3.2.2. Green

As expected, the green samples appear to be indigo overdyed onto yellow—either dyed or naturally yellowish—yarns. Indigotin (and usually indirubin, depending on the sample) was observed in the chromatographic profiles of all the green yarns, and was detected as the M- ion (formed by direct Penning ionization) as well as the [M-H]<sup>−</sup> ion at *m/z* 262.08 and 261.07, respectively, in negative ion mode by DART-MS. Indigo was detected in all the green samples, as well, both by DART-MS and by HPLC. Separation of the indigoid

isomers by HPLC showed that indirubin was a minor component, and in several cases, was not detected. Thus, the peak at *m/z* 262.08 in the negative ion DART spectra can be attributed primarily to indigotin, the major constituent. The yellow components in the green yarns were, as with the yellow yarns, difficult to interpret. The Paracas sample of mantle threads from 382-10 05904 indicated a luteolin-based dye by both direct mass spectrometry and by HPLC. Unfortunately, it did not directly correlate with any of the yellow reference dyes. The Paracas poncho ground cloth yarns from 382-68 02929 indicated a different source of yellow lacking evidence of luteolin, with a unique chromatogram. The DART mass spectra (negative ion mode) also did not indicate the presence of any luteolin, but peaks at *m/z* 343.07 and 373.09 correlate to the [M-H]− ions of eupatorin or of cirsilineol and gardenin D, respectively. The corresponding MH<sup>+</sup> ions were observed in the positive ion PS mass spectra of this sample, as well. With no reference standards for these compounds, their presence could account for the unique chromatographic profile observed. According to Cardon [10], these compounds are found in some species of Baccharis, though the lack of luteolin, also expected in dyes prepared from Baccharis, complicates the identification.

The Nazca samples were all extremely small, as they were obtained from intact objects. Only negative ion DART-MS and HPLC analyses were performed on the green samples, showing little in addition to indigo. Luteolin and its methyl ether were observed in two of the samples, the snake band and the hummingbird border, by DART-MS. Luteolin-like components, differing significantly in retention time from luteolin and luteolin methyl ether standards, were observed by HPLC. The snake band sample also showed traces (less than 1% relative abundance) of the same ions observed in the green Paracas poncho ground cloth. A tentative identification of Baccharis seems probable for this Nazca sample. The yellow component in the green yarn from the anthropomorphic human "bean people" border was significantly different from the other two Nazca samples and may be completely oxidized.

Similarly, the green samples from the Wari and Lambayeque textiles showed no significant evidence of yellow dyes. Only the yellow-green sample from the frayed Wari band (2003.40.5) showed evidence of quercetin and kaempferol by HPLC; neither was detected by negative ion DART-MS. Both of these compounds have been proposed to oxidize to form the hydroxybenzoic acids described by Zhang et al. [5], all of which were found in the Wari and Lambayeque green samples based on the presence of molecular ions of the same mass in the negative ion DART mass spectra. The most likely explanation is that the yellow component of the greens in these textiles has decomposed over time. As the objects sampled at the Carlos Museum textiles came from collectors, it is reasonable to presume that the objects were displayed and may have experienced at least some light exposure, leading to photo-oxidation. It is also possible that the green coloration was obtained by applying indigo to naturally yellowish or buff-colored yarns, which may themselves contain these hydroxybenzoic acids. Further studies on naturally colored wools are needed.

#### 3.2.3. Orange

The orange yarn from the Lambayeque textile and the salmon fibers from the Wari tunic contained carminic acid. All of the other orange samples had components characteristic of plant reds from *Relbunium* in both the negative ion DART mass spectra and by the presence of purpurin in the chromatograms. Of the Paracas samples investigated, only the orange ground cloth from the skirt in 382-49 (23808) 03174 showed sufficient evidence to suggest that *Bidens* may be the source of the yellow, based on the negative ion DART mass spectra showing a small peak at *m/z* 287.06, consistent with the [M-H]− ion of okanin. Confirmation with HPLC was not possible due to the low signal intensity. The yellow component in the only orange sample from the Nazca textiles is likely to be oxidized, as was the case with the green.

The salmon and orange fibers from the Wari tapestry tunic differed in their source of red dye, with the former being carminic acid and the latter consistent with *Relbunium* plant red. Neither showed any evidence of yellow dyes. Mordants or the dyeing process (e.g., temperature, pH, etc.) may account for the different shades of color observed without the addition of a yellow dye. The HPLC of the Lambayeque orange yarn showed primarily carminic acid but little evidence for yellow dyes. The negative ion DART-MS showed a peak at *m/z* 301.05, consistent with a number of possible flavonoids, along with the decomposition products expected for flavonols. PS-MS failed to detect carminic acid in this sample.

#### 3.2.4. Browns

Brown yarns may be naturally colored fibers in shades of brown, or they may have been dyed with tannins, which are difficult to identify and can be sourced from a variety of different plants. Indeed, the brown yarn from the Wari tapestry weave tunic appears to be undyed, as none of the methods showed any evidence of dye chromophores. The brown fragments from the Paracas textile 421-132 02096, on the other hand, have a composition similar to that of the gold sample from 382-54 02846 described above and consistent with the reference samples of *Bidens andicola*. The presence of both butein and okanin, identified by their [M-H]− ions in negative ion DART, could not be confirmed by HPLC due to the low signal intensity; monitoring the chromatogram at 375 nm, a trace of butein may be present at 7.7 min, as shown in Supplementary Figure S22.

Both the brown warp yarn (consisting of two plies, one dyed brown and one possibly natural, Figure 2b) and the golden brown yarn from the Lambayeque textile had a peak in the HPLC at 7.4 min with a UV-vis spectrum consistent with that of one of the laccaic acids found in Kerria lacca, which is native to Asia and not found in South America (Figure 3). None of the laccaic acids were detected in the paper spray mass spectra of the extracts from these yarns, though peaks at *m/z* 331.05 and 353.04, consistent with the MH<sup>+</sup> and [M+Na]+ ions for kermesic acid, were observed in the PS mass spectra of the EDTA/DMF extracts of both of these samples, though no carminic acid was present. Degano and Colombini [15] observed laccaic acids in red yarn from the belt of a child mummy from the transitional period between the Wari and Chancay cultures, overlapping the time period of the Lambayeque, as well. These findings further emphasize the importance of developing a database of known reference dyes available to the textile artists of ancient Peru.

**Figure 3.** Unusual chromatogram (at 500 nm) and UV-vis spectrum (inset) for peak at 7.402 min in the brown warp yarn (MCCM 2002.64.1) shown in Figure 2b.
