Determination of residual manganese in Mn porphyrin-based superoxide dismutase (SOD) and peroxynitrite reductase mimics

The awareness of the beneficial effects of Mn porphyrin-based superoxide dismutase (SOD) mimics and peroxynitrite scavengers on decreasing oxidative stress injuries has increased the use of these compounds as mechanistic probes and potential therapeutics. Simple Mn2+ salts, however, have SOD-like activity in their own right both in vitro and in vivo. Thus, quantification/removal of residual Mn2+ species in Mn-based therapeutics is critical to an unambiguous interpretation of biological data. Herein we report a simple, sensitive, and specific method to determine residual Mn2+ in Mn porphyrin preparations that combines a hydrometallurgical approach for separation/speciation of metal compounds with a spectrophotometric strategy for Mn determination. The method requires only common chemicals and a spectrophotometer and is based on the extraction of residual Mn2+ by bis(2-ethylhexyl)hydrogenphosphate (D2EHPA) into kerosene, re-extraction into acid, and neutralization followed by UV–vis determination of the Mn2+ levels via a Cd2+-catalyzed metallation of the H2TCPP4− porphyrin indicator. The overall procedure is simple, sensitive, specific, and amenable to adaptation. This quantification method has been routinely used by us for a large variety of water-soluble porphyrins.