Determination of Free Fatty Acids from Soil and Bryophyte by HPLC with Fluorescence Detection and Identification with Mass Spectrometry
Abstract A simple and sensitive method for the determination of free fatty acids using acridone-9-ethyl- p -toluenesulfonate (AETS) as tagging reagent by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed. Studies on derivatization conditions indicate that free fatty acids react rapidly and smoothly with AETS in the presence of K 2 CO 3 catalyst in N , N -dimethylformamide (DMF) solvent at 85°C to give the corresponding sensitive fluorescence derivatives. The maximal derivatization yields are close to 100% with a six fold molar reagent excess. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. Separation of the derivatized fatty acids exhibits a good baseline resolution in combination with a gradient elution on a reversed-phase Eclipse XDB-C 8 column. The identification of 19 fatty acid derivatives was carried out by online post-column mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive-ion mode. The contents of 19 free fatty acids in soil and three kinds of bryophytes ( Tree Moss, Entodon macropodus, Dicranum caesium ) were determined. The results indicate that the bryophyte plants enrich an abundance of free fatty acids from soil. The fluorescence excitation and emission wavelengths of derivatives were 404nm of λ ex and 440 nm of λ em , respectively. Excellent linear responses were observed with the linear correlation coefficients over 0.9996, and detection limits (at signal-to-noise of 3:1) are 12.3–43.7 fmol. The proposed method is sensitive and reproducible for the determination of free fatty acids from real samples with satisfactory results.