RESUMEN
Amino acids are important biomolecules and contribute to essential biological processes. Liquid chromatography tandem mass spectrometry (LC-MS) now is a powerful tool for the analysis of amino acid metabolites; however, the structural similarity and polarity of amino acids can lead to the poor chromatographic retention and low detection sensitivities. In this study, we used a pair of light and heavy isotopomers of diazo probes, d0/d5-2-(diazomethyl)-N-methyl-N-phenyl-benzamide (2-DMBA/d5 -2-DMBA) to label amino acids. The paired MS probes of 2-DMBA and d5 -2-DMBA carry diazo groups that can efficiently and specifically react with the carboxyl group on free amino acid metabolites under mild conditions. Benefiting from the transfer of the 2-DMBA/d5 -2-DMBA to carboxyl group on amino acids, the ionization efficiencies of amino acids presented great enhancement during LC-MS analysis. The results suggested that the detection sensitivities of 17 amino acids increased by 9-133-fold upon 2-DMBA labeling, and the obtained limits of detection (LODs) of amino acids on-column ranged from 0.011 fmol-0.057 fmol. With the application of the developed method, we successfully achieved the sensitive and accurate detection of the 17 amino acids in microliter level of serum sample. Moreover, the contents of most amino acids were different in the serum from normal and B16F10-tumour mice, demonstrating that endogenous amino acids may play important roles in the regulation of tumors development. This developed method of chemical labeling of amino acids with diazo probes assisted LC-MS analysis provides a potentially valuable tool to investigate the relationships between amino acids metabolism and diseases.