高效液相(HPLC)荧光法同时测定人血浆中4种硫醇物的浓度

Simultaneous determination of four aminothiols in human plasma by high-performance liquid chromatography (HPLC) with fluorimetric detector

目的建立同时测定人血浆中半胱氨酸（cysteine，Cys）、同型半胱氨酸（homocysteine，Hcy）、半胱氨酰甘氨酸（cysteinylglycine，CysGly）和谷胱甘肽（glutathione，GSH）等4种硫醇物的高效液相色谱法（high-perfofinance liquid chromatography，HPLC）。方法血浆经磷酸盐缓冲液（phosphate buffered saline，PBS）稀释后，用三（2-羧乙基）膦盐酸盐[tris-（2-carboxyethyl）-phosphine hydrochloride，TCEP]还原，用三氯乙酸（trichloroacetic acid，TCA）溶液进行蛋白沉淀，再用7-氟苯呋咱-4-硫酸铵盐（7-fluorobenzofurazan-4-sulfonic acid ammonium salt，SBD-F）进行衍生化反应，荧光检测器检测。色谱条件：色谱柱为Kromasil C18（250mm×4．6mm，5μm），柱温29℃；流动相为甲醇：0．1mol／L醋酸盐缓冲液（pH=4．5，3．5：96．5，V／V），流速为o．8mL／min，等度洗脱。激发波长385nm，发射波长515nm。采用外标法定量。结果Cys、Hcy、CysGly和GSH的线性范围分别为50-800μmol／L、4～64／μmol／L10～160／μmol／L和2．5～40μmol／L。Cys、Hcy、CysGty和GSH的最低检测浓度分别为2．5／μmol／L、1.0μmol／L1．0／lmol／L和1．0μmol／L。各组分日内、日间精密度RSD均〈12％。平均回收率为95．01％～116．17％。结论该方法具有检测时间短、灵敏性高、精密度和准确度好的特点，适用于临床人血浆中硫醇物浓度的常规检测。

Objective To develop a simple high-performance liquid chromatography （HPLC） with fluorimetric dectetor method for simultaneously determining total concentrations of four aminothiols including aminothiols, homocysteine （Hcy）, cysteine （Cys）, cysteinylglycine （CysGly） and glutathione （GSH） in human plasma. Methods The human plasma was diluted by phosphate buffered saline （PBS）, reduced by tris-（ 2-carboxyethyl ）-hosphine hydrochloride （ TCEP ）, derivated by 7-fluorobenzofurazan-4-sulfonic acid ammonium salt （SBD-F）,and detected by fluorimetric detector. The HPLC separation was carried out using a Kromasil C,8 （250 mm x 4.6 mm, 5 gm）. The mobile phase was consisted of methanol：0.1 mol/L acetate buffer （pH = 4.5,3.5：96.5 ,V/V） at a flow rate o{ 0.8 mL/min, delivered isocratically. The fluorimetrie detector was set at excitation and emission wavelengths of 385 nm and 515 nm,respectively. The assay utilizes a quantification method of external standard. Results The calibration graphs were linear over 50 - 800 μmol/L,4 - 64 μmol/L, 10 - 160μmol/L and 2.5 -40 μmol/L for Cys, Hcy, CysGly and GSH, respectively. The lowest limits of quantification for Cys, Hcy, CysGly and GSH were 2. 5 μmol/L, 1. 0 μmol/L, 1.0 μmot/L,and 1. 0 gmol/L,respectively. The intra-and inter-day precision of RSD was below 12%. The average recovery was ranged from 95.01% to 116.17 M. Conclusions The method was proved to be rapid,sensitive, precise, accurate, and suitable for routine clinical determination of aminothiols in human plasma.