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 30 pmol/10 μl 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 2 μl for each run; also, 5 samples could be analyzed within 10 min.