采用氟代和荧光衍生化的5-羟基吲哚选择性灵敏液相色谱测定法

Selective and sensitive liquid chromatographic determination method of 5-hydroxyindoles with fluorous and fluorogenic derivatization.

作者信息

Sakaguchi Yohei, Ikenaga Jun, Yoshida Hideyuki, Hayama Tadashi, Itoyama Miki, Todoroki Kenichiro, Imakyure Osamu, Yamaguchi Masatoshi, Nohta Hitoshi

机构信息

Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan.

出版信息

J Pharm Biomed Anal. 2015 Oct 10;114:348-54. doi: 10.1016/j.jpba.2015.06.003. Epub 2015 Jun 6.

DOI:10.1016/j.jpba.2015.06.003

PMID:26112924

Abstract

A liquid chromatographic (LC) method with improved selectivity for the simultaneous determination of 5-hydroxyindoles (5-HIs; 5-hydroxytryptophan, 5-hydroxytryptamine, N-acetyl-5-hydroxytryptamine, 5-hydroxyindole-3-acetic acid, and 5-hydroxytryptophol) is described. This method involves precolumn derivatization with 4-(3',3',4',4',5',5',6',6',7',7',8',8',9',9',10',10',10'-heptadecafluorodecyl)benzylamine (HFBA) and separation of the derivatives using a fluorous LC column. In this study, stable benzoxazole derivatives of 5-HIs with HFBA have been obtained by a simple derivatization procedure; their fluorescent properties enabled highly sensitive detection. In addition, only the HFBA derivatives of 5-HIs has been selectively retained on the fluorous LC column via fluorous interaction whereby perfluoroalkyl compounds show affinities with each other, while the non-fluorous compounds did not. The HFBA derivatives were separated within 30 min and the detection limits for 5-HIs in a 20-μL injection volume were 1.2-14 fmol (S/N=3). Furthermore, this method was applied to the analysis of 5-HIs in the human plasma from healthy subjects.

摘要

本文描述了一种具有更高选择性的液相色谱（LC）方法，用于同时测定5-羟吲哚（5-HIs；5-羟色氨酸、5-羟色胺、N-乙酰-5-羟色胺、5-羟吲哚-3-乙酸和5-羟色醇）。该方法包括用4-（3′，3′，4′，4′，5′，5′，6′，6′，7′，7′，8′，8′，9′，9′，10′，10′，10′-十七氟癸基）苄胺（HFBA）进行柱前衍生，并使用含氟液相色谱柱分离衍生物。在本研究中，通过简单的衍生程序获得了5-HIs与HFBA的稳定苯并恶唑衍生物；它们的荧光特性实现了高灵敏度检测。此外，5-HIs的HFBA衍生物仅通过含氟相互作用选择性保留在含氟液相色谱柱上，全氟烷基化合物之间表现出亲和力，而非含氟化合物则没有。HFBA衍生物在30分钟内分离，进样体积为20μL时5-HIs的检测限为1.2 - 14 fmol（S/N = 3）。此外，该方法应用于健康受试者人血浆中5-HIs的分析。