4-(1-Methylphenanthro[9,10-d]imidazol-2-yl)-benzohydrazide as derivatization reagent for carboxylic acids in high-performance liquid chromatography with conventional and laser-induced fluorescence detection

Abstract

4-(1-Methylphenanthro[9,10-d]imidazol-2-yl)-benzohydrazide has been developed as a highly sensitive and selective fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatography (HPLC). The reaction conditions were optimized with C₁₆–C₂₀ linear, saturated fatty acids. The derivatization reaction proceeded in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at mild temperatures. The resulting fluorescent derivatives were separated by reversed-phase (C₁₈) liquid chromatography with aqueous methanol and were detected with conventional fluorescence detection at 460 nm with excitation at 325 nm. The detection limits (signal-to-noise ratio = 3) for the acids are 2–12 fmol for an injection volume of 10 µl. The fluorescent derivatives display an excitation maximum at 325 nm, which coincides closely with the light emission of the helium–cadmium laser. Hence, HPLC with the reagent was combined with helium–cadmium laser-induced fluorescence detection. Using this system, attomole detection limits (70–100 amol on-column) were achieved for various carboxylic acids.