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

Abstract

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.

Lactonization of the Oncometabolite D-2-Hydroxyglutarate Produces a Novel Endogenous Metabolite

“…R-711, a lactonase that acts on D-2-HG-lactone, has been described, but no lactone formation has been detected [37]. Lactone formation during the derivatization of 2-HG has already been described by others as a side-product of the derivatization reaction or due to the acidification of the sample [21,30,31]. We observed a stable ratio of 4:1 (lactone/2-HG) of the two derivatives when standard solutions were analyzed.…”

“…Recently, Strain et al observed non-negligible amounts of R-2-HG-lactone (IUPAC: R-5-Oxo-2-tetrahydro-furancarboxylic acid), the intramolecular ester of R-2-HG (=D-2-HG), in urine specimens from IDH1/2-mutated brain tumor patients after the derivatization of 2-HG with (trimethylsilyl)diazomethane for enantioselective GC-MS analysis [21]. Here, we confirmed the endogenous occurrence of 2-HG-lactone in various biological specimens in the presence of IDH1/2 mutations and investigated possible mechanisms for its formation.…”

Separation and quantification of the urinary enantiomers of 2‐hydroxyglutaric acid by capillary electrophoresis with capacitively coupled contactless conductivity detection: Application to the diagnosis of D‐ and L‐2‐hydroxyglutaric aciduria

“…Given that L-2-HG and D-2-HG are related to different acidurias, identification of the enantiomers of 2-HG plays an important role in the diagnosis of these diseases. Different analytical methods for the separation and determination of the enantiomers of 2-HG in urine samples have been reported, including 1 H and 13 C NMR [11], use of potentiometric membrane electrodes [12], HPLC-MS [13,14], GC-MS [15][16][17][18], and CE-MS [19]. However, methods that involve MS systems require tedious sample pretreatment and derivatization steps, long analysis times, and expensive systems.…”