Determination of 2-Hydroxyglutarate in Glioma Primary Cell Cultures by HPLC-MS/MS Using In-Line SAX Coupled with Ion Pair Reverse Phase Chromatography

摘要

Accurate quantitation of 2HG in cell lysates and cell growth media was achieved by ~(13)C_(4)-2HG calibration and linear ion trap MRM detection. (1) Lower limit of detection 0.16 pmol on column. (2) Lower limit of quantitation 136 nM (2.7 pmol on column). (3) 2 HG is stable in media and methanol lysate for at least 48 h at 10 deg C. The validated assay was used to show that 2HG levels are not altered by the previously unreported IDH alterations V178I and Y183C, only by the well-known R132 mutations. Future work will include the study of 2HG's effects on surrounding cells over time, optimization of glioma primary cells' culture conditions and studying glioma xenografts in an immunodeficient murine model. Establishing 2HG as a biomarker in brain tumors would provide a noninvasive diagnostic tool for early detection. This work is supported by a VA MERIT award to MKC.