Separation and determination of the enantiomers of lactic acid and 2-hydroxyglutaric acid by chiral derivatization combined with gas chromatography and mass spectrometry

Lactic acid and 2-hydroxyglutaric acid are chiral metabolites that have two distinct D- and L-enantiomers with distinct biochemical properties. Perturbations of a single enantiomeric form have been found to be closely related to certain diseases. Therefore, the ability to differentiate the D and L enantiomers is important for these disease studies. Herein, we describe a method for the separation and determination of lactic acid and 2-hydroxyglutaric acid enantiomers by chiral derivatization (with L-menthol and acetyl chloride) combined with gas chromatography and mass spectrometry. The two pairs of above-mentioned enantiomers exhibited linear calibration curves with a correlation coefficient (R-2) exceeding 0.99. The measured data were accurate in the acceptable recovery range of 88.17-102.30% with inter- and intraday precisions (relative standard deviations) in the range of 4.23-17.26%. The limits of detection for D-lactic acid, L-lactic acid, D-2-hydroxyglutaric acid, and L-2-hydroxyglutaric acid were 0.13, 0.11, 1.12, and 1.16 mu M, respectively. This method was successfully applied to analyze mouse plasma. The D-lactic acid levels in type 2 diabetes mellitus mouse plasma were observed to be significantly higher (P < 0.05, t-test) than those of normal mice, suggesting that D-lactic acid may serve as an indicator for type 2 diabetes mellitus.