Differentiation of 2-hydroxyglutarate enantiomers and its lactones by gas chromatography/electron ionization tandem mass spectrometry

Rationale: 2-Hydroxyglutarate (2-hg) exists as enantiomers and can readily undergo cyclization to its lactone. Gas chromatography/electron ionization mass spectrometry (GC/EI-MS) has been used to separate 2-hg enantiomers in bodily fluids but the assay cannot simultaneously measure cyclic and acylic 2-hg enantiomers. Furthermore, the assignment of ion structures was not verified by complementary MS data. Methods: GC/EI-MS and product ion analysis were used to obtain MS and MS/MS spectra of 2-hg, deuterated and ¹³C-labeled 2-hg, and 2-hg lactone. Ion structures and EI fragmentation mechanisms were determined by fragmentation pattern and isotopologue comparisons. Using the EI data, a GC/MS/MS assay was developed to separate and detect 2-hg enantiomers and 2-hg lactone enantiomers in blood and urine using a cyclodextrin capillary column. Results: A new ion structure was predicted for the 85 m/z fragment than what was previously hypothesized, and the 117 m/z ion was the only fragment unique to the linear 2-hg compound. MS/MS data suggested that the majority of the fragments were the result of secondary fragmentation. Finally, separation of serum and urine 2-hg and 2-hg lactone enantiomers was achieved, and the acyclic 2-hg compound was found to be the major compound detected, though the amount of lactone detected was considerable in a number of samples. Conclusions: Unique EI fragmentation pathways for both 2-hg and the 2-hg lactone have been described. Subsequently, the GC/MS/MS assay presented herein has significant potential as a novel clinical assay as it separates and detects both 2-hg enantiomers and the 2-hg lactone enantiomers, a capability which has not been previously demonstrated by any other assay to date.