Reversed phase hplc

Reversed‐phase HPLC (Jasco, Tokyo, Japan) was performed with an aSymmetry C18 column (100 Å, 5 μm, 3.9 × 150 mm; Waters, Milford, Massachusetts) containing dimethyloctadecylsilyl‐bonded amorphous silica to detect the presence of PAHs in the black inks. A PAH identification mixture and a benzo[a]pyrene standard were obtained from Sigma Chemical. To prepare the benzo[a]pyrene standard, the chemical was dissolved in methanol to a concentration of 10 μg/mL. Each black ink (1 mL) was extracted overnight with 5 mL of dinitrochloromethane (Biosolve, Valkenswaard, The Netherlands), and dried in a vacuum concentrator (RVC‐2‐25 Co plus; M. Christ, Osterode, Germany). Subsequently, ink samples were dissolved in methanol. Samples were filtered over a polytetrafluoroethylene 0.2‐μm membrane filter, and elution was performed with a linear gradient from 40% to 85% acetonitrile (Actu‐ALL chemicals, Oss, The Netherlands) containing 0.1% trifluoroacetic acid (Biosolve) for 45 minutes at a flow rate of 1 mL/min. The PAH identification mixture was used for comparison with peaks obtained from the tattoo inks. For identification of benzo[a]pyrene, 20 μL of 10 μg/mL benzo[a]pyrene standard was analysed separately, and 90 μL of 100 μg/mL benzo[a]pyrene standard was then spiked with 10 μL of the extracted Intenze Sculpting Black tattoo ink fraction.