Impurities in active pharmaceutical substances are unwanted substances that remain from the manufacturing process or that result from the active substance storage. Every ingredient of a pharmaceutical substance, which is not defined as a pharmaceutical substance, and every ingredient of a pharmaceutical product, which is not a pharmaceutical product or an excipient, is considered to be an impurity, and its presence is problematic due to undesirable pharmacological effects, toxic properties and reduced effectiveness of the pharmaceutical. 2,2'-(methanediyldisulfinyl)diacetamide occurs as an impurity of one active pharmaceutical substance. Since the mechanism of its emergence is unknown, one of its intermediates was synthesized by this research, and converted in 2,2'-(methanediyldisulfinyl)diacetamide by oxidization. The final product and intermediate are characterized by high performance liquid chromatography, applying the same methods used for analysis during the manufacturing processes of active pharmaceutical substances, by which the retention times to be used for identification during the analysis of the following batches were determined. The structures of the compounds were confirmed by liquid chromatography in tandem with mass spectroscopy and they correspond to proposed structures. The analysis of the results showed that a small excess of water in the production process leads to the formation of this impurity.