An impurity present in some samples of SIN-1 oxidizes it to nitric oxide in anaerobic solutions

N-Morpholino-N-nitrosoaminoacetonitrile (SIN-1), a nitrovasodilator metabolite of the drug, molsidomine, is widely used in studies on the pharmacology and toxicology of nitric oxide (NO) because solutions of SIN-1 'spontaneously' release NO in a pathway involving molecular oxygen. Preliminary results, however, suggested that SIN-1 could react with hemoglobin in anaerobic solutions to release NO and form NO-hemoglobin. Electron paramagnetic resonance (EPR) studies showed that heme(III) of methemoglobin was not being reduced, thereby not serving as the oxidant in the reaction generating NO-hemoglobin. When anaerobic solutions of SIN-1 and hemoglobin kept in the light and in the dark were compared, substantially more NO-hemoglobin was eventually generated in the dark, indicating that SIN-1 did not undergo photochemical decomposition to NO under the conditions used. Solutions of NO-hemoglobin were equally stable under these same conditions of light and dark. The initial pH (7.0) of stirred, unbuffered solutions of SIN-1 decreased at nearly the same rate whether or not oxygen was present. Anaerobic and aerobic solutions plateaued at the same pH, namely 5.4. Anaerobic solutions of SIN-1 in phosphate buffer, pH 7.4, released NO to the gas phase, where it was identified by trapping it with hemoglobin on agarose beads and deriving the characteristic NO-hemoglobin EPR spectrum. High pressure liquid chromatography revealed the presence of an unknown species with a retention time between that of SIN-1 and molsidomine. Samples from two different lots of SIN-1 contained this impurity which appears to oxidize SIN-1 to products that release NO in the absence of oxygen. This unknown impurity may be unstable toward light.