Methodology research of determination of thiols in plasma by high performance liquid chromatogra-phy with fluorescence / 新乡医学院学报

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 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.