An on-line heart-cutting two-dimensional liquid chromatography method for intracellular 2-hydroxyglutarate enantiomers

d-2-Hydroxyglutarate (d-2-HG) is an oncometabolite that induces cancer cell survival and growth. d-2-HG is produced by mutations in isocitrate dehydrogenases 1 and 2. l-2-HG has different roles than the d-form, and chiral discrimination is important for elucidating the exact roles of the 2-HG enantiomers. In this study, an analytical method for 2-HG enantiomers was developed using on-line heart-cutting two-dimensional liquid chromatography (2D-LC) with fluorescence detection. Fluorescence derivatization of 2-HG with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was performed using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, a hydrophilic condensing reagent, at 70 degrees C for 30 min. The first dimension on the octadecylsilyl column was aimed at separating NBD-PZ-2-HG from other compounds obtained via derivatization or from biological fluids. The NBD-PZ-2-HG peak was fractionated into a sample loop and automatically injected into the second dimension. In the second dimension, a CHIRALPAK IC column separated NBD-PZ-d- and l-2-HG with a resolution of 2.14. The limits of quantification were 0.25 pmol per injection for NBD-PZ-d-2-HG and-l-2-HG. The precision values were below 6.58%, and the accuracies were 88.2-92.8%. The intracellular concentrations of d-2-HG and l-2-HG in the cancer cells were 13.5 +/- 0.4 and 9.9 +/- 0.3 pmol per 1.0 x 10(6) cells, respectively. The developed method will be useful for elucidating the role of 2-HG enantiomers in cancer cells.