Determination of free fatty acids in blood, tagged with 4-(2-carbazoylpyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl )- 2,1,3-benzoxadiazole, by high-performance liquid chromatography with fluorescence detection.

The free fatty acids in blood were determined by high performance liquid chromatography (HPLC) after pre-column tagging with 4-(2-carbazoylpyrrolidin-1-yl)- 7-(N,N-dimethylaminosulphonyl)-2,1,3-benzoxadiazole [sequence: see text] (DBD-ProCZ). The tagging conditions were optimized with palmitic acid (C16:0) and linoleic acid (C18:2) as representative free fatty acids, saturated and unsaturated, respectively. Under the mild reaction conditions of room temperature for 90 min in dimethylformamide (DMF) containing 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC, 0.2 M)/pyridine (2%), all the fatty acids tested were tagged with the DBD-ProCZ to produce highly fluorescent derivatives which emit light at 550 nm (excitation at 450 nm). The fluorescence wavelengths were essentially the same for all fatty acids, whereas the intensities were different for individual fatty acids. The derivatives obtained from ten free fatty acids were completely separated by reversed-phase chromatography with two isocratic elution conditions. The on-column detection limit (signal-to-noise ratio of 3) with proposed HPLC separation and fluorescence detection is in the range of 19 (palmitic acid)--176 fmol (palmitoleic acid). The free fatty acids in rat serum and human plasma were successfully determined using the present methods.