5,6-Dimethoxy-2-(4′-Hydrazinocarbonylphenyl)benzothlazole as a Highly Sensitive and Stable Fluorescence Derivatization Reagent for Carboxylic Acids in High Performance Liquid Chromatography

Abstract

Abstract 5,6-Dimethoxy-2-(4-hydrazinocarbonylphenyl)benzothiazole (BHBT-hydrazide) was found to be a highly sensitive and stable fluorescence derivatization reagent for carboxylic acids in liquid chromatography. Its reactivity was investigated for various C18:0-C22:6 saturated and unsaturated fatty acids. The reagent readily reacted with the fatty acids in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 37°C to produce the corresponding fluorescent derivatives. The derivatives were separated on a reversed-phase column, TSK-gel ODS 120T, with gradient elution using 40–100% (v/v) aqueous acetonitrile, and were detected spectrofluorimetrically at 447 nm with excitation of 365 nm. Calibration curves were linear over the range 10 fmol–5.0 pmol per 20-μl injection (r=0.992−0.999). The relative standard deviations for ten replicated determinations did not exceed 2.0% for any of the fatty acids (1.0 nmol/ml). The detection limits (signal-to-noise ratio=3) for the acids were 1–2 fmol for an injection volumn of 20 μl. Further, the BHBT derivative of stearic acid was synthesized to examine the fluorescence properties.