Chiral Analysis of 2-Hydroxyglutarate in IDH-mut Glioma Tissue using the Kinetic Method

Abstract

Abstract In almost all biological systems, stereochemistry is an important aspect of the molecular structure. Aberrant molecular geometry can be a reflection of the disease state which is then manifested through effects on cellular metabolism. A well-known example occurs in isocitrate dehydrogenase (IDH)-mutant (mut) cancers, where the mutant IDH enzyme produces a unique chiral molecule, d-2-hydroxyglutarate (d-2HG). d-2HG accumulates in IDH-mut tumors and disrupts cellular pathways, including epigenetic regulation. Here, we apply the kinetic method to direct chiral analysis of 2HG in human glioma tissue. A trimeric cluster ion, [Ni(II)(Ac-l-Phe)2(l-2HG)−H]+, is formed and fragmented during tandem mass spectrometry (MS/MS). Cluster ions containing different 2HG geometry show distinctive fragment intensities. Calibration curves spanning from −100% to +100% enantiomeric excess (ee) were constructed, and the effects of potential interferences, including glutamate (Glu) and glutamine (Gln), were assessed. Using these data, we developed a chromatography-free method to determine the ee of 2HG in the glioma tissue. Chiral analysis of IDH-mut glioma tissue samples using the kinetic method was validated using LC−MS, with ee values agreeing within < 12%. Detection of predominantly d-2HG in tissue samples from (IDH)-mutant cancer is consistent with the neomorphic gain of function associated with the IDH mutation. This separation-free workflow enables rapid chiral analysis of 2HG in IDH-mut cancers and may provide complementary information to support intraoperative decision-making and precision medicine applications.