High-performance liquid chromatography and sensitive detection of amino acids derivatized with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole

7-Fluoro-4-nitrobenzo-2-oxa-1,3-diazole is used as a precolumn fluorescent labeling reagent for high-performance liquid chromatography of amino acids, including proline and hydroxyproline. The reaction is run at pH 8.0 at 60°C for 5 min. The fluorophors (Asp, Glu, Hyp, Ser, Gly, Thr, Ala, Pro) are separated on a reversed-phase column (μBondapak C₁₈) with 0.1 m phosphate buffer (pH 6.0) containing 6.75% methanol and 1.8% tetrahydrofuran, and are detected at the level of 10 fmol with excitation at 470 nm and emission at 530 nm.