新型荧光试剂柱前衍生高效液相色谱定量分析小鼠脑脊液中氨基酸类神经递质  

Determination of amino acid neurotransmitters in mouse cerebrospinal fluid using novel fluorescent reagent by high performance liquid chromatography with pre-column derivatization

作　　者：李金淑[1,2] 张华山[2] 王红[2] 

机构地区：[1]中央民族大学生命与环境科学学院,北京100081 [2]武汉大学化学与分子科学学院,武汉430072

出　　处：《国际药学研究杂志》2012年第1期65-70,共6页Journal of International Pharmaceutical Research

基　　金：国家自然科学基金项目(30770550);国家民委科研项目(09ZY011);中央民族大学青年教师科研基金项目(MZ2008033)

摘　　要：目的采用新合成的荧光试剂柱前衍生氨基酸类神经递质,建立其测定方法,并测定小鼠脑脊液中氨基酸类神经递质的含量。方法以1,3,5,7-四甲基-8-苯基-（4′-O-（N-琥珀酰亚胺乙酸酯））-二氟化硼-二吡咯甲烷（TMPAB-OSu）作为柱前荧光衍生试剂,在pH 8.8的硼酸-硼砂缓冲溶液中,30℃衍生反应5 min后获得稳定的荧光衍生产物,在反相色谱柱上,采用梯度洗脱进行5种氨基酸类神经递质标准品衍生物的分离检测,荧光激发波长为497 nm,检测波长为509 nm。结果5种氨基酸类神经递质的线性范围为0.01~1.2μmol/L,线性回归系数均大于0.998 1,检出限为1 nmol/L（S/N=3∶1）。小鼠脑脊液中天门冬氨酸、谷氨酸、谷氨酰胺、γ-氨基丁酸和丙氨酸的含量分别为0.06、0.20、0.26、0.03和0.07μmol/L,标准加入回收率为96%~104%,RSD为0.7%~2.9%。结论该方法的灵敏度高、重现性好,为小鼠脑脊液中氨基酸类神经递质的分析检测提供了一种新方法。Objective To establish a method for amino acid neurotransmitters content determination using novel fluorescent reagent with pre-column derivatization and determine the content of amino acid neurotransmitters in cerebrospinal fluid of the mouse.Methods Amino acids were separated with a gradient elution after derivatization with 8-phenyl-（4-oxy-acetic acid N-hydroxysuccinimide ester）-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene（TMPAB-OSu）.Optimum derivatization,giving the corresponding stable fluorescent derivatives,was obtained by reaction of amino acids with TMPABOSu at 30℃ for 5 min in borate buffer（pH 8.8）.A simple and sensitive method for the quantification of amino acid neurotransmitters in cerebrospinal fluid of the mouse using HPLC with fluorescence detection at 509 nm（excitation wavelength 497 nm） was developed.Results The linear range of 5 amino acid neurotransmitters were 0.01-1.2 μmol/L,all correlation coefficients 0.998 1,detection limit was 1 nmol/L（at signal to noise 3∶1）.It is indicated that the contents of asparagic acid,glutamic acid,glutamine,γ-aminobutyric acid and alanine in cerebrospinal fluid of the mouse were 0.06,0.20,0.26,0.03 and 0.07 μmol/L,respectivey.The average recoveries of amino acids were 96%-104%.The precisions were 0.7%-2.9%.Conclusion The established method exhibits high sensitivity and excellent reproducibility,and provides a new technology for the determination of amino acids in cerebrospinal fluid of the mouse.

关 键 词：高效液相色谱 柱前衍生 荧光试剂 氨基酸类神经递质 分析检测 

分 类 号：R96[医药卫生—药理学]