Determination of fatty acids in bio-samples based on the pre-column fluorescence derivatization with 1,3,5,7-tetramethyl-8-butyrethylenediamine-difluoroboradiaza-s-indacene by high performance liquid chromatography

• We have designed and synthesized TMBB-EDAN as a new fluorescent labeling reagent for fatty acids (FAs).
• TMBB-EDAN has high fluorescence quantum yields (Φ = 0.70) and high reactivity with FAs.
• An HPLC-fluorescence method was developed for the detection of twelve FAs by using TMBB-EDAN.
• The derivatization of TMBB-EDAN with FAs could be completed at room temperature in 30 min.
• The detection limits for FAs were in the 0.2–0.4 nM range.

1,3,5,7-Tetramethyl-8-butyrethylenediamine-difluoroboradiaza-s-indacene (TMBB-EDAN) has been designed and synthesized as a highly fluorescent labeling reagent for carboxylic acids. By using TMBB-EDAN, a sensitive and rapid method based on high performance liquid chromatography-fluorescence detection for the determination of twelve fatty acids (FAs) in bio-samples has been developed. Under optimized conditions, these FAs were tagged with TMBB-EDAN in the presence of 1-ethyl-3-(3-dimethyla-minopropyl) carbodiamide at 20 °C for 30 min and then the baseline separation was achieved on a C18 column with a linear gradient elution in 26 min. With fluorescence detection at λex/λem = 490 nm/510 nm, the linear ranges of FAs were from 3.0 to 300 nM and the detection limits with a signal-to-noise ratio of 3 were in the 0.2–0.4 nM range. The proposed method offers advantages of milder derivatization condition and much better sensitivity for the determination of FAs, when compared to the reported fluorescence derivatization-based methods.