人血浆中高半胱氨酸水平的测定方法研究

HPLC determination of homocysteine in the human plasma

目的研究一种改良后HPLC法测定人血浆中高半胱氨酸(homocysteine,HCY)水平的方法。方法取人血浆100μL加10μL水、100μL正戊醇以及单溴二胺在50℃水浴反应12min,经调pH、离心分离后,取10μL进样分析测定。色谱条件采用NovaPakC18分离柱,流速1.00mL·min-1,梯度洗脱,荧光法检测,激发波长385nm,发射波长515nm。结果高半胱氨酸在4.00～40μmol·L-1浓度(C)与峰面积(Y)呈良好的线性关系,线性方程为Y=1.9237×106C-4.2714×10-2(r=0.9993)。本法测定高半胱氨酸最低限浓度为2.67μmol·L-1,平均回收率RSD为4.23%,日内、日间平均RSD分别为3.84%和4.54%。结论本法操作快速、分析时间短,可用于人血浆中高半胱氨酸水平的测定。

OBJECTIVE To recommend a new determination method for homocysteine in the human plasma by HPLC. METHODS Plasma samples (100 μL) reacted with n-amyl alcohol and monobromobimane for 12 min at 50 ℃.After centrifugation and pH adjustment,10 μL of the sample was analyzed by HPLC on Nova-pak C_ 18 column,0.05 mol·L~ -1 KH_2PO_4 with gradient mobile phase for methanol. The flow rate was 1.00 mL·min~ -1. The active contents were detected with the excitation wavelength at 385 nm and the emission wavelength at 515 nm.RESULTS The calibration curves were linear in the range of 4.00～40.00 μmol·L~ -1 for homocysteine in plasma Y=1.923 7×10~6C-4.271 4×10~ -2 (r=0.999 3). The detection limit of homocysteine in plasma was 2.67 μmol·L~ -1. The average RSD of recovery was 4.23%.The intra-day and inter-day RSD were 3.84% and 4.54% respectively. CONCLUSIONS The method is simple rapid,and suitable for the determination of homocysteine in the human plasma.