The α-ketoglutarate metabolite, 2-hydroxyglutarate (2-HG), has emerged as an important mediator in a subset of cancers and rare inherited inborn errors of metabolism. Because of potential enantiospecific metabolism, chiral analysis is essential for determining the biochemical impacts of altered 2-HG metabolism. We have developed a novel application of chiral liquid chromatography-electron capture/atmospheric pressure chemical ionization/mass spectrometry, which allows for the quantification of both (R)-2-HG (D-2-HG) and (S)-2-HG (L-2-HG) in human cell lines. This method avoids the need for chiral derivatization, which could potentially distort enantiomer ratios through racemization during the derivatization process. The study revealed that the pesticide rotenone (100 nM), a mitochondrial complex I inhibitor, caused a significant almost 3-fold increase in the levels of (S)-2-HG, (91.7 ± 7.5 ng/10(6) cells) when compared with the levels of (R)-2-HG (24.1 ± 1.2 ng/10(6) cells) in the SH-SY5Y neuronal cells, a widely used model of human neurons. Stable isotope tracers and isotopologue analysis revealed that the increased (S)-2-HG was derived primarily from l-glutamine. Accumulation of highly toxic (S)-2-HG occurs in the brains of subjects with reduced L-2-HG dehydrogenase activity that results from mutations in the L2HGDH gene. This suggests that the observed stereospecific increase of (S)-2-HG in neuronal cells is due to rotenone-mediated inhibition of L-2-HG dehydrogenase but not D-2-HG dehydrogenase. The high sensitivity chiral analytical methodology that has been developed in the present study can also be employed for analyzing other disruptions to 2-HG formation and metabolism such as those resulting from mutations in the isocitrate dehydrogenase gene.