Highly Selective Fluorometric Determination of Polyamines Based on Intramolecular Excimer-Forming Derivatization with a Pyrene-Labeling Reagent

基于芘试剂4-（1-芘）丁酸N-羟基琥珀酰亚胺酯（PSE）的分子内激基体形成荧光衍生化，建立了一种高选择性、高灵敏度的多胺反相高效液相色谱（HPLC）测定方法。分子中含有2 ~ 4个氨基的多胺与PSE反应（100℃，20 min），转化为相应的二芘-四芘标记衍生物。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PSE、其水解物和单芘标记的单胺衍生物发射的单体（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。在C8柱上，采用反相高效液相色谱线性梯度洗脱，可分离腐胺（Put）、尸胺（Cad）、亚精胺（Spd）和精胺（Spm）4种多胺的PSE衍生物。多胺的检测限（信噪比为3）分别为1.0（Put）、1.0（Cad）、4.7（Spd）和7.0（Spm）fmol。此外，本方法是如此的选择性，生物单胺在色谱图中没有峰。

A highly selective and sensitive method was developed to determine polyamines, based on intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene) butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed phase high performance liquid chromatography (HPLC). Polyamines having two to four amino moieties in a molecule, were converted to the corresponding dipyrene-to tetrapyrene-labeled derivatives by reaction (100℃ ,20min) with PSE. The derivatives afforded intramolecular excimer fluorescence (450-520 nm) which can clearly be discriminated from monomer (normal) fluorescence (360-420 nm) emitted from PSE, its hydrolysate and monopyrene-labeled derivatives of monoamines. 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 PSE derivatives of four polyamines [putrescine (Put), cadaverine (Cad), spermidine (Spd) and spermine (Spm)] 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 polyamines were 1.0 (Put), 1.0 (Cad), 4.7 (Spd) and 7.0 (Spm) fmol on column. Furthermore, the present method was so selective that biogenic monoamines gave no peak in the chromatogram.