Resolution of carboxylic acid enantiomers by high-performance liquid chromatography with peroxyoxalate chemiluminescence detection

Abstract

The peroxyoxalate chemiluminescence (CL) detection of carboxylic acid enantiomers, combined with high-performance liquid chromatography (HPLC), is described. The CL reaction is influenced by various factors ( e.g., cluent pH, type of aryl oxalate, relative concentrations of aryl oxalate and hydrogen peroxide and reaction time). Good linearity between CL intensity and injected amounts (5 fmol-5 pmol) of authentic-derivatives, DBD-Apy-Nap and ABD-APy-Nap, which were synthesized by the reaction with naproxen (Nap) with (+)-4-(N,N-dimethylaminosulphonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole [(+)-DBD-APy] and (+)-4-(aminosulphonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole [(+)-ABD-APy)], were obtained with the proposed procedures. The reproducibility of the CL intensity during 6 h was also excellent, and no peak decrements were observed. The detection limits (signal-to-noise ratio = 2) of authentic DBD-APy-Nap, ABD-APy-Nap and NBD-APy-Nap {synthesized from naproxen and (+)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole [(+)-NBD-APyl]} with the bis[4-nitro-2-(3,6,9-trioxadecyloxy)phenyl] oxalate (TDPO)-hydrogen peroxide (H 2O 2) system after separation by HPLC were 0.49, 1.9, and 15 fmol, respectively, whereas those with bis(2,4,6-trichlorophenyl_ oxalate (TCPO)-H 2O 2 were 0.74, 2.8 and 29 fmol, respectively. Some carboxylic acid enantiomers were converted on reaction with (+)-DBD-APy into the corresponding fluorescent diastereomers after 2 h at room temperature in the presence of 2,2′-dipyridyl disulphide and triphenylphosphine, activating agents for carboxylic acids. The diastereomers derived from each pair of enantiomers of anti-inflammatory drugs and N-acetylamino acids were efficiently resolved by reversed-phase chromatography with on ODS column and a 0.1 M imidazole-NO 3 (pH 7.0)-acetonitrile mixture as the mobile phase. The applicability of the proposed procedure was also evaluated for the detection of racemic ibuprofen (anti-inflammatory drug) added to rat plasma and human urine.