RESOLUTION OF CARBOXYLIC-ACID ENANTIOMERS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH PEROXYOXALATE CHEMILUMINESCENCE DETECTION

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.. eluent 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 (H2O2) system after separation by HPLC were 0.49, 1.9 and 15 fmol, respectively, whereas those with bis(2,4,6-trichlorophenyl) oxalate (TCPO)-H2O2 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-NO3 (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.