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 13 C‐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.