Since it is important to produce hyperpolarized fumarate in high concentration and with high C polarization, we performed the chemical reaction with varied bubbling duration to find an optimum. In each experiment, after the chemical reaction and magnetic field cycle, the C-polarized fumarate solution was transferred to a benchtop NMR spectrometer for C signal acquisition using a 𝜋/2 pulse. After the hyperpolarized NMR signals had relaxed, a thermal equilibrium 1 H NMR spectrum was acquired on each sample to quantify the fumarate concentration. The results of these experiments are shown in Fig. , and further experimental details are provided in the Materials and Methods section. The concentration of fumarate increases approximately linearly with bubbling duration up to 150 mM after 60 s, but the rate of fumarate formation seems to slow beyond this, and 180 mM is produced after 90 s. The polarization decreases as bubbling duration is increased due to nuclear spin relaxation of the fumarate 1 H singlet state.