Fluorogenic reagents: 4-aminosulphonyl-7-hydrazino-2,1,3-benzoxadiazole, 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole and 4-hydrazino-7-nitro-2,1,3-benzoxadiazole hydrazine for aldehydes and ketones

Abstract

Fluorogenic reagents for aldehydes and ketones, viz., 4-aminosulphonyl-7-hydrazino-2,1,3-benzoxadiazole (ABD-H) and 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H) and also purified 4-hydrazino-7-nitro-2,1,3-benzoxadiazole hydrazine (NBD-H.NH₂NH₂) were synthesised. These reagents are not fluorescent; however, their reaction products with aldehydes and ketones fluoresce at wavelengths from 548 to 580 nm with excitation from 450 to 470 nm. Both ABD-H and DBD-H exhibited similar reactivity and were more reactive than NBD-H.NH₂NH₂. The respective pseudo-first-order reaction rate constants for the production of the hydrazone of propionaldehyde with ABD-H, DBD-H and NBD-H.NH₂NH₂ were 8.9 × 10^–2, 7.2 × 10^–2 and 4.2 × 10^–2 min^–1(the reaction was carried out in 0.0025% trifluoroacetic acid in acetonitrile at room temperature, 22 °C). The detection limits using the manual method (i.e., measurement of fluorescence intensity) for the hydrazones of aldehydes and ketones with ABD-H, DBD-H and NBD-H.NH₂NH₂ were in the µM range. The substrate blank fluorescence with ABD-H was half of that with DBD-H and NBD-H.NH₂NH₂. The reaction products were separated and analysed by reversed-phase high-performance liquid chromatography (HPLC) with spectrofluorimetric detection. The detection limits for propionaldehyde were 1040, 120 and 35.0 fmol with ABD-H, DBD-H and NBD-H.NH₂NH₂, respectively, and those for heptan-4-one were 2690, 560 and 673 fmol, respectively. Of the three reagents, DBD-H is recommended for the sensitive detection of ketones and NBD-H.NH₂NH₂ for the detection of aldehydes. The detection limits for aldehydes and ketones by HPLC were in the sub-pmol to pmol range.