Du Xiao-Lan, Zhang Hua-Shan, Guo Xiao-Feng, Deng Ying-Hua, Wang Hong
Department of Chemistry, Wuhan University, Wuhan 430072, China.
J Chromatogr A. 2007 Oct 26;1169(1-2):77-85. doi: 10.1016/j.chroma.2007.08.038. Epub 2007 Aug 21.
A new fluorescein-based fluorescent derivatizating reagent, 6-oxy-(acetyl piperazine) fluorescein (APF), has been designed, synthesized and developed for carboxylic acid labeling. It was used as a pre-column derivatizing reagent for the determination of seven free fatty acids (lauric acid, myristic acid, arachidonic acid, linoleic acid, palmitic acid, oleic acid, and stearic acid) with high-performance liquid chromatography (HPLC). The derivatization reaction of APF with seven fatty acids was completed at 60 degrees C for 1 h using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as the condensing reagent. On a C18 column, the derivatives of APF with seven free fatty acids could be separated completely in 22 min using a mobile phase of methanol-water (88:12, v/v) containing 7 mmol L(-1) pH 6.5 Na2HPO4-H3Cit3 buffer with fluorescence detection at lambdaex/lambdaem=467/512 nm. The detection limits could reach 0.1-6.4 nmol L(-1) (signal-to-noise=3). This reagent was applied to the determination of the free fatty acids in human serum samples with satisfying recovery efficiencies varying from 93 to 105%.
一种新型的基于荧光素的荧光衍生化试剂，6-氧代-（乙酰哌嗪）荧光素（APF），已被设计、合成并开发用于羧酸标记。它被用作柱前衍生化试剂，通过高效液相色谱（HPLC）测定七种游离脂肪酸（月桂酸、肉豆蔻酸、花生四烯酸、亚油酸、棕榈酸、油酸和硬脂酸）。以1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDC）作为缩合剂，APF与七种脂肪酸的衍生化反应在60℃下1小时内完成。在C18柱上，使用含有7 mmol L(-1) pH 6.5 Na2HPO4-H3Cit3缓冲液的甲醇-水（88:12，v/v）流动相，在λex/λem=467/512 nm处进行荧光检测，APF与七种游离脂肪酸的衍生物可在22分钟内完全分离。检测限可达0.1-6.4 nmol L(-1)（信噪比=3）。该试剂应用于人体血清样品中游离脂肪酸的测定，回收率令人满意，在93%至105%之间。
