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RESEARCH PROFILES Extraction of tattoo pigments from human skin
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down. But clinicians and scientists have no idea whether to be concerned about the concentrations of carcinogenic
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Next time you’re mesmerized by a large tattoo on a burly man’s bicep, take a moment to question the concentration of pigments buried in his skin. Tattoo pigments are extremely stable, stubbornly insoluble, and involve some strange chemistry. Analyzing them is a challenge, but in the Sept 15 issue of Analytical Chemistry (pp 6440–6447), Wolfgang Bäumler and colleagues at the University of Regensburg (Germany) and the U.S. Food and Drug Administration describe a method to quantitatively extract tattoo pigments and their cleavage products from human skin. Tattoo pigments are not regulated by U.S. and European federal agencies. In the late 1990s, Bäumler and colleagues discovered that pigments in tattoos were identical to the pigments used as paints on cars, in printer inks, and in colors on consumer goods. “The reason why people in the tattoo studios are using these dyes or pigments is because [the pigments] are brilliant and chemically very stable,” says Bäumler. “If you puncture them into the skin, you get a very nice image.” There is reason to be troubled about the use of these pigments. In Europe, Pigment Red 22 (PR22) is banned in cosmetics, such as lipsticks, because the pigment and its cleavage products, which are carcinogenic amides, can be absorbed by the skin. But Bäumler says tattoo artists can inject PR22 into skin because “tattoos are not considered to be cosmetics, because you don’t apply them topically to the skin.” No evidence exists to show whether the concentrations of tattoo pigments injected into the skin are toxic. The laser removal of tattoos is also an issue. During the procedure, a highenergy laser beam is focused onto the pigment in the skin. The laser beam heats the pigment to 800 °C, and the pigment crystals literally explode into smaller pieces. Some tattoo pigments, like azo dyes, are known to produce carcinogenic amides as they break
Investigators have developed a way to quantitatively extract tattoo pigments and their cleavage products from human skin.
amides, because they don’t know anything about the initial concentrations of pigments in the skin. To get a handle on the concentration question, Bäumler and colleagues developed a quantitative method to extract PR22, Pigment Red 9 (PR9), and their laser-induced cleavage products—2methyl-5-nitroaniline, 4-nitrotoluene, 2,5 dichloraniline, and 1,4-dichlorobenzene (1,4-DCB)—from aqueous solutions and human skin that wasn’t tattooed. The investigators added a known concentration of pigment or cleavage product to a solution or to a suspension of human skin and tested to see whether
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they could completely recover the molecules and measure them by HPLC. There were problems in the measurements. First, some of the cleavage products were volatile and hard to maintain in solution. Second, because the pigments were so insoluble, only a handful of solvents were useful for extraction. One of the most effective was methylene chloride with Diglyme, a keeper that retains volatile compounds in solution. Bäumler and colleagues found that this combination, when heated to 60 °C, got the pigments and their cleavage products into solution. The third problem was that the pigment crystals absorbed into the interior of the cells, so the investigators had to not only break down the skin’s macrostructure but also lyse the cells in a way that didn’t destroy the pigments. The degradation of skin had to be performed in several steps. Proteins were first denatured at high temperatures. Enzymes were added next: collagenase broke down collagen; elastin released skin cells from the extracellular matrix; and proteinase K cleaved peptide bonds, puncturing the cell membranes. A special buffer was also added to help with the lysis. From their HPLC measurements of the extracted molecules, the investigators determined that they could recover >92% of PR22 and PR9 from aqueous solutions and >94% from lysed skin. They also recovered up to 100% of the cleavage products, except for 1,4-DCB, which was too volatile, from both aqueous solution and human skin. Bäumler and colleagues would now like to test the recovery of pigments from tattooed skin. They’ve gotten a tattoo machine that’s used in the studios and are adding pigments to human skin excised during surgeries or to pig skin, which happens to be very similar to human skin. Bäumler says the investigators would also like to analyze tattooed skin obtained from forensic medicine. a —Rajendrani Mukhopadhyay
