Determination of Free Fatty Acids from Soil and Bryophyte by HPLC with Fluorescence Detection and Identification with Mass Spectrometry

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 K2CO3 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-C8 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 λex and 440 nm of λem, 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.