6-(N-(3-Propionohydrazino)thioureido)benzo(g) -phthalazine-1,4(2H,3H)-dione as a Derivatization Reagent for Fatty Acids in High-Performance Liquid Chromatography with Chemiluminescence Detection.

Abstract

6-[N-(3-Propionohydrazino)thioureido]benzo[g]-phthalazine-1,4(2H,3H)-dione was found to be a sensitive chemiluminescence (CL) derivatization reagent for fatty acids in high-performance liquid chromatography. The reagent readily reacted selectively with fatty acids at room temperature in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and pyridine in aqueous solution to form the corresponding acid hydrazide derivatives, which produce chemiluminescence by reacting with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in an alkaline solution. The CL derivatives of seven fatty acids (myristic, linolenic, palmitoleic, linoleic, palmitic, oleic and stearic acids) were separated within 55 min by reversedphase (C18) liquid chromatography with isocratic elution, and were detected with CL detection after mixing with the oxidizing reagents. Calibration curves were linear up to at least 66.7 pmol per 20 µl injection volume (r=0.996 to 0.999). The detection limits (signal-to-noise ratio=3) for the acids were 11.8–64.4 fmol for an injection volume of 20 µl.