Development of Ultra-sensitive HPLC Determination Method for Histamine Based on Intramolecular Excimer-forming Fluorescence Derivatization and Its Applications to Clinical and Neuroscience Investigations

基于分子内准分子形成荧光衍生化，建立了组胺相关化合物灵敏、选择性的高效液相色谱检测方法。首先筛选了合适的试剂，包括新试剂的设计，并对衍生化和高效液相条件进行了优化，以组胺标准进行初步检测。经验证，该方法比以往的方法具有更高的选择性和灵敏度，且前处理步骤繁琐。为此，开展了以下应用研究：(1)人尿，建立的方法无需任何前处理即可准确测定L尿液中仅1μ的组胺含量。(2)大鼠血浆和组织：用准分子荧光衍生化方法测定大鼠血浆和组织(腹部皮肤、肺、胃、心脏和肝脏)经简单预处理后的组胺水平。血浆和组织的测定值与其他工作人员的测定值吻合较好。(3)大鼠脑组织的微透析标本与微透析技术相结合，可对大鼠脑组织中组胺进行实时监测。研究了下丘脑、前额叶皮质、海马体和纹状体等脑区组胺含量与药物影响的关系，建立了基于分子内准分子形成荧光衍生法的组胺和组胺前体组氨酸的同时测定方法。该方法具有较高的灵敏度和选择性，适用于临床和食品卫生领域。总之，本衍生化技术可以简便、选择性和灵敏地测定组胺相关化合物。

Sensitive and selective HPLC determination methods for histamine related compounds based on intramolecular excimer-forming fluorescence derivatization have been developed. At first, the suitable reagent was screened including the design of novel reagents, and derivatization and HPLC conditions were optimized for the preliminary examinations using histamine standard. By the validation tests, the proposed method is more selective and sensitive than the previous methods with tedious pretreatment procedure. Therefore, the following application studies were carried out.(1) Human urine ; the amounts of histamine in only 1 μL of human urine were determined by the established method with high accuracy without any pretreatment. (2) Rat plasma and tissues; the histamine levels in rat plasma and tissues (abdominal skin, lung, stomach, heart and liver) were measured using excimer fluorescence derivatization method after a simple pretreatment. The obtained values in plasma and tissues were in good agreements with those reported by other workers. (3) Microdialysis samples from rat brain ; real-time monitoring of histamine in rat brain could be carried out by combined with microdialysis techniques. The relationships between the amounts of histamine in some regions of brain (hypothalamus, prefrontal cortex, hippocampus and striatum) and the influence of drugs were investigated.At last, simultaneous determination method of histamine and histidine (precursor of histamine) based on intramolecular excimer-forming fluorescence derivatization was developed. This method was highly sensitive and selective, and useful for clinical and food sanitary fields.In conclusion, the present derivatization technique allows simple, selective and sensitive determination of histamine related compounds.