摘要
建立了测定血浆中总同型半胱氨酸的柱前衍生、高效液相色谱荧光检测的分析方法。以Bromobimane作荧光剂,对巯基进行衍生。同型半胱氨酸的最低检测浓度为0.5μmolL,线性浓度范围是2.5～80.0μmolL,回收率为94.0%～112.0%,批内、批间相对标准偏差都小于5.6%。
This article report a highly sensitive method specific for the determination of homocysteine in plasma by high performance liquid chromatography with fluorescence detection. Half mL plasma with 100 μL 0.11 mol/L sodium borohydride in 50 mmol/L Tris\|HCl (pH 9.0) was kept at 30 ℃ for 30 min, and then 0.5 mL 0.5 mol/L perchloric acid was added. After the mixture was kept at room temperature for 10 min and centrifuged at 15 000 r/min for 10 min, 0.5 mL aliquots of the supernatant solution was pipetted into another vial containing 0.1 mL 3 mmol/L Bromobimane in 1.0 mol/L Na 2EDTA (pH 7.0) and 0.7 mL of 90 mmol/L ammonium bicarbonate buffer containing 1.43 mol/L Na 2EDTA, pH 8.0. The content was mixed, kept at 37 ℃ for 30 min and centrifuged at 15 000 r/min for 10 min. Then 10 μL aliquots of the supernatant solution was injected into a high performance liquid chromatograph with fluorescence detector. The chromatographic conditions were as follows: an ODS column (4.6 mm i.d.×150 mm ,5 μm), was eluted with a flow rate of 1.0 mL/min. The fluorescence detector was operated at λ ex 365 nm and λ em 475 nm. Mobile phase frompump A was 3% methanol containing 0.25% acetic acid. In gradient elution program the methanol from pump B was as follows: 0\|8 min, 5%; 8\|15 min, 5%\|12%; 15\|20 min, 12%; 20\|30 min, 12%\|20%; 30\|32 min, 20%; 32\|35 min, 20%\|100%; 35\|40 min, 100%; 40\|43 min, 100%\|5%; 43\|45 min, 5%. The method proved to be linear in the range of 2.5\|80.0 μmol/L with a regression coefficient of 0.998 8. The minimum detection limit was 0.5 μmol/L, the recoveries were 94.0%\|112.0%, and the RSD values were less than 5.6%.
出处
《色谱》
CAS
CSCD
北大核心
2000年第1期49-51,共3页
Chinese Journal of Chromatography
基金
广东省科委攻关项目!资助 (962 2 0 5 2 0 1)
关键词
高效液相色谱法
荧光检测
同型半胱氨酸
血浆
high performance liquid chromatography
fluorescence detection
homocysteine
