高效液相色谱法测定尿液中的5-羟色醇：一种新型柱后衍生荧光检测法的应用

Determination of 5-hydroxytryptophol in urine by high-performance liquid chromatography: application of a new post-column derivatization method with fluorometric detection.

作者信息

Helander A, Löwenmo C, Beck O

机构信息

Karolinska Institute, Department of Clinical Neuroscience, St Görans Hospital, Stockholm, Sweden.

出版信息

J Pharm Biomed Anal. 1995 Apr;13(4-5):651-4. doi: 10.1016/0731-7085(95)01287-u.

DOI:10.1016/0731-7085(95)01287-u

PMID:9696582

Abstract

The aim of the present study was to develop a high-performance liquid chromatographic (HPLC) method for determination of the serotonin metabolite 5-hydroxytryptophol (5HTOL) in human urine. 5HTOL was liberated from its conjugated form by enzymatic hydrolysis and isolated by a sample clean-up procedure on a small Sephadex G-10 column. The eluate was injected onto an isocratically eluted C18 reversed-phase column and 5HTOL was converted into a fluorescent oxazole derivative by on-line post-column reaction with benzylamine in the presence of potassium hexacyanoferrate(III). The limit of detection was about 10 nM and the intra-assay coefficients of variation were below 4% with urine samples and standard solutions. The results indicate that the method can be used as a screening method to discriminate between normal and elevated levels of total (free + conjugated) 5HTOL in urine.

摘要

本研究的目的是开发一种高效液相色谱（HPLC）方法，用于测定人尿中血清素代谢物5-羟色醇（5HTOL）。5HTOL通过酶促水解从其共轭形式中释放出来，并通过在小型Sephadex G-10柱上的样品净化程序进行分离。将洗脱液注入等度洗脱的C18反相柱，在六氰合铁（III）酸钾存在下，5HTOL通过与苄胺的在线柱后反应转化为荧光恶唑衍生物。检测限约为10 nM，尿样和标准溶液的批内变异系数低于4%。结果表明，该方法可作为一种筛选方法，用于区分尿中总（游离+共轭）5HTOL的正常水平和升高水平。