Sensitive Bromine-Labeled Probe D-BPBr for Simultaneous Identification and Quantification of Chiral Amino Acids and Amino-Containing Metabolites Profiling in Human Biofluid by HPLC/MS

A novel bromine-isotope probe named D-BPBr with stereodynamic chiral recognition characteristics was developed for the labeling, separation, and detection of trace chiral amino acids and amino containing metabolites. Fourteen enantiomeric pairs of amino acids could be successfully separated and quantified on a reverse-phase C18 column with an HPLC-MS/MS system after D-BPBr labeling. The chromatographic resolution for D,L-amino acid enantiomers ranged from 1.14 to 8.83 with the L-amino acid derivative always eluting prior to the corresponding D-enantiomer. Meanwhile, D-BPBr showed strong chiral selectivity on D-amino acids, and the ratio of mass spectrometric response for D-BPBr labeled D-amino acids to that of L-enantiomers ranged from 1.31 to 12.87 under the same condition. The D-BPBr labeling method was also demonstrated to be highly efficient and selective in separation and quantification of chiral amino acids especially for trace level D-amino acids in human biofluids including urine and plasma, and in total, 11 L-amino acids and 10 D-amino acids in urine and 11 L-amino acids and 6 D-amino acids in plasma were detected and quantified. Based on the characteristic 2-Da mass difference of precursor ions and the nearly 1:1 peak intensity ratio originated from Br-79 and Br-81 natural isotopes, as well as their dissociation features, 119 amino-containing metabolites were also rapidly detected in urine and plasma samples. Our work indicated that D-BPBr may be a potentially promising tool for the detection of D-amino acid-type biomarkers in disease diagnosis.