3.1.1. Blue

Indigo, derived from *Indigofera* species, including *I. suffructosa* and *I. truxillensis*, *Cybistax antisyphilitica*, or other such species native to South America, are the most likely sources of the blue colorants indigotin and indirubin in all of the investigated samples, which is consistent with previous studies of pre-Columbian Andean textiles [6–8,14,15,17,18]. DART-MS cannot differentiate between these two isomers, and the result is a single peak at *m/z* 262.07 for the M- ion in negative ion mode. DART-MS is extremely sensitive to indigoids, and this peak (or the MH<sup>+</sup> in positive ion mode at *m/z* 263.08 [34]) often dominates the spectra of the samples in which it is present. Due to solubility issues, the signal is quite variable in paper spray mass spectra, making this approach less than ideal for identification of indigoids. Only HPLC-DAD was able to separate the isomers and clearly identify each of them based on their different UV-visible spectra. While the ratio of indigotin to indirubin has been suggested to be indicative of the dyeing process or even the altitude at which the dyes were prepared due to the lower concentration of atmospheric oxygen content at high

altitudes [14], the peak areas for the isomers are variable depending on the wavelength used for integration. Further, we observed that the dark blue Lambayeque sample extracted into dimethyl sulfoxide (one of the few solvents in which indigoids are readily soluble) changed color from blue to reddish-purple during a delay between preparation and HPLC analysis, indicating some shift from indigotin to indirubin in solution. Indeed, HPLC analysis of this sample showed only indirubin, while a re-extracted sample showed both isomers in a ratio indicating more of the indigotin and less indirubin, as expected for the dark blue solution. Based on these observations, the ratio of the isomers should not be interpreted as particularly meaningful. Both the DMSO and methanol/HCl extractants were appropriate for the HPLC analyses of the indigoids. Example chromatograms, UV-vis spectra, and mass spectra for a blue sample are shown in Supplementary Figure S15.
