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Qualitative and quantitative analysis of N-glycans by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is hampered mainly by the low ionization efficiency of analytes and their poor cocrystallization with traditional organic acid matrices. In the present study, a combination strategy of reactive and catalytic matrices (CSRCM) was proposed for the on-target derivatization and detection of reducing N-glycans: a novel reactive matrix, i.e., 2,5dihydroxybenzohydrazide (DHBH), having a skeleton structure similar to that of DHB, was designed and synthesized, and this reactive matrix was mixed with catalytic matrix DHB to form a rationally combinatorial matrix (DHB/DHBH). Qualitatively, DHB/DHBH could improve the ionization efficiency of reducing carbohydrates significantly. Quantitatively, the acid−base chemistry of DHB/DHBH leads to a uniform cocrystallization of analytes-matrix mixtures. Consequently, CSRCM provides accurate quantitation for N-glycans with high derivatization efficiency and good linearity (R 2 > 0.99) within 2 orders of magnitude on the basis of an internal standard method. Furthermore, the CSRCM was successfully applied to evaluating Nglycan in serum samples of colorectal cancer patients, thus showing potential in clinical applications for biomarker discovery.

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 from79Br and 81Br 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.

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