[1]班艳娜,甄乾娜,傅惠佳.高效液相色谱荧光法检测血浆中硫醇物的方法学研究[J].新乡医学院学报,2018,35(6):459-463.[doi:10.7683/xxyxyxb.2018.06.003]
　BAN Yan-na,ZHEN Qian-na,FU Hui-jia.Methodology research of determination of thiols in plasma by high performance liquid chromatography with fluorescence[J].Journal of Xinxiang Medical University,2018,35(6):459-463.[doi:10.7683/xxyxyxb.2018.06.003]
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高效液相色谱荧光法检测血浆中硫醇物的方法学研究
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
35
期数:
2018年6
页码:
459-463
栏目:
基础研究
出版日期:
2018-06-05

文章信息/Info

Title:
Methodology research of determination of thiols in plasma by high performance liquid chromatography with fluorescence
作者:
班艳娜1; 甄乾娜2; 傅惠佳1
(1.重庆医科大学附属第一医院生殖健康与不孕症专科,重庆　400016;2.重庆医科大学附属第一医院内分泌科,重庆　400016)
Author(s):
BAN Yan-na1; ZHEN Qian-na2; FU Hui-jia1
（1.Department of Laboratory of Health Reproduction and Infertility,the First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China;2.Department of Laboratory Endocrinology,the First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China）
关键词:
尿毒症; 硫醇物; 高效液相色谱法
Keywords:
uremia; aminothiols; high performance liquid chromatography
分类号:
R33-33
DOI:
10.7683/xxyxyxb.2018.06.003
文献标志码:
A
摘要:
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、10.0~1 500.0 和3.0~240.0 μmol·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0.01~4.00 μmol·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。
Abstract:
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 cysteinylglycine (CysGly).Methods　HPLC fluorescence detection method was established under the below conditions.The axcitation 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 method 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 1.0-120.0,2.0-80.0,10.0-1 500.0 and 3.0-240.0 μmol·L-1 respectively;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 method 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.Conclusion　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.

参考文献/References:

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更新日期/Last Update: 2018-06-05