Determination of Trace Amounts of Chiral Amino Acids in Complicated Biological Samples Using Two-Dimensional High-Performance Liquid Chromatography with an Innovative “Shape-Fitting” Peak Identification/Quantification Method

Abstract

A new peak identification/quantification method, i.e., “shape-fitting” method, has been devised and applied to the determination of chiral amino acids in human plasma and urine. The shape fitting method enables the determination of target analytes using a part of the peak by mathematically predicting the whole peak even when the baseline is not clear. Using this method, D-glutamic acid (Glu) and D-proline (Pro) as well as their L-enantiomers in the plasma and urine were determined. The calibration lines of 4 target enantiomers were linear with correlation coefficients higher than 0.9998; the RSD values of the intra-day precision and inter-day precision were less than 6.70%. In the human plasma, trace amount of D-Pro (0.47 nmol/mL) was observed, and in the urine, 2.97 nmol/mL of D-Glu and 0.08 nmol/mL of D-Pro were successfully determined.