Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan.
J Chromatogr A. 2011 Aug 19;1218(33):5581-6. doi: 10.1016/j.chroma.2011.05.076. Epub 2011 May 27.
A liquid chromatographic (LC) derivatization method for simple and selective determination of catecholamines and indoleamines in human urine has been developed. This method uses "fluorous interaction" in which perfluoroalkyl compounds show affinity with each other. The amino groups of native fluorescent analytes are precolumn derivatized with a non-fluorescent fluorous isocyanate, 2-(perfluorooctyl)ethyl isocyanate, and the fluorous-labeled analytes are retained in the fluorous LC column, whereas underivatized substances are not. Only the retained fluorous-fluorescent analytes are detected fluorometrically at appropriate retention times, and retained amines without fluorophores are not detected. In this study, 3,4-dihydroxyphenylalanine, dopamine, norepinephrine, epinephrine, and metanephrine were used as the representative of catecholamines. Tryptophan, 5-hydroxytryptophan, and 5-hydroxytryptamine were used as the representative indoleamines. This method was applied to determine eight biogenic amines in urine from healthy humans. The fluorous-labeled amines could be separated by fluorous LC column under conditions of isocratic elution within 35 min and simultaneously determined without interference from contaminants in biological samples. The detection limits for eight biogenic amines were 31-640 fmol on column. Calibration curves of them were linear over the range of at least 10-100 nmol/mL urine (r² > 0.9989) with good repeatability.
已开发出一种用于简单、选择性测定人尿液中儿茶酚胺和吲哚胺的液相色谱（LC）衍生化方法。该方法利用“氟相互作用”，其中全氟烷基化合物彼此之间具有亲和力。天然荧光分析物的氨基在预柱上用非荧光氟异氰酸酯 2-（全氟辛基）乙基异氰酸酯衍生化，氟标记的分析物保留在氟 LC 柱上，而未衍生的物质则不保留。只有保留的氟荧光分析物在适当的保留时间处被荧光法检测到，而没有荧光团的保留胺则不被检测到。在这项研究中，3,4-二羟基苯丙氨酸、多巴胺、去甲肾上腺素、肾上腺素和间甲肾上腺素被用作儿茶酚胺的代表。色氨酸、5-羟色氨酸和 5-羟色胺被用作吲哚胺的代表。该方法应用于测定健康人体尿液中的八种生物胺。在等度洗脱条件下，氟标记的胺可通过氟 LC 柱分离，并且可以在没有生物样品中污染物干扰的情况下同时进行测定。八种生物胺的检测限为 31-640 fmol 柱上。它们的校准曲线在线性范围内至少为 10-100 nmol/mL 尿液（r²>0.9989），具有良好的重复性。
