Survey of Chemical Substances in Consumer Products, 61 – Colorants in transferable picture tattoos for the skin

Colorants in transferable picture tattoos for the skin

3 Analysis

3.1 Chemicals and reagents
3.2 Sample preparation
3.3 High performance liquid chromatography (HPLC)
3.4 UV-Visible spectra library of reference colorants
3.5 Identification and semi-quantitative determination

3.1 Chemicals and reagents

All-in all 129 reference colorants were obtained through various suppliers, as described in an earlier study (5) on the contents of colorants in cosmetic products. Acetonitrile (ACN, Lichroslov, for chromatography) and tetrabutyl ammonium hydroxide (TBAH, 40%) was purchased from E. Merck, tetrahydrofuran (THF, HPLC grade) from Rathburn, and hydrated citric acid was purchased from M&B.

HPLC Buffer: In a one litre volumetric flask, dissolve 1.4 g citric acid, 6.0 g TBAH in water, adjust pH to 9.0 with ammonia and fill up to the mark with water.

HPLC solvent: HPLC buffer/ACNl/THF (75:12,5:12,5, v/v/v)

3.2 Sample preparation

Weigh the picture tattoos, including the paper, in a centrifuge glass, add 5 ml HPLC solvent and ultrasonicate the mixture for 15 min. Scrape off carefully the coloured material, which still remains on the paper, into the solvent. The paper is removed, dried overnight at room temperature, and finally weighed. The extract is filtered through a Whatman No. 2 filter paper and analysed by HPLC. To the filter paper with undissolved coloured material, 2 ml THF is added and the solution is filtered again through a Whatman No. 2 filter paper, and analysed by HPLC.

3.3 High performance liquid chromatography (HPLC)

All sample extracts and calibration standard solutions are analysed in duplicate by HPLC as described below:

Instrument

HPLC pump	Gradient pump (Waters 616 pump with 600 controller)
Autosampler	Autosampler/Visp (Waters 717)
Detector	PDA detector (Waters 996)
PC-Software	Waters EMPOWER software for PC-control of HPLC system
HPLC column	Precolumn PLRP-S Guard cartridge, 5 mm x 3 mm and analytical column PLRP-S 100 Å, 5 µm, 150 mm x 4,6 mm from Polymer laboratories.
Column temperature	25°C

HPLC analysis

Run	Gradient
Flow	0,8 ml/min
Mobile phase	As described in the table below
Injections volume	20 µl
Analysis time	35 min
Data collection	In the range of 275 nm-760 nm, 1 spectrum/s, resolution 4.8 nm

Gradient program

Time	Flow ml/min	% HPLC buffer	% ACN	% THF	Curve
0	0,8	75	12	13
2,5	0,8	75	12	13
25	0,8	5,0	47	48	Linear
30	0,8	5,0	47	48
35	0,8	75	12	13	Linear

3.4 UV-Visible spectra library of reference colorants

HPLC analysis of all reference colorants dissolved in a suitable solvent (5) is performed as described above. Chromatographic data on each colorant is treated with EMPOWER software to generate a ”maxplot” chromatogram, where all substances eluted from the HPLC column show a chromatographic peak at their respective λ_max.

3.5 Identification and semi-quantitative determination

A maxplot chromatogram of each sample t is prepared from the chromatographic data on respective sample extract. Identification of colorants in the sample extracts is performed by matching the retention times and spectra of the HPLC peaks with the retention times and spectra of reference colorants analysed under the same conditions (5).

The content of the identified colorants is determined using one point calibration curve for each colorant. The concentrations of the colorants used for the preparation of calibration curves, 0.5-3.0 ppm, varied depending upon the chromatographic response of the respective colorants.