高效液相色谱-程序波长紫外检测法同时测定血浆中的色氨酸及其代谢物

摘要

A high performance liquid chromatographic (HPLC) method with programmed wavelength ultraviolet (UV) detection was established to simultaneously determine tryptophan (Trp) and its key metabolites kynurenine (Kyn) and 5-hydroxytryptamine (5-HT). A BDSHypersil-C8 column (150 mm ×4. 6 mm, 5 μm) was used for the analysis at 25 ℃. The separation was carried out with the mobile phase consisting of 10 mmol/L sodium acetate-acetic acid ( pH 4. 5 ) and acetonitrile (94: 6, v/v) using theophylline as internal standard (IS) at a flow rate of 0. 6 mL/min. The eluates were monitored by the programmed wavelength UV detection at 360 nm for Kyn and IS, 220 nm for 5-HT and 302 nm for Trp. The mean recoveries were in the range of 87％ to 113％. The linearities were from 3.97 to 400 μmol/L for Trp, 0. 421 to 20. 2 μmol/L for Kyn and 4. 36 to 980. 5 nmol/L for 5-HT. The detection limits were 0. 134 μmol/L for Trp, 0. 016 μmol/L for Kyn and 2.03 nmol/L for 5-HT. Fifteen plasma samples of patients with depression and fifteen plasma samples of healthy controls were tested, and the results demonstrated that the metabolism of Trp in the plasma from the depression group was significantly different from that of the control group.%建立了一种高效液相色谱-程序波长紫外检测法同时测定血浆中色氨酸(Trp)及其主要代谢产物犬尿氨酸(Kyn)和5-羟色胺(5-HT).以茶碱为内标(IS),采用BDS-Hypersil-C8柱(150 mm×4.6 mm,5 μm)分离.流动相为10 mmol/L醋酸钠缓冲液(pH 4.5)-乙腈(94:6,v/v),流速为0.6 mL/min;柱温为25℃;紫外检测波长设定:Kyn和IS为360 nm,5-HT为220 nm,Trp为302 nm.3种物质的平均回收率为87%～113%;线性范围分别为3.97～400 μmol/L(Trp),0.421～20.2 μmol/L(Kyn),4.36～980 nmol/L(5-HT);检出限分别为0.134 μmol/L(Trp),0.0160 μmol/L(Kyn),2.03 nmol/L(5-HT).利用该方法对15例抑郁症患者和15例健康志愿者的血浆进行测定,结果表明两组间Trp的代谢存在显著的差异.