Correct assignment of lipophilic dye mixtures? A case study for high-performance thin-layer chromatography–mass spectrometry and performance data for the TLC–MS Interface

• First detailed performance data for the TLC–MS Interface.
• Potential of quantitation by HPTLC–ESI-MS.
• Discovery of incorrect compound assignments in commercially available dye mixtures.
• Gain in information by HPTLC–MS.
• Photooxidative degradation study of dyes stored on plates in daylight.

The TLC–MS Interface, the successor of the ChromeXtract, has been available for elution head-based coupling of high-performance thin-layer chromatography with mass spectrometry (HPTLC–MS) since 2009, and is meanwhile widespread in use, mainly for compound confirmation. Until now, quantitative performance data has not been reported in detail and thus were investigated in this study. The performance data of HPTLC–electrospray ionization (ESI)-MS via the TLC–MS Interface showed good mean precisions (%RSD, n = 5) for 6 dyes in a commercially available dye mixture investigated at two different concentrations (7.3% for the 1:8 dilution, and 10.1% for the 1:16 diluted) in a selected worst case scenario. The respective mean precisions of absorbance measurements were ≤1.3%. For calibrations by HPTLC–ESI-MS, the mean determination coefficient was 0.9975 for the 6 dyes (versus 0.9997 for absorbance measurement). HPTLC–MS analysis revealed the incorrect assignment of components in two commercially available dye mixtures. Using an additional software (MassWorks) that delivered a 100 times increased mass accuracy, the proposal of molecular formulae was shown to be obtainable under certain conditions with a low resolution single quadrupole mass spectrometer and in the case of helpful information such as the double bond equivalents. This enabled the identification of the incorrectly assigned unknown dyes and clearly demonstrated the benefit of using HPTLC–MS for zone confirmation.