Measurement of 2-hydroxyglutarate enantiomers in serum by chiral gas chromatography-tandem mass spectrometry and its application as a biomarker for IDH mutant gliomas

(R)-2-hydroxyglutarate (R-2-hg) is a metabolite produced under physiologic conditions but also by tumors harboring isocitrate dehydrogenase (IDH) mutations. Detection is challenging as it must be distinguished from its enantiomer (S)-2-hydroxyglutarate (S-2-hg), which is also produced in the body but can increase under hypoxic conditions. A chiral gas chromatography-tandem mass spectrometry (GC–MS/MS) assay was developed, which separated enantiomers using a chiral column and quantified levels using a stable-isotope internal standard. The assay improves upon current methods by avoiding chiral derivatization and implementing a simplified sample extraction procedure. The assay was validated and serum 2-hg levels from healthy patients were measured, establishing a new, comprehensive reference range for normal levels of each enantiomer. Differences in basal levels were observed between races, but not sex. Age also correlated with S-2-hg levels, but not R-2-hg levels. Finally, serum levels of 2-hg enantiomers were measured in a pilot study of 11 patients with and without IDH mutant gliomas. An increase in R-2-hg levels was observed in 2/3 patients with actively growing IDH mutant gliomas. S-2-hg levels were increased in 4/11 patients, irrespective of IDH status. The results presented demonstrate the feasibility of a GC–MS/MS assay for measurement of 2-hg enantiomer levels for clinical use.