柱前衍生高效液相色谱-荧光检测法测定血浆中同型半胱氨酸

建立了一种柱前衍生高效液相色谱-荧光检测法用于测定血浆中同型半胱氨酸(Hcy)。使用三(2-羧乙基)膦盐酸盐(TCEP)为还原剂,N-(1-芘)马来酰亚胺(NPM)为衍生剂进行样品预处理,Agilent Hypersil C-18柱(250 mm×4.0 mm, 5 μm)进行分离,流动相为15 mmol/L醋酸钠-乙腈-混合酸(300 mL水中含1 mL醋酸和1 mL磷酸)混合溶液,采用梯度洗脱,荧光检测激发波长为330 nm,发射波长为380 nm。Hcy的回收率为(102.08±4.94)%。线性范围为0.500～100 μmol/L,检出限（以信噪比为3计）为0.016 μmol/L。日内与日间相对标准偏差均小于5%。利用该方法对7例高血压患者和7例健康志愿者的血浆进行了测定,结果表明两组间的Hcy含量存在显著的差异(p＜0.05)。本方法简单、快速、灵敏、特异,适用于血浆Hcy的临床定量测定。

Determination of homocysteine in plasma by precolumn derivatization-high performance liquid chromatography with fluorescence detection

A precolumn derivatization-high performance liquid chromatographic method for the determination of homocysteine(Hcy) in plasma was established.Tris(2-carboxyethyl)phosphine hydrochloride(TCEP) and N-(1-pyrenyl)maleimide(NPM) were used as the reduced reagent and derivatization reagent,respectively.The separation was carried out on an Agilent Hypersil C-18 column(250 mm×4.0 mm,5 μm) in gradient elution mode.The mobile phase consisted of A(15 mmol/L sodium acetate solution),B(acetonitrile) and C(300 mL water containing 1 mL acetic acid and 1 mL phosphoric acid).The eluate was monitored by the fluorescence detector at an excitation wavelength of 330 nm and an emission wavelength of 380 nm.The mean recovery of Hcy was(102.08±4.94)%.The linear range was from 0.500 μmol/L to 100 μmol/L,with a detection limit of 0.016 μmol/L.The intra-day and inter-day relative standard deviations(RSDs) for Hcy were less than 5%.Seven plasma samples of patients with hypertension and seven plasma samples of healthy controls were tested,and the results demonstrated that the Hcy in the plasma from the hypertension group was significantly different from that of the control group(p<0.05).The developed method is simple,fast,accurate,and suitable for clinical measurement.