LC/MS/MS Analysis of DL-d-Hydroxyglutaric Acid Enantiomers on Astec^® CHIROBIOTIC^® R

Chiral 2-hydroxyglutarates (2-OHG) are important molecular signatures of both healthy and diseased biological cells, their specific biochemical pathways and inborn errors of metabolism. The chiral differentiation and quantification of D-2-OHG (R(-)-2-OHG) and L-2-OHG (S(+)-2-OHG) is key for characterizing neurometabolic disorders like the 2-hydroxyglutaric acidurias that cause neurological impairment early in life. The ability to distinguish and quantify the enantiomers of 2-OHG is therefore important to be able to track its disposition throughout the metabolic pathway of normal vs. mutated cells. We show here a rapid and sensitive method to separate and measure D-2-OHG and L-2-OHG using LC-MS/MS detection. An Astec^® CHIROBIOTIC R (ristocetin chiral selector) column was run under polar ionic mobile phase conditions. Using tandem MS eliminated two fundamental analytical challenges with these compounds: the lack of a UV-absorbing chromophore in the 2-OHG molecule, and the abundance of competing low molecular weight acids in urine. The upper panel in the chromatogram shows the separation of D-2-OHG and L-2-OHG obtained during method development. The lower panel shows the pure L-2-hydroxyglutaric acid.
Besides providing the necessary enantioselectivity, Astec^® CHIROBIOTIC columns have the advantage of operating in aqueous and polar organic mobile phases that are amenable to polar drugs and metabolites. Their ionic interactions promote analyte ionization which enhances sensitivity in ESI detection. In addition to the CHIROBIOTIC columns, Sigma-Aldrich also provides the high purity mobile phase agents, racemic and pure chiral 2-OHG enantiomers and other compounds in the isocitrate dehydrogenase (IDH) pathway.

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