Research Of A Novel Chiral Amino Acid Probe D-BPBr And Its Application

Amino acids are important small molecule metabolites with chirality,and their concentrations and alteration are closely related to the physiological activities and the occurrence and development of diseases.Recent studies have found that some D-amino acids presented significant changes in many disease states.However,due to their trace levels,accurate quantitative analysis of D-amino acids in complex biological samples is quite challenging.Based on the previous research of our group,we designed a chiral aldehyde probe named D-BPBr with bromine isotopes,and carried out a series of studies on chiral amino acids and amino-containing metabolites by HPLC-MS/MS.The detailed contents are presented in the following three parts:1.A novel chiral aldehyde probe D-BPBr,was designed and synthesized,and 14 enantiomeric pairs of amino acids could be labeled,separated and quantified on a reverse-phase C18 column with HPLC-MS/MS after D-BPBr labeling.The chromatographic resolution(Rs)for D,L-amino acid enantiomers ranged from 1.14 to 8.83.At the same time,D-amino acids products gave higher response in mass spectrometry in comparison to L-amino acids products upon the D-BPBr labeling,and its chiral selectivity value(Cs)ranged from 1.31 to 12.87.This method was applied for quantitative analysis of 23 amino acids and showed excellent quantitative results for method validation.The D-BPBr labeling method was also demonstrated to be highly efficient and selective in separation and quantification of chiral amino acids in human biofluids including urine and plasma.2.Targeted analysis of chiral amino acids in urine samples from 55 healthy people and 52 gastric cancer patients was applied based on the probe D-BPBr method.Orthogonal partial least squares discriminant analysis(OPLS-DA)was utilized to characterize important variables,and binary logistic regression analysis was appiled to elucidate potential biomarkers and establish a diagnostic model.The results showed that these two types of urine samples presented good separation and clustering effects.Six biomarkers in gastric cancer patients were significantly higher than those in the healthy control group.The accuracy of the established diagnostic model was up to 92.5%.3.Based on the characteristic 2 Da mass difference of precursor ions and the nearly 1:1 peak intensity ratio originated from79 Br and 81 Br natural isotopes,as well as their dissociation features,119 amino-containing metabolites were rapidly detected in the non-targeted analysis,including 108 amino-containing metabolites in urine samples and 54 amino-containing metabolites in plasma samples.