Interference-free quantification of 2-hydroxyglutarate enantiomers in cells using an optimized 2D-LC method

D-2-Hydroxyglutarate (D-2-HG) is an oncometabolite produced by mutant isocitrate dehydrogenases (IDH), and accurate quantification of 2-hydroxyglutarate (2-HG) enantiomers is significant for disease diagnosis and functional analysis of D-2-HG. We have previously developed an analytical method for the determination of 2-HG enantiomers in cell samples. In this method, 2-HG is derivatized with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) and analyzed using heart-cutting two-dimensional liquid chromatography with fluorescence detection. However, although the developed system was used for the analysis of cell samples, an interfering peak overlapping with the NBD-PZ-D-2-HG peak was observed, potentially compromising the accurate determination of D-2-HG. Accordingly, in this study, we re-evaluated the separation conditions to eliminate this interference. We examined the derivatization procedure and the mobile phases used in the first and second dimensions. In the first dimension, NBD-PZ-2-HG was separated from most endogenous compounds under reversed-phase conditions, using a mobile phase of 0.05% trifluoroacetic acid in water/acetonitrile (75/25, v/v). In the second dimension, NBD-PZ-D-2-HG and NBD-PZ-L-2-HG were separated using a chiral column. Evaluation of the methanol to acetonitrile ratio showed that a 95:5 (v/v) mixture successfully separated the interfering compound from NBD-PZ-D-2-HG, achieving a chromatographic resolution of 1.51. The improved analytical method showed excellent linearity (R 2 =0.9999), precision (≦4.81%), and accuracy (94.2–113.0%). Furthermore, this method was successfully applied to the determination of 2-HG enantiomers in non-transduced cells and IDH2-mutant cells. By resolving the interference, the improved method provides greater reliability for D-2-HG determination in non-transduced cells, which is significant for disease diagnosis.