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A new fluorescent labeling reagent has been developed for the determination of fatty acids (FAs) by HPLC with fluorescence detection. The derivatization conditions including the amount of derivatization reagent, temperature, and type of catalyst were investigated, the results indicated that the reaction proceeded within 30 min at 90°C in the presence of K₂CO₃ catalyst. The maximal yield was obtained with a four- to fivefold molar reagent excess. The derivatives exhibited strong fluorescence with an excitation maximum at <i>λ</i><sub>ex</sub> = 245 nm and an emission maximum at <i>λ</i><sub>em</sub> = 410 nm. Twenty-five FA derivatives were well separated by RP-HPLC on a Hypersil BDS C₈ column in combination with gradient elution. All FAs were found to give excellent linear responses with correlation coefficients >0.9992. The method gave a low LOQ of 0.85-5.5 ng/mL (S/N of 10). The developed method was employed to analyze free FAs (FFAs) composition in pomegranate samples without any purification. FFAs in samples were doubly identified by HPLC retention time and protonated molecular ion corresponding to <i>m</i>/<i>z</i> [M+H]⁺. This newly developed method allows a highly sensitive determination of trace FFAs from pomegranate and other foodstuffs.