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

Abstract

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 °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 × 10⁶ cells, respectively. The developed method will be useful for elucidating the role of 2-HG enantiomers in cancer cells.