Resolving challenges in detection and quantification of D-2-hydroxyglutarate and L-2-hydroxyglutarate via LC/MS

D-2-hydroxyglutarate and L-2-hydroxyglutarate (D-2HG/L-2HG) are typically metabolites of non-specific enzymatic reactions that are kept in check by the housekeeping enzymes, D-2HG /L-2HG dehydrogenase (D2HGDH/L2HGDH). In certain disease states, such as D-2HG or L-2HG aciduria and cancers, the accumulation of these biomarkers interferes with the activity of oxoglutarate-dependent enzymes that regulate bioenergetic metabolism, histone methylation, post-translational modification, and protein expression. The complex role of D-2HG in tumorigenesis has driven metabolomics investigations in cancer research. Meanwhile, L-2HG is produced by non-specific action of malate dehydrogenase and lactate dehydrogenase under acidic or hypoxic environments. Characterization of the divergent effects of D-2HG/L-2HG on the activity of specific enzymes in diseased metabolism depends on their accurate quantification via mass spectrometry. Despite advancements in high-resolution mass spectrometry (HRMS), challenges are typically encountered when attempting to resolve isobaric and isomeric metabolites such as D-2HG/L-2HG for quantitative analysis. Herein, D-2HG/L-2HG derivatization and liquid chromatography (LC) MS quantification methods were examined. This outcome led to the development of a high-throughput LC-HRMS approach that permits concomitant quantification of the D-2HG and L-2HG enantiomers. This approach also provided the added benefit of quantifying other intermediates that are dysregulated within interconnecting pathways. A calibration curve was obtained over the linear range of 0.8-104 nmol/mL with R² ≥ 0.995 for each enantiomer. The LC/MS-based assay had an overall precision with intra-day CV % ≤ 8.0 and inter-day CV % ≤ 6.3 across the quality control level for commercial standard and pooled biological samples; relative error % ≤ 2.7 for accuracy; and resolution, Rs = 1.6 between 2HG enantiomers (m/z 147.030), D-2HG and L-2HG (at retention time of 5.82 min and 4.75 min, respectively) following chiral derivatization with diacetyl-L-tartaric anhydride (DATAN). Our methodology was applied to disease-relevant samples to illustrate the implications of proper enantioselective quantification of both D-2HG and L-2HG. The method allows scaling to large cohorts of clinical samples in the future.