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Tattooing has become increasingly popular throughout society. Despite the recognized issue of adverse reactions in tattoos, regulations remain challenging with limited data available and a missing positive list. The diverse chemical properties of mostly insoluble inorganic and organic pigments pose an outstanding analytical challenge, which typically requires extensive sample preparation. Here, we present a multimodal bioimaging approach combining micro X-ray fluorescence (μXRF) and laser desorption ionization-mass spectrometry (LDI-MS) to detect the elemental and molecular composition in the same sample. The pigment structures directly absorb the laser energy, eliminating the need for matrix application. A computational data processing workflow clusters spatially resolved LDI-MS scans to merge redundant information into consensus spectra, which are then matched against new open mass spectral libraries of tattoo pigments. When applied to 13 tattoo inks and 68 skin samples from skin biopsies in adverse tattoo reactions, characteristic signal patterns of isotopes, ion adducts, and in-source fragments in LDI-MS 1 scans yielded confident compound annotations across various pigment classes. Combined with μXRF, pigment annotations were achieved for all skin samples with 14 unique structures and 2 inorganic pigments, emphasizing the applicability to larger studies. The tattoo-specific spectral libraries and further information are available on the tattoo-analysis.github.io website.

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