高效液相色谱柱后光照射荧光法测定人尿液和血清中的凯林

Fluorometric determination of khellin in human urine and serum by high-performance liquid chromatography using postcolumn photoirradiation.

作者信息

Mawatari Ken-ichi, Mashiko Satoshi, Watanabe Mitsuo, Nakagomi Kazuya

机构信息

Department of Analytical Chemistry, Faculty of Pharmacutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan.

出版信息

Anal Sci. 2003 Jul;19(7):1071-3. doi: 10.2116/analsci.19.1071.

DOI:10.2116/analsci.19.1071

PMID:12880095

Abstract

For the determination of khellin in urine and serum, fluorometry using HPLC-postcolumn photoirradiation has been developed. Khellin and visnagin of similar structure were separated on a column of Capcell Pak C8. The mobile phase consisted of 40%(v/v) ethanol containing 75 mmol l(-1) H2O2. The postcolumn reagent, 70 mmol l(-1) KH2PO4-NaOH buffer (pH 12.7) containing 50%(v/v) ethanol, were mixed with the mobile phase, which was irradiated with ultraviolet light to induce fluorescence. The fluorescence was monitored with excitation at 378 nm and emission at 480 nm. The calibration graph for khellin was linear over the range of 65 - 2620 ng ml(-1) using an injection volume of 20 microl. The pretreatment of the urine or serum samples consisted of diluting steps or deproteinizing steps using perchloric acid, respectively.

摘要

为了测定尿液和血清中的凯林，已开发出使用高效液相色谱柱后光照射的荧光分析法。结构相似的凯林和紫铆因在Capcell Pak C8柱上分离。流动相由含75 mmol l(-1) H2O2的40%（v/v）乙醇组成。柱后试剂为含50%（v/v）乙醇的70 mmol l(-1) KH2PO4-NaOH缓冲液（pH 12.7），与流动相混合，用紫外光照射以诱导荧光。在激发波长378 nm和发射波长480 nm处监测荧光。使用20微升进样体积时，凯林的校准曲线在65 - 2620 ng ml(-1)范围内呈线性。尿液或血清样品的预处理分别包括稀释步骤或使用高氯酸的脱蛋白步骤。