使用含荧光试剂的流动相通过高效液相色谱法荧光测定血清中的链霉素。

Fluorometric determination of streptomycin in serum by high-performance liquid chromatography using mobile phase containing fluorogenic reagent.

作者信息

Kubo H, Li H, Kobayashi Y, Kinoshita T

出版信息

Anal Biochem. 1987 Apr;162(1):219-23. doi: 10.1016/0003-2697(87)90030-3.

Abstract

A new postcolumn derivatization method for the fluorometric determination of streptomycin in serum by high-performance liquid chromatography is described. The serum was treated with 3.5% perchloric acid to precipitate proteins and the supernatant was directly injected into the chromatograph. Streptomycin was separated by reversed-phase, ion-pair chromatography with a mobile phase containing ninhydrin as a fluorogenic reagent, octanesulfonate, and 1,2-ethanedisulfonate as counterions, and was detected by fluorescence using continuous-flow, postcolumn derivatization in an alkaline stream with ninhydrin in the mobile phase. This method is sensitive to 1.0 microgram/ml using only 100 microliter of serum. Comparison with a fluorescence polarization immunoassay gave a good correlation coefficient of 0.976.

摘要

描述了一种通过高效液相色谱荧光法测定血清中链霉素的柱后衍生新方法。血清用3.5%的高氯酸处理以沉淀蛋白质，上清液直接注入色谱仪。链霉素通过反相离子对色谱法分离，流动相含有茚三酮作为荧光试剂、辛烷磺酸盐和1,2 - 乙二磺酸盐作为抗衡离子，并通过在碱性流中使用连续流柱后衍生、流动相中的茚三酮进行荧光检测。该方法仅使用100微升血清时对1.0微克/毫升敏感。与荧光偏振免疫测定法比较，得到良好的相关系数0.976。