A Two-Step Pyrolysis-Gas Chromatography Method with Mass Spectrometric Detection for Identification of Tattoo Ink Ingredients and Counterfeit Products

Tattoo inks and other inks are complex mixtures of ingredients. Our method provides a quick and easy way to analyze multiple components at the same time. Pyrolysis and then coupled to gas chromatography with mass spectrometric detection allows the analysis of nonvolatile and volatile compounds in two consecutive runs.

In addition to pure analysis of ink ingredients, this method can also be used to differentiate original and counterfeit products. In general, this method can be used for all kind of liquid samples. The data evaluation approach may also be used for any other kind of pyrolysis data.

As with most analytical techniques, data interpretation of pyrograms is the Achilles Heel of this method. Therefore we provide libraries of pyrograms and single-analyzed spectra to ease interpretation of the data. First hold a 25-millimeter hollow glass pyrolysis tube with specialized tweezers and insert the necessary amount of quartz wool into the tube with pointed tweezers.

Insert two steel sticks at each side of the pyrolysis tube and compress the wool into a one-to two-millimeter-thick stopper. The stopper must be positioned at the lower third of the pyrolysis tube to achieve adequate heating during the pyrolysis process. Ignite a gas burner and bake the pyrolysis tube and filling for two to three seconds from each side to remove contaminants.

Next, manually shake the tattoo ink bottles vigorously for one minute to ensure homogeneity. Dip a two-microliter microcapillary tip in the ink and aspirate about one microliter of ink by filling half the capillary. Insert the capillary into the pyrolysis tube and stain the quartz wool stopper with the ink.

A clear color staining must be visible without adding too much ink to the sample. Using the specialized tweezers, attach the prepared pyrolysis tube to a steel transport adaptor for the automated injection unit, and check that the pyrolysis tube is perfectly vertical and does not fall off during shaking. Place the transport adaptor in the tray of the automated injection unit at the desired position for pyrolysis GCMS analysis.

For data evaluation of volatile compounds, start the GCMS analysis MS library searching software, and open the chromatogram of the desorption run. Select commercial libraries by clicking on Spectrum"and select Library. Then load the library of interest.

Select Integration Parameters, and perform a library search by clicking on Spectrum"and Library Search Report. For pyrolysis data evaluation, open the chromatogram of the pyrolysis run. Mark the whole chromatogram in the GCMS evaluation software.

With the right mouse button, press down to obtain an average mass spectra, or AMS. In order to establish a self-made library, click on Spectrum"and Edit Library. Select the library of interest, followed by Add New Entry, and fill in all information of interest.

Generate an AMS of the investigated ink pyrogram and use the library search for comparison to the self-made AMS library. Exclude masses from column bleed or other column noises. To identify nonvolatile compounds with specialized pyrogram evaluation software, build a folder with all pyrogram entries that should serve as a library, such as a pigment pyrogram library for pigment identification, or pyrograms of an original ink, to compare it to putative counterfeit products.

Load the unknown pyrogram in the tab library search by clicking on Browse. Next, load the library folder and select only MS matching"and RT matching"in the search options, since the overall abundance will vary compared to pyrogram of reference pigments. Click on Advanced"in the search options.

Select an RT window width of interest, an area threshold of 0.1%and allow multiple matching. In the Mass Spec"options, select the parameter use only peaks with specified MS spectra, and use a fit threshold of 850. Load the file pigment_search_spectra.

spf, or click on Add"to save specified MS spectra from each pyrogram of reference pigments or polymers from the library in the advanced search options. Press OK"to return to the main window. Then click on Search"to start the comparison.

If needed, go to the chromatogram match tab and select a compound under the chromatogram match tab. Right-click and select Search spectrum in NIST"to forward the spectra to the MS library software and identify the compound. For manual data evaluation for nonvolatile compounds, start the GCMS analysis MS library searching software and open the chromatogram of the pyrolysis run.

Select commercial and pyrolysis libraries by clicking on Spectrum"and Select Library. Load all libraries of interest. Integrate the pyrogram in the GCMS evaluation software and consider all peaks with an area not less than 0.2%of the total area.

Next, start the library search by clicking on Spectrum"and Library Search Report. Manually compare all library matches to specific pigment and polymer decomposition products in the text protocol, or fragments stated in literature. Well-produced inks with highly pure ingredients and a limited number of components resulted in chromatograms easy to interpret with standard libraries, since most peaks can be identified.

But even in the high-quality inks, non-declared ingredients such as propylene glycol are often found in addition to the declared glycerol. Inks containing multiple ingredients and impurities will result in a pyrogram that is difficult to interpret. Most peaks occurring in the second run may not be baseline separated from each other, making identification difficult.

Some substances might also result in peaks below the threshold set during data evaluation. A solution to interpreting complex data might be a step-wise approach using 400, 600, and 800 degrees Celsius in consecutive pyrolysis steps for the same sample. Some pigment decomposition products may descend from multiple pigments.

A positive result for counterfeit product identification is demonstrated for three lemon inks purchased from different vendors. Using a forward match factor above 0.9, the chromatogram from the first desorption run and the pyrogram from the second run of the original ink were compared against three independent acquisitions of the original ink, and the two counterfeit products using pyrogram evaluation software. Pyrolysis is useful to identify multiple compounds with just one analytical method.

Qualification of specific compounds can be performed afterwards with more specialized methods.