Use of 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole as a labelling reagent for peroxyoxalate chemiluminescence detection and its application to the determination of the β-blocker metoprolol in serum by high-performance liquid chromatography

Abstract

Fluorogenic reagents having a benzofurazan moiety, viz., 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 7-fluoro-4-nitro-2,1,3-benzoxadiazole and 4-(aminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole, were compared for the sensitive analysis of their derivatives by high-performance liquid chromatography with peroxyoxalate chemiluminescence detection. Of the proline derivatives, DBD-proline was the most sensitive with a detection limit of 2 fmol. The optimum concentrations of bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate and H₂O₂ for the post-column reaction were 0.5 and 75 mmol dm^–3 respectively, and amino acids and β-blockers derivatized with DBD-F were detected in the range 0.2–40 fmol (signal-to-noise ratio = 3) using the proposed method. The lower detection limit of metoprolol (a β-blocker having an isopropylamino group) spiked in serum was 0.8 ng ml^–1 using 20 µl of serum (signal-to-noise ratio = 5).