Validation of a routine gas chromatography mass spectrometry method for 2-hydroxyglutarate quantification in human...

High circulating levels of 2-hydroxyglutarate (2HG) have been reported in patients with determinate isocitrate dehydrogenase (IDH) mutated tumors. Recent studies indicate that in malignancies such as acute myeloid leukemia (AML), measurements of 2HG in serum provide useful diagnostic and prognostic information and improve patient selection and monitoring of IDH-targeted treatments. In the current study, we validated a sensitive and specific gas chromatography mass spectrometry (GC-MS) method specifically intended to quantify serum levels of 2HG in routine clinical laboratories. Extraction was liquid-liquid with ethyl acetate, and derivatization was reduced to 3 min of microwave irradiation. The analytical method was linear over a wide dynamic range, presenting acceptable intraday and day-to-day precision and accuracy. The limit of quantification was 10 ng/mL, process efficiency ranged between 38% and 49%, and recovery of added 2HG was 99-105%. 2HG was found to be stable in serum for up to 48 h at both 4 °C and at ambient temperature, and after three freeze-thaw cycles. Microwave derivatizated extracts in the autosampler were found to be stable for up to 120 h. In summary, the present method is useful for quantification of 2HG serum levels in patients with IDH mutated malignancies in clinical laboratories.