (450-520 nm) which can clearly be discriminated from the monomer (normal) fluorescence (360-420 nm) emitted from PBH and monopyrene-labeled derivatives of monocarboxylic acid. The structures of the derivatives were confirmed by HPLC with mass spectrometry, and the emission of excimer fluorescence could be proved by spectrofluorometry and time-resolved fluorometry. The PBH derivatives of four dicarboxylic acids [glutaric acid (GA), adipic acid (AA), suberic acid (SubA) and sebacic acid (SebA)] could be separated by reversed phased HPLC on C8 column with linear gradient elution. The detection limits (signal-to-noise ratio of 3) for the dicarboxylic acids were 22 (GA), 25 (AA), 31 (SubA) and 54 (SebA) fmol on column. Furthermore, the present method was so selective that biogenic monocarboxylic acids gave no peak in the chromatogram.

在1-乙基-3-（3-二甲氨基丙基）碳二亚胺存在下，芘试剂4-（1-芘）丁酸酰肼（PBH）与二羧酸形成分子内准分子荧光衍生，反相高效液相色谱（HPLC）测定二羧酸。在分子中具有两个羧酸部分的二羧酸通过与PBH反应（40 ℃，60分钟）转化为相应的联芘标记的衍生物。衍生物提供分子内激基缔合物荧光（450-520 nm），其可以清楚地与从PBH和单芘标记的单羧酸衍生物发射的单体（正常）荧光（360-420 nm）区分开。衍生物的结构经高效液相色谱-质谱联用确证，激发态荧光光谱和时间分辨荧光光谱证实其发射。采用反相高效液相色谱法，在C8柱上，线性梯度洗脱，可分离戊二酸（GA）、己二酸（AA）、辛二酸（SubA）和癸二酸（SebA）4种二元羧酸的PBH衍生物。二羧酸的检测限（信噪比为3）分别为22（GA）、25（AA）、31（SubA）和54（SebA）fmol。此外，本方法是如此的选择性，生物源的一元羧酸在色谱图中没有峰。