6-Oxy-(acetyl piperazine) fluorescein as a new fluorescent labeling reagent for free fatty acids in serum using high-performance liquid chromatography

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 °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 λex/λem = 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%.