A readily 16O-/18O-isotopically-paired chiral derivatization approach for the quantification of 2-HG metabolic panel by liquid chromatography-Tandem mass spectrometry

Abstract

With the rapid development of immunometabolism, 2-hydroxyglutarate (2-HG) is being promoted as a key immunometabolite to regulate the immune system. Based on the well-established crosstalk between 2-HG and other immunometabolites, here we firstly constructed a 2-HG metabolic panel by mapping the related metabolic pathways. Quantitative methods to globally monitor 2-HG metabolic panel are of great importance for immunometabolism study. However, the existence of enantiomer hampers the accurate measurement of these immunometabolites. This study addressed an original isotopically-paired chiral derivatization approach for UPLC-MS/MS quantification of 2-HG metabolic panel. To achieve better chromatographic separation, N-(p-toluenesulfonyl)-L-phenylalanyl chloride (TSPC) was utilized as an optical resolving reagent to form diastereomers. For accurate quantitation, an ¹⁸O₂-labeled-TSPC reagent was designed and readily synthesized to produce one-to-one internal standards. The developed approach enabled an accurate quantification of 13 immunometabolites in 2-HG metabolic panel with good linearity (R² > 0.99) and high sensitivity (0.5-120 fmol for LLOQ). With this method, we were able to simultaneously monitor the specific alterations of 2-HG metabolic panel in collagen-induced rheumatoid arthritis (CIA) rats. The measured levels of this panel ranged from 0.02 to 85.14 μg g^-1 for synovium tissue and 0.012 to 87.75 μmol L^-1 for serum samples. We envisage that the present isotopically-paired chiral derivatization approach will be practicable for different bio-samples to quantitatively profile the amino- and hydroxyl acids submetabolome, especially for the endogenous enantiomers. By virtue of the low cost of reagents and the simple procedure used in the assay, this method could be readily implemented.

Keywords: (18)O-isotopic labeling; Chemical derivatization; Enantiomers; Immunometabolites; Mass spectrometry analysis.