Fluorescence and chemiluminescence detection of oxazole-labelled amines and thiols.

Abstract

Fluorescence and chemiluminescence analyses of amino acids and thiols derivatized with 2-fluoro-4,5-diphenyloxazole (DIFOX) and 2-chloro-4,5-bis(p-N,N-dimethylaminosulphonylphenyl)oxazole (SAOX-Cl) were investigated. Thirteen diphenyloxazole (DIOX)-derivatized amino acids were separated within 38 min by a linear gradient elution from 100% A [0.05 M phosphate (pH 7.0): CH3CN (75:25)] to 100% B [0.05 M phosphate (pH 7.0):CH3CN (1:1)] over 30 min and an isocratic elution of 100% B for 30 min. The detection limits (S/N = 2) with fluorescence detection were in the range of 19-64 fmol. Thiols derivatized with SAOX-Cl were separated by an isocratic elution using 0.1 M H3PO4:CH3CN (65:35) and detected fluorimetrically. The detection limits (S/N = 2) of reduced glutathione, N-acetylcysteine, 2-mercaptopropionylglycine, cysteine, homocysteine and captopril were 1.2, 1.5, 1.9, 5.7, 6.4 and 7.9 fmol, respectively. Peroxyoxalate chemiluminescence (CL) intensities of sulphonyl-5-N,N-dimethylaminonaphthalene (DNS), SAOX and DIOX derivatives were compared using three different oxalate esters (DFPO, TCPO and TDPO) by flow injection analysis. The relative chemiluminescence intensity (RCL) of SAOX-proline and DIOX-proline were 76-80% and 19-25% of DNS-proline (100%), respectively. Other SAOX and DIOX derivatives showed lower CL intensities (< 12%). Extremely low CL intensities were obtained for the fluorescent tagging reagents (< 0.11%) and their hydrolysis products (< 0.80%). Secondary amino acids and peptides, derivatized with DIFOX in aqueous media at room temperature for 1 h, were detected using DFPO/H2O2. TCPO/H2O2 and TDPO/H2O2 after separation by high performance liquid chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)