Working with technical purity: simulation of red tattoo pigment metabolism by online-liquid chromatography-electrochemistry-mass spectrometry

Tattooing is a popular form of body art that has evolved from ancient times into being part of modern society. The understanding of biotransformation processes of coloring tattoo pigments in human skin is limited although skin reactions to tattoos with unknown culprits occur. Electrochemistry coupled to mass spectrometry (EC-MS) has widely been used as a tool for a purely instrumental approach to simulating the enzymatic biotransformation of xenobiotics. For the application to pigments, however, clean standards are lacking. Hence, metabolic studies in general as well as simulation approaches like direct EC-MS are hindered because transformation products cannot be assigned unequivocally to the pigment or its impurities. This can be addressed by online separation prior to electrochemical transformation. An online-liquid chromatography (LC)-EC-MS approach was developed to simulate the metabolism of Pigment Red 5 (PR 5). The electrochemical cell was coupled to the chromatographic column and was operated in oxidative and reductive mode. Distinct retention times allowed the electrochemical transformation products to be linked to their parent compound. Oxidative and reductive azo cleavage of PR 5 were observed. Two different PR 5 formulations were investigated, yielding the same transformation products and showcasing the applicability to individual PR 5 samples with different impurity profiles. The presented LC-EC-MS approach overcomes the lack of clean standards that hampers the metabolic studies of tattoo pigments in general and using direct EC-MS without prior chromatographic separation. Applying the purely instrumental online separation approach, first ideas on possible transformation routes of pigments frequently identified in adverse skin reactions are provided.