Development of a Highly Sensitive Analytical Method of d and l-Amino Acids Constructed by Multiple Regression Prediction and Novel Chiral Derivatization Reagents for UHPLC-ESI-MS/MS Analysis

Abstract

Several years have passed since the dynamics of d-amino acids (AAs) in the living body of mammals have drawn attention as potential biomarkers of several diseases. So far, researchers have developed an enantiomer-separable method using liquid chromatography and mass spectrometry (LC-MS). Using these excellent methods for dl-AA analysis, much knowledge was obtained about d-AAs. In our previous study, (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine-2-carboxylate (DMT-(S)-Pro-OSu: DMT(S)), which was a chiral derivatization reagent for LC-MS analysis, showed high sensitivity that enabled detection of dl-AAs at the low concentration level of biological samples. However, DMT(S) required five different elution conditions and two columns for 19 dl-AA analysis with resolution (Rs) > 1.5. Herein, we report the development of novel derivatization reagents, which have a modified structure based on DMT(S) to enhance enantiomeric separation. A comparison of the separation efficiency of developed reagents and DMT(S) was higher for DMT(S) and the isopropoxy-modified reagent (i.e., DiPT(S)). In addition, a multiple regression model for the prediction of Rs was also developed. Our binary mobile phase system prediction model showed high accuracy with only five condition test data sets. The resulting analytical method, using parallel derivatization (DMT(S) and DiPT(S)) and an elution condition predicted by the developed model, enabled simultaneous analysis of 19 dl-AAs in 45 min LC run by stepwise elution. These techniques contribute to the efficient and speedy development of an analysis method with chiral derivatization for dl-AAs and other chiral metabolites.