4-(AMINOSULPHONYL)-2,1,3-BENZOXADIAZOLE DERIVATIVES AS PRECOLUMN FLUOROGENIC TAGGING REAGENTS FOR CARBOXYLIC-ACIDS IN HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Three 4-(aminosulphonyl)-2,1,3-benzoxadiazole amine derivatives (ABD-amines), 4-(aminosulphonyl)-7-(1-piperazinyl)2, 1,3-benzoxadiazole (ABD-PZ), 4-(aminosulphonyl)-7-(5-aminopentylamino)-2,1,3-benzoxadiazole (ABD-AP) and 4-(aminosulphonyl)-7-N-(2-aminoethylamino)-2,1,3-benzoxadiazole (ABD-AE), have been synthesized as fluorogenic reagents for carboxylic acids. All three reacted with fatty acids at room temperature (20-30-degrees-C) in the presence of diethyl phosphorocyanidate (DEPC) to produce fluorescent adducts. The maximum wavelengths of arachidic acid labelled with ABD-PZ, ABD-AP and ABD-AE were 580 nm (excitation, 440 nm), 570 nm (excitation, 438 nm) and 573 nm (excitation, 429 nm), respectively. The rate of derivatization of ABD-PZ with arachidic acid was the slowest compared with those of ABD-AP and ABD-AE. However, the fluorescence intensity with ABD-PZ after a reaction time of 75 min was the highest among the three benzoxadiazoles. The ABD-amine reagents are applicable to the determination of carboxylic acids. Eight saturated free fatty acids derivatized with ABD-amines were completely separated with a linear elution gradient by use of a reversed-phase high-performance liquid chromatography column. The detection limits (signal-to-noise ratio = 3) that could be obtained for eight fatty acids were in the 10-50 fmol range.