HomeSmall Molecule HPLC2-Hydroxyglutarates Analysis by LCMS

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

Hydroxyglutaric Acid Metabolism

Figure 1. Hydroxyglutaric Acid Metabolism

Cancer Cell 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.


Kranendijk M, Struys EA, Salomons GS, Van der Knaap MS, Jakobs C. 2012. Progress in understanding 2-hydroxyglutaric acidurias. J Inherit Metab Dis. 35(4):571-587.
Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM, Fantin VR, Jang HG, Jin S, Keenan MC, et al. 2009. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature. 462(7274):739-744.
Yen KE, Bittinger MA, Su SM, Fantin VR. 2010. Cancer-associated IDH mutations: biomarker and therapeutic opportunities. Oncogene. 29(49):6409-6417.