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

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 C-16-C-20 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 (C-18) 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 mu l. 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.