**Figure 3. 2HG levels in AML patient samples** AML patient peripheral blood or bone marrow samples were analyzed for intracellular metabolites. Cells had organic acids extracted, purified, and derivatized with MTBSTFA before analysis by GC-MS. Patient samples were prescreened for IDH genotypic status prior to metabolite analysis. **(A)** Quantitation of 2HG signal intensity relative to glutamate was determined using the TIC for each sample. Dot plot with means reported as crossbars. One-sample t-tests on log10 transformed data comparing 2HG/glutamate level of samples to a constant of 0.01 (1% 2HG/glutamate level). Samples with an IDH1 mutation compared to constant: *t*(3) = 2.60, uncorrected *p* = .080, Bonferroni corrected *p* = .241. Samples with an *IDH2* mutation compared to constant: *t*(3) = 6.07, uncorrected *p* = .0090, Bonferroni corrected *p* = .027. Samples with an *IDH1* or *IDH2* mutation compared to constant: *t*(7) = 5.44, uncorrected *p* = .00097, Bonferroni corrected *p* = .0029. **(B)** **Representative TIC from samples without an *IDH* mutation (top panel), samples with an *IDH1* mutation (middle panel), and samples with an *IDH2* mutation (bottom panel). The derivatized organic acids eluting between 31.4 and 34.4 min are shown, including aspartate (31.7 min), 2HG (33.1 min), and glutamate (34.3 min) based on spectra of derivatized commercial standards. Additional details for this experiment can be found at [https://osf.io/smdfr/](2). ** **(B) Representative TIC image files for samples are as follows and can be found along with all other chromatograms for this protocol [here](1):** - TIC Scan Inj48_HR_02 (Wild Type) - TIC Scan Inj46_HR_11 (IDH1) - TIC Scan Scan Inj56_HR_06 (IDH2) --- **Figure 3 - figure supplement 1. 2HG levels in AML patient samples based on EIC analysis** This is the same experiment as in Figure 3, but with quantitation of 2HG signal intensity relative to glutamate determined using the EIC for each AML patient sample. **(A)** Dot plot with means reported as crossbars. One-sample *t*-tests on log10 transformed data comparing 2HG/glutamate level of samples to a constant of 0.01 (1% 2HG/glutamate level). Samples with an *IDH1* mutation compared to constant: *t*(3) = 2.55, uncorrected *p* = .084, Bonferroni corrected *p* = .253. Samples with an *IDH2* mutation compared to constant: *t*(3) = 5.96, uncorrected *p* = .0094, Bonferroni corrected *p* = .028. Samples with an *IDH1* or *IDH2* mutation compared to constant: *t*(7) = 5.34, uncorrected *p* = .0011, Bonferroni corrected *p* = .0032. **(B)** Representative EIC from samples without an IDH mutation (top panel), samples with an IDH1 mutation (middle panel), and samples with an IDH2 mutation (bottom panel) with aspartate, glutamate, and 2HG shown. Additional details for this experiment can be found at [https://osf.io/smdfr/](3). ** **(B) Representative EIC image files for samples are as follows and can be found along with all chromatograms for this protocol [here](1):** - EIC Scan Inj55_HR_03 (Wild Type) - EIC Scan Inj46_HR (IDH1) - EIC Scan Inj56_HR_06 (IDH2) [1]:https://osf.io/dp9nk/ [2]:https://osf.io/smdfr/ [3]:https://osf.io/smdfr/