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

• Published in: Journal of Chromatography Open Vol. 1; p. 100005
• Main Authors: Ohtawa, Takuma, Hattori, Ayuna, Isokawa, Muneki, Harada, Masashi, Funatsu, Takashi, Ito, Takahiro, Tsunoda, Makoto
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.11.2021; Elsevier
• Subjects: Cancer; Enantiomer separation; Fluorescence; Oncometabolite; Fluorescence; Oncometabolite; Enantiomer separation; Cancer
• ISSN: 2772-3917; 2772-3917
• DOI: 10.1016/j.jcoa.2021.100005

•2-HG underwent lactonization before forming a fluorescent derivative with NBD-PZ.•A 2D liquid chromatography system with an ODS column and a chiral column established.•2-HG Enantiomers in U937 cells successfully quantified.•IDH mutant cells had more D-2-HG than wild-type cells. 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. [Display omitted]