反相高效液相色谱柱前衍生荧光检测法测定游离多胺

Determination of Polyamines by High Performance Liquid Chromatography-Mass Spectrometry with Fluorescence and Atmospheric Pressure Chemical Ionization Detection

合成新型荧光试剂1,2-苯并-3,4-二氢咔唑-9-异丙基氯甲酸酯(BCIC)作为柱前衍生化试剂,在Eclipse XDB-C8色谱柱上,通过梯度洗脱对5种多胺进行了分离和在线质谱定性。在乙腈中,以硼酸钠缓冲液(pH9)为催化剂,40℃下衍生反应10min后获得稳定的荧光产物。激发和发射波长分别为λex=333nm,λem=390nm。采用大气压化学电离源(APCI)正离子模式,实现了人尿中游离多胺的定性及定量测定。多胺的线性相关系数大于0.9996,检出限为5.3～9.9fmol。

A simple and highly sensitive method was developed based on the derivatization of polyamines utilizing1,2-benzo-3,4-dihydrocarbazole-9-isopropyl chloroformate（BCIC） as the novel pre-column derivatization reagent followed by high performance liquid chromatography with fluorescence detection and online mass spectrometric identification.Optimum derivatization was obtained by the reaction of polyamines with BCIC at 40 ℃ for 10 min in the presence of sodium borate（pH 9.0） in acetonitrile solvent.Derivatives were sufficiently stable to be efficiently analyzed by high performance chromatography without pretreatment.On a reversed-phase XDB-C8 column,five kinds of polyamines derivatives were separated in conjunction with a gradient elution with a good baseline resolution.The precise determination of polyamines from urine sample was obtained by post-column online mass spectrometric identification with atmospheric pressure chemical ionization source under positive mode detection.The established method exhibited excellent reproducibility and recovery.Linear responses were observed with the correlation coefficients of 〉0.9996.The detection limits（at signal-to-noise of 3∶1） were 5.3～9.9 fmol.