2-Hydroxyglutarates Analysis by LCMS
We have developed an application for the separation and identification of S-2-hydroxyglutaric acids and R-2-hydroxyglutaric acids by chiral LC-MS, using a CHIROBIOTIC® R Column (covalently bonded glycopeptide - ristocetin A). Chiral 2-hydroxyglutarates provide important molecular signatures of both healthy and diseased biological cells, their specific biochemical pathways, and inborn errors of metabolism.
The chiral differentiation and quantification of S-2-hydroxyglutaric acids and R-2-hydroxyglutaric acids is key for characterizing neurometabolic disorders like 2-hydroxyglutaric acidurias, which cause neurological impairment at a young age.1 In human brain tumor patients mutations in the enzyme cytosolic isocitrate dehydrogenase 1 (IDH1) are found in approximately 80% of grade II-III gliomas and secondary glioblastomas. The demonstration that cancer-associated IDH1 mutations result in a new ability of the enzyme to catalyze the NADPH-dependent reduction of α-ketoglutarate to the oncometabolite R(−)-2-hydroxyglutarate represents a milestone event in cancer biology. Cancer-associated IDH mutations in the IDH1 and IDH22 enzyme across gliomas, in addition to several hematologic malignanicies, have become of prognostic interest; as well as for biomarkers and therapeutic opportunities.2,3
Figure 1. Hydroxyglutaric Acid Metabolism
Figure 2. Cancer Cell Metabolism
We provide the racemic and the pure chiral 2-hydroxyglutarates as well as the corresponding high-performance products and tools for their analysis.
Figure 3.Method - Supelco® CHIROBIOTIC R, 5 μm particle size, L × I.D. 25 cm × 4.6 mm (13024AST) Mobile Phase: 75% EtOH/MeOH (3/1), 25% Water / 0.1% TEAA pH=4.5, Flow: 0.4 mL/min, Temp: 20° C. Upper chromatogram shows the separation of the racemate used to develop the method. Lower chromatogram depicts L-Hydroxy Glutaric Acid enantiomer purified from a real sample. Purity was determined to be 99.6% compared to D-Hydroxy Glutaric acid enantiomer, and identification was distinguished by optical rotation.
Figure 4.Racemate sample; MS scan of each enantiomer. Upper scan 9.00 - 10.60 min.; lower scan 10.61 - 12.38 min.