A double surrogate approach for the quantitation of 2-Hydroxyglutarate - An oncometabolite in human brain tumors via LC-MS/MS.

We are reporting a validated LC-MS/MS assay for the quantitation of 2-Hydroxyglutarate (2-HG), an endogenous oncometabolite, in human brain tumors. To address the challenges of endogenous levels of 2-HG in biological matrix and the limited availability of human brain tumors, a surrogate analyte in conjunction with a surrogate matrix approach was used (stable isotope labeled 2-HG in monkey brain homogenate). Human brain tumor homogenate samples were spiked with stable isotope labeled internal standard, processed by protein precipitation, and analyzed using LC-MS/MS. The mass spectrometer was optimized to ensure equal molar response between 2-HG - the authentic analyte and C 2-HG - the surrogate analyte. Parallelism was successfully demonstrated between human brain tumor homogenate (the authentic matrix) and monkey brain homogenate (the surrogate matrix). The linear analytical range of the assay was set at 0.15-150 μM. This validated LC-MS/MS method demonstrated excellent accuracy and precision. For C 2-HG in monkey brain homogenate and human brain tumor homogenate, the overall accuracy was between 98.4 % and 102 %, and the intra- and inter-assay precision (%CV) were less than 5.7 %. 2-HG was found to be stable in human brain tumor homogenate for at least 26 h at room temperature, 454 days when stored at 70 °C and after five freeze/thaw cycles. The assay has been successfully applied to human brain tumor samples to support clinical studies. Novel Aspect: A surrogate matrix and surrogate analyte approach to quantify endogenous 2-HG concentrations in human brain tumors.