高效液相色谱荧光法检测血浆中硫醇物的方法学研究

摘要

Objective To establish a rapid high performance liquid chromatography(HPLC)method for simultaneous determination of the concentration of total,free and reduced homocysteine (Hcy),glutathione(GSH),cysteine (Cys)and cys-teinylglycine (CysGly). Methods HPLC fluorescence detection method was established under the below conditions. The axci-tation and emission wavelengths was 330 nm and 380 nm respectively;the separation of thiols was achieved by using a C-18 column and the column temperature was 25 ℃;the mobile phase was gradient eluted with the three carboxyl ethyl phosphine (TCEP)as reducing agent and N-1- phenyl maleimide (NPM)as derivatization agent. The HPLC fluorescence detection meth-od was used to measure the thiol concentration in plasma of uraemia patients and healthy people. Results The linear range of total and free Hcy,GSH,Cys and CysGly were 1. 0 - 120. 0,2. 0 - 80. 0,10. 0 - 1500. 0 and 3. 0 - 240. 0 μmol·L - 1 respec-tively;the linear range of reduced Hcy,GSH,Cys and CysGly was 1. 25 - 50. 00,0. 10 - 8. 00,1. 25 - 50. 00 and 0. 01 -4. 00 μmol·L - 1 respectively. The intra-and inter-day ralative standard deviation were less than 5％;the recovery of this meth-od was 80. 1％ - 111. 7％ . The newly established HPLC fluorescence detection method was successfully applied to determine the total,free and reduced concentration of GSH,Cys,Hcy and CysGly in 34 uraemia patients and 32 healthy people. Conclu-sion A new HPLC fluorescence detection method for the determination of Hcy,GSH,Cys,and CysGly in plasma is developed and this method is accurate and reliable.%目的 建立一种可同时测定血浆中同型半胱氨酸(Hcy)、谷胱甘肽(GSH)、半胱氨酸(Cys)及半胱氨酸甘氨酸(CysGly)的总量及其游离型和还原型浓度的高效液相色谱(HPLC)荧光检测方法.方法 采用C-18柱分离,柱温25℃,激发波长330 nm,发射波长380 nm,流动相采用梯度洗脱,以三羧基乙基膦(TCEP)为还原剂,N-1-苯基-马来酰亚胺(NPM)为衍生剂建立HPLC荧光检测方法.将建立的HPLC荧光检测方法用于测量尿毒症患者和健康人群血浆中各种类型硫醇物浓度.结果 总量和游离型Hcy、GSH、Cys和CysGly线性范围分别为1.0～120.0、2.0～80.0、10.0～1500.0和3.0～240.0μmol·L-1,还原型Hcy、GSH、Cys和CysGly线性范围分别为1.25～50.00、0.10～8.00、1.25～50.00和0.01～4.00μmol·L-1;日内和日间变异均小于5％,回收率在80.1％ ～111.7％.该方法成功应用于34例尿毒症患者和32例健康人血浆中GSH、Cys、Hcy和CysGly的总量、游离型及还原型浓度的测定.结论 建立了一种新的可同时测定血浆中Hcy、GSH、Cys及CysGly的HPLC荧光检测方法,该方法准确可靠.