柱前衍生-高效液相色谱分离测定及质谱鉴定脂肪胺

Pre-column Derivatization-High Performance Liquid Chromatography for the Determination of Aliphatic Amines with Fluorescence Detection and Mass Spectrometric Identification

采用新型荧光衍生试剂2-（2-苯基-1-氢-菲[9,10-d]咪唑）-乙酸（PPIA）进行柱前衍生,经荧光检测实现了脂肪胺的高效液相色谱（HPLC）分离测定及柱后质谱鉴定.60℃下在乙腈溶剂中用N-乙基-N′-[ （3-二甲氨基） 丙基 ]碳二亚胺盐酸盐（EDC）做缩合剂,衍生反应15 min可获得稳定的荧光产物.脂肪胺衍生物荧光检测波长为380 nm（激发波长为260 nm）.在Eclipse XDB-C8 色谱柱上,采用梯度洗脱对12种脂肪胺衍生物进行了优化分离,测定了造纸厂废水、大鼠端脑和酸奶中脂肪胺的含量.经柱后在线质谱大气压化学电离源（APCI Source）正离子模式实现了各种脂肪胺衍生物的质谱鉴定,借助对活性中间体的质谱解析确定了衍生反应的反应机理.该方法具有良好的重现性和回收率,多数脂肪胺的线性回归系数大于0.9996;检出限为3.1～18 fmol （S/N＝3∶ 1）.

A simple and highly sensitive method based on the derivatization of aliphatic amines with 2-（2- phenyl-1H-phenanthro-[9,10-d] imidazole-1-yl）-acetic acid （PPIA） followed by high performance liquid chromatography with fluorescence detection and on-line post-column mass spectrometric identification has been developed. Optimum derivatization, giving the corresponding stable fluorescent derivatives, was obtained by reacting of aliphatic amines with PPIA at 60℃ for 15 min in the presence of N-ethyl-N＇-（ 3-dimethylamino propyl） carbodiimide hydrochloride （EDC） catalyst in acetonitrile solvent. The fluorescence detection wavelength of PPIA derivatives was 380 nm （excitation wavelength 260 nm）. Separation of the derivatized amines with a good baseline resolution in conjunction with a gradient elution was carried out on a reversed phase Eclipse XDB-C8 column （4.6 mm × 150 mm, 5 μm）, in this way, the content of free fatty amines in waste water of paper mill, telencephalon tissue of rat and acidophilus milk was determined. The identification of aliphatic amine derivatives was carried out by post-column online mass spectrometry with atmospheric pressure chemical ionization source under positive-ion mode detection. The established method exhibits excellent reproducibility and recovery. Excellent linear responses are observed with regression coefficients 〉 0. 9996, detection limits （ at signal-to-noise of 3： 1 ） are 3.1 - 18 fmol.