N-(bromoacetyl)-N′-[5-(dimethylamino)naphthalene-1-sulphonyl]piperazine as a sensitive labelling reagent for the determination of carboxylic acids by liquid chromatography with peroxyoxalate chemiluminescence and fluorescence detection

Abstract

The synthesis and application of a labelling reagent for the column liquid chromatographic determination of carboxylic acids using chemiluminescence (CL) detection is described. Dansyl chloride is first reacted with an excess of piperazine and then with bromoacetic acid. The resulting label, N-(bromoacetyl)-N′-[5-(dimethylamino)naphthalene-1-sulphonyl]piperazine (dansyl-BAP), which contains a reactive bromoacetamide group, is stable for at least 6 months. Pre-column derivatization is carried out in polar aprotic media in 30 min at room temperature for aliphatic acids, and in 30 min at 55 °C for aromatic acids, in the presence of 18-crown-6 and potassium hydrogen carbonate as a base catalyst. After derivatization, excess of dansyl-BAP is converted into a relatively polar product by reaction with thymine and is subsequently separated from the derivatized analytes on a silica cartridge. High-performance liquid chromatography is performed on C₁₈ and C₈ columns with acetonitrile–water mixtures as the eluent. Fluorescence detection is carried out at an excitation wavelength of 255 nm using a 470 nm emission cut-off filter. Chemiluminescence detection is carried out by adding bis(2-nitrophenyl) oxalate and hydrogen peroxide dissolved in acetonitrile to the column effluent. The fluorescence detection limit is 0.8–1 pmol for acids such as substituted benzoic acids and the drugs naproxen and ibuprofen; the CL detection limit is 25 fmol for retinoic acid, a vitamin A precursor.