Simultaneous Determination of D and L Enantiomers of 2-Hydroxyglutarate by UHPLC-MS/MS Method in Human Biological Fluids and its Clinical Application.

Abstract

2-hydroxyglutarate has limited abundance in normal tissues but a high level under certain pathologic conditions. To clarify the diagnostic value of two chiral isomers of 2-hydroxyglutarate in plasma and urine of Chinese cancer patients, an ultra-high performance liquid chromatography-tandem mass spectrometric method was developed for simultaneous quantification of D-/L-2-hydroxyglutarate. The selected D-/L-2-hydroxyglutarate-d5 as internal standards were added to samples before the SPE on Waters Oasis® MAX 96-Well plate (30μm, 60mg). A derivatization step with (+)-O,O'-diacetyl-L-tartaric anhydride permitted the chromatography separation of D-/L-2-hydroxyglutarate on an ACQUITY UPLC-HSS T3 column (50 × 2.1mm, i.d. 1.8μm) with acetonitrile and water (containing 0.1% formic acid and 10mmol ammonium acetate) as the mobile phase. The calibration curves showed good linearity (R ≥ 0.99) over the concentration ranges of 200-5,000ng/mL and 500-20,000ng/mL for analysis of D-/L-2-hydroxyglutarate in plasma and urine samples, respectively. Intra- and inter-run precision were ≤ 12.33%, and the accuracy was within the range of -10.44 to 13.90%. This method was further successfully applied to clinical sample analysis in isocitrate dehydrogenase 1/2 mutated Chinese cancer patients.