高效液相色谱法同时测定血浆同型半胱氨酸及其相关硫醇物浓度方法的建立

Simultaneous determination of homocysteine and other related thiols in plasma by high performance liquid chromatography

目的建立一种柱前衍生反相高效液相色谱荧光检测法同时测定血浆同型半胱氨酸(homocysteine,Hcy)、半胱氨酸(cysteine,Cys)、半胱氨酰甘氨酸(cysteinylglycine,CysGly)、谷胱甘肽(glutathione,GSH)的方法。方法以tris(2carboxylethyl)phosphine(TCEP)为还原剂,7fluorbenzo2oxa1,3diazole4sulfonate(SBDF)为衍生剂,N乙酰半胱氨酸(Nacetylcystine,NAC)为内标,C8柱分离。流动相为甲醇磷酸盐缓冲液(25mmol/L,pH3.0),梯度洗脱,激发波长(λex)380nm,发射波长(λem)510nm,内标标准曲线法定量。结果Hcy、Cys、CysGly、GSH的线性范围分别为1.0～160.0μmol/L,30.0～1040.0μmol/L,2.0～300.0μmol/L,1.0～130.0μmol/L,具有良好的相关性,r为0.9994～0.9999。最低检测限为0.1～2.0μmol/L。日内精密度不超过3.0%,日间精密度除GSH为13.21%外均小于6.0%。平均回收率为87.24%～114.99%。结论本方法准确、灵敏、快速、特异,适合于实验室研究和临床常规检测。

Objective A method for the determination homocysteine （Hcy）, cysteine （Cys）, cysteinylglycine （CysGly）, glutathione （GSH） in plasma by reverse phase high performance liquidchromatography and Fluorescence Detection by pre-column derivatization was developed. Methods Thiols inplasma and internal standard N-acetylcysteirea were reduced by tfis-（2-carboxylethyl）-phosphine （TCEP）,then derivated by 7-fluorbenzo-2-oxa-1,3-diazole-4-sulfonate （SBD-F）. The derivatives were separated using a methanol/phosphate buffer solution （25 mmol/L, pH 3.0） gradient on Cs column and detected at ）rex 380nm, gem 510 nm. Results The linearitys of the assay of Hey, Cys, CysGly, GSH were 1.0-160. 0μmol/L, 30. 0-1040.0μmol/L, 2. 0-300. 0μmol/L, 1.0-130.0μmol/L respectively, the regressions ranged from0. 999 4 to 0. 999 9, for the thlols, the detection limit ranged from 0. 1 to 2. 0μmol/L, the withln-dayprecision were 〈 3.0%, The between-day precision were 〈 6.0% except the GSH was 13.21%, theaverage recoveries were 87.24%-114.99%. Conclusion This method is accurate, sensitive, rapid,specific and suitable for the routine clinical practice and the research practice.