一种新的柱前衍生法测定血浆同型半胱氨酸

A new precolumn derivatization method for the determination of homocysteine in plasma by high performance liquid chromatography

目的 :建立一种新的高效液相柱前衍生法 ,准确、快速测定血浆同型半胱氨酸 (HCY)浓度。方法 :血中结合态的HCY经三丁基膦还原为游离HCY后 ,与衍生试剂 7 氟苯唑 2 氧 1,3 二唑 4 磺酸胺 (ABD F)进行柱前衍生反应 ,衍生物经反相C18色谱柱 ,用 0 .15mol/L磷酸盐缓冲液 (pH =4.2 )与甲醇按 85∶15配制的混合液洗脱。荧光检测激发λ =385nm ,发射λ =5 15nm。结果 :在 3.12 5～ 10 0 .0 0 μmol/L范围内 ,HCY浓度与荧光强度的线性关系良好 ,γ =0 .9998,测定的批内、批间变异系数分别为 3 .13 %和 5 .10 % ,高低 2种浓度的回收率分别为 98.5 %和 92 .7% ,健康人血浆HCY的参考值为 8.12± 2 .72 μmol/L。 结论 :本法测定血浆HCY准确快速、特异性高、分离效果好 ,适用于临床血浆HCY测定的常规需要。

Objective:To establish a new precolumn derivatization method with high performance liquid chromatography for rapid and accurate determination of the concentration of homocysteine in human plasma.Methods:After homocysteine was liberated from its protein bound by tri n butylphosphine,all free form homocysteine were precolumn derivatized with 7 fluorobenzo 2 oxa 1,3 diazole 4 sulfonamide(ABD F)and eluted with 0.15 mol/L phosphate buffer(pH=4.2)containing 15% methanol.The analyses,carried out on a reversed phase C 18 column,were based on spectrofluorimetric detection.Excitation at 385 nm and emission at 515 nm.Results:The assay was linear from 3.125 to 100.00 μmol/L homocysteine.Correlation factor was 0.9998,within and between was 3.18% and 5.15%,the mean recovery of homocysteine added to two plasma samples was 98.5% and 92.7%,and the normal values in health adults were 8.12±2.72 μmol/L.Conclusion:This method was accurate,rapid and specific and suitable for clinical measurement of homocysteine.