A Sensitive and Rapid FIA with an Immobilized Enzyme Column Reactor and Peroxyoxalate Chemiluminescence Detection for the Determination of Total d-Amino Acids in Human Plasma

Abstract

A sensitive and rapid FIA using an immobilized enzyme column reactor was developed for the determination of D-amino acids in human plasma. This method is based on a specific enzymic reaction with D-amino acid oxidase and a sensitive peroxyoxalate chemiluminescence (PO-CL) detection with bis(2, 4, 6-trichlorophenyl)oxalate and 2, 4, 6, 8- tetrathiomorpholinopyrimido[5, 4-d]pyrimidine as CL reagents. Eleven kinds of D-amino acids could be independently detected quantitatively (r=0.983-1.000). The detection limits of each D-amino acid ranged from 0.4 to 30pmol/10μl injection at a signal-to-noise ratio of 3. The average concentration level of D-amino acids in human plasma was found to be 14.6±4.7 (in the range of 5.0-24.3, n=18)nmol/ml plasma. The correlation between the results obtained by the proposed FIA-PO-CL method and the colorimetric method was satisfactory (r=0.900). Moreover, this method is advantageous, since the net volume of the plasma sample needed was 2μl for each run; also, 5 samples could be analyzed within 10min.