PRECOLUMN FLUORESCENCE DERIVATIZATION OF CARNITINE AND ACYLCARNITINES WITH 4-(2-AMINOETHYLAMINO)-7-NITRO-2,1,3-BENZOXADIAZOLE PRIOR TO HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

4-(2-Aminoethylamino)-7-nitro-2,1,3-benzoxadiazole (NBD-ED) was synthesized as a precolumn fluorescence derivatization reagent for the high-performance liquid chromatographic determination of carnitine and acylcarnitines. Carnitine and acylcarnitines were reacted with NBD-ED (2.0 mM) and 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (35 mM) in pyridine-dimethylformamide (1:4) at room temperature for 2 h. The NBD-ED derivatives of carnitine and acylcarnitines were separated by gradient elution with water-acetonitrile containing 10 mM trifluoroacetic acid on a reversed-phase column and the eluate was monitored with excitation at 470-485 nm and emission at 530-540 nm. Carnitine and sixteen acylcarnitines were determined within 45 min. The detection limits (signal-to-noise ratio=3) for the carnitine-related compounds were 10-100 fmol. Four other NBD-alkyldiamines were also studied.