LC-MS/MS analysis of fumarate, succinate, and total 2HG

Chromatographic separation of oncometabolites (succinate, fumarate and 2HG) and other TCA cycle metabolites (α-ketoglutarate, glutamate, and malic acid) was achieved on a Phenomenex Synergi^TM Polar-RP column (150 × 2 mm, 4 µm) using a gradient elution consisting of mobile phase A (0.03% formic acid in water) and mobile phase B (0.03% formic acid in acetonitrile), at the flow rate of 0.25 mL/min. The gradient was programmed as: 0.3 min, 0% B; 5 min, 18.1% B; 7 min, 95% B; 7.5 min, 0% B; 13 min, 0%. The column oven was maintained at 35 °C.

To eliminate potential carryover, external and internal washes were implemented prior to and post the injection for both the auto-sampler syringe and injection port. The external wash was performed with 50% methanol (for R3) with 1 second rinse dip and 500 µL volume. The internal wash was performed at the sequence R1 to R0 to R2, with isopropanol – acetonitrile – methanol - water (1:1:1:1) for R1 and water with 0.03% FA for R0 and R2.

Column eluents were monitored under negative and positive electrospray ionization mode using the multiple reaction monitoring (MRM) on an AB Sciex QTRAP 6500 mass spectrometer. Mass spectrometric parameters (including ionization polarity, product ion, collision energy, declustering potential, and cell exit potential) were optimized to obtain the most sensitive and specific mass transitions for individual metabolites by direct infusion of the standard solutions into the ion source with a syringe pump. Succinate, fumarate, and 2HG were monitored at the mass transitions m/z, 117.0 > 73.0, 114.9 > 71.0, and 147.0 > 128.9, respectively. Their respective stable isotope-labeled internal standards, succinate-d6, fumarate-1,4-¹³C2, 2,3-d2 and (RS)-2-hydroxyglutarate-2,3,3-d3 were monitored at the mass transitions m/z, 121.0 > 76.9, 119.0 > 74.0, and 150.0 > 131.9, respectively. α-ketoglutarate, 1,2,3,4-¹³C4 α-ketoglutarate (internal standard), glutamate, and malic acid were monitored at the mass transitions m/z, 144.9 > 101.0, 149.0 > 104.9, 148.0 > 84.0, and 132.9 > 114.9, respectively. Other optimized mass spectrometric parameters were as follows: ion spray potential, −4500 V; nebulizer gas (GS1) and bath gas (GS2), 50 psi; curtain gas, 35 psi; collision gas, medium level; and source temperature, 475 °C. The dwell time for each MRM transition was 100 milliseconds.