Parahydrogen-induced polarization is a hyperpolarization method for enhancing nuclear magnetic resonance signals by chemical reactions/interactions involving the para spin isomer of hydrogen gas. This method has allowed for biomolecules to be hyperpolarized to such a level that they can be used for real time in vivo metabolic imaging. One particularly promising example is fumarate, which can be rapidly and efficiently hyperpolarized at low cost by hydrogenating an acetylene dicarboxylate precursor molecule using parahydrogen. The reaction is relatively slow compared to the timescale on which the hyperpolarization relaxes back to thermal equilibrium, and an undesirable 2nd hydrogenation step can convert the fumarate into succinate. To date, the hydrogenation chemistry has not been thoroughly investigated, so previous work has been inconsistent in the chosen reaction conditions in the search for ever-higher reaction rate and yield. In this work we investigate the solution preparation protocols and the reaction conditions on the rate and yield of fumarate formation. We report conditions to reproducibly yield over 100 mM fumarate on a short timescale, and discuss aspects of the protocol that hinder the formation of fumarate or lead to irreproducible results. We also provide experimental procedures and recommendations for performing reproducible kinetics experiments in which hydrogen gas is repeatedly bubbled into an aqueous solution, overcoming challenges related to the viscosity and surface tension of the water
