Asymmetry c18 column

AST in the blood plasma was measured by high-performance liquid chromatography (HPLC)
(LC10, Shimadzu, Kyoto, Japan). Blood plasma (2 ml) was mixed with 0.1
ml acetone, 1.0 ml water and 1.0 mlethanol containing 0.025% butylhydroxytoluene (BHT), and extracted with 5
ml n-hexane/dimethyleter (1:1) three times. After drying with nitrogen
gas, the residue of the extract was resuspended in 0.1 mlethanol/chloroform (9:1) containing 0.025% BHT. Reversed-phase HPLC was performed using a
Symmetry C18 column (bead size: 5 µm, diameter ×length: 4.6 × 250 mm)
(Waters, Milford, MA, U.S.A.) in methanol containing 0.04% phosphoric acid /
t-butylmethyleter (83:17) at a flow rate of 0.5 ml/min. The absorbance
wavelength was 474 nm. AST concentrations in samples were determined using a calibration
curve made from the measured values of standard AST (racemic type) mixed with control
plasma. This method cannot distinguish between isomers of AST.

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.