Determination of intracellular 2-hydroxyglutarate enantiomers using two-dimensional liquid chromatography

Abstract

In this study, a heart-cutting two-dimensional liquid chromatography (2D-LC) method for the determination of intracellular 2-hydroxyglutarate (2-HG) enantiomers is proposed. D-2-HG is an oncometabolite produced by tumors harboring isocitrate dehydrogenase (IDH) mutations. Hence, achieving accurate quantification of the 2-HG enantiomers in cancer cells is important. In this study, a fluorescence derivatization reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ), was used. We found that 2-HG was successfully derivatized with NBD-PZ after lactonization. The enantiomers of 2-HG derivatized with NBD-PZ were quantified using the heart-cutting 2D-LC method. NBD-PZ-2-HG was stable for 1 week and was separated from endogenous compounds using an octadecyl silica column before enantiomeric separation on a chiral column. The developed heat-cutting 2D-LC method was validated and successfully applied to cell samples. The amount of D-2-HG and L-2-HG in U937 cells were 9.8 and 8.3 pmol/5.0 × 105 cells, respectively. Furthermore, high amounts of D-2-HG were measured in U937 cells expressing mutant IDH proteins as expected. This method can therefore be applied to further elucidate the role of 2-HG in cancer cells, particularly those harboring isocitrate dehydrogenase mutations.