**1. Introduction**

The ancient weavers of Peru are known for their complex multicolored textiles, preserved in burial contexts primarily due to the dry and cold conditions of these high-altitude locations. These textiles, which span a wide range of time and geography across the Andes, have long been admired for their intricate workmanship and complex designs. Determining the sources of the dyes used to achieve the colors in these priceless objects has been the object of a great amount of study over the years, dating back to work by dye chemists Fester [1] and later Saltzman [2,3] in the mid-20th century.

The characterization of dyes in ancient textiles is challenging for numerous reasons, including difficulty in obtaining material in sufficient quantity for analysis while minimizing damage to objects. The high tinting strength of most dyestuffs means that while the color of the sample may be readily visible, the actual amount of colorant present may be below the limits of detection of many analytical approaches. Degradation may complicate the identification of dye colorants, particularly those present in the fugitive yellow dyes,

**Citation:** Campos Ayala, J.; Mahan, S.; Wilson, B.; Antúnez de Mayolo, K.; Jakes, K.; Stein, R.; Armitage, R.A. Characterizing the Dyes of Pre-Columbian Andean Textiles: Comparison of Ambient Ionization Mass Spectrometry and HPLC-DAD. *Heritage* **2021**, *4*, 1639–1659. https:// doi.org/10.3390/heritage4030091

Academic Editor: Diego Tamburini

Received: 29 June 2021 Accepted: 3 August 2021 Published: 7 August 2021

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due to photo-oxidation [4] or other decomposition pathways [5] that may occur. Further, decomposition—either during burial or after excavation—may complicate determining color, as what appears blue today may have once been green, but the yellow colorants have decomposed. Colorants, or dye chromophores, are the molecules that come from the dye preparation to impart color to a textile. It should be noted, though, that some mordants and additives can also alter the apparent color of the dye. In some cases, identifying a colorant also identifies the dye source, as in the case of carminic acid, which is indicative of cochineal from *Dactylopius coccus* and various subspecies. Other colorants, such as luteolin and quercetin, are found in numerous plant dye sources. Thus, characterizing the dyes in textile samples to identify the colorants may or may not identify the dye sources that were used by their makers.

Yellow dyes have long been considered the most difficult to identify because the sources of yellow dyes are myriad and have many overlapping colorants in common. A wide variety of different plants have been suggested as possible sources of yellow dyes in pre-Columbian Peruvian textiles by previous researchers [6–8]: *Bidens andicola* and other Asteraceae (including commercially available *Coreopsis* and *Dahlia*); *Baccharis* species, including *B. floribunda* and *B. genistelloides*; and *Alnus jorullensis*. Previous work has also investigated the composition of *Kageneckia lanceolata* and *Hypericum larcifolium* [9]. The Asteraceae contain the chalcone colorants butein and okanin and their glycosides, while the others, along with *Chlorophora tinctoria* (locally called *insira*), are primarily flavonoid dyes. The flavones luteolin and apigenin and the flavonols quercetin and kaempferol are the most abundant colorants, along with their corresponding methyl ethers [10]. *Bocconia pearcei* contains the benzoisoquinoline alkaloids sanguinarine and chelerythrine [11,12]. *Chuquiraga espinosa* and *Dicliptera hookeriana* are not yet well characterized, but both appear to be flavonoids, as well.

Early work on the identification of Andean dyestuffs relied predominantly on UVvisible spectroscopy, and occasionally on separations by thin layer chromatography [8,13]. More recently, the bulk of research on the identification of dyes in textiles has been carried out with chromatographic separations, primarily by high-performance liquid chromatography (HPLC), considered the "gold standard" method for such materials. Wouters and Rosario-Chirinos [6] reported an extensive study in which they used HPLC with diode array detection (DAD) to separate, quantify, and identify all components of purple, red, and blue dyes in textiles from a range of samples from pre-Columbian to Inca period civilizations. Niemeyer et al. [14] performed HPLC dye analysis on South American red and blue textiles, finding evidence of trade throughout the region due to the presence of non-local dye sources. Degano and Colombini [15] described the analysis of blue and red dyes in 11th and 13th century materials using HPLC with a variable wavelength UV-vis detector to determine what dyes were present in textiles associated with a mummy from a pre-Columbian site in Peru. Price et al. [16] described the identification of dyes in materials from a pre-Columbian weaver's toolkit. Most recently, Boucherie et al. [17] reported on HPLC analyses of Nazca textile dyes, and Sabatini et al. [7] combined HPLC-MS/MS with X-ray fluorescence to characterize both the mordants and dyes in Paracas textiles. HPLC is the most effective way to separate components of dyes. Using UV-vis spectroscopy alone to identify the components can be difficult, particularly when reference standards of the pure colorants are unavailable, as is often the case.

Mass spectrometry (MS) has the advantage of identifying components based on their molecular masses and, in some cases, fragmentation patterns, which are generally more specific than UV-vis spectra. Michel et al. [18] utilized high-resolution mass spectrometry to characterize red, blue, and purple textiles from the Atacama desert of northern Chile. They found evidence that the Pachacamác people used both shellfish purple and combinations of red and blue dye sources to color their textiles. This early work required a relatively large 1-centimeter-long sample to be destructively extracted in hot dimethyl sulfoxide for direct exposure electron impact mass spectrometry. The potential of some spectroscopic methods for non-invasive identification of dyes in Andean textiles is also of note, particularly applications of fiber-optic reflectance spectroscopy and Raman spectroscopy [19–21]. With secure contexts for the materials being characterized, a great deal can be learned about the culture and technology of the textile artists, as demonstrated by Boytner [22].

Another method that shows potential for dye analysis is direct analysis in real time mass spectrometry (DART-MS) [23] with a time-of-flight mass analyzer. Armitage et al. [24] undertook analysis of the dyes in red textile samples from the Paracas Necropolis period, which had been previously identified as *Relbunium*. A major limitation of all soft ionization mass spectrometric methods is the inability to distinguish structural isomers, such as indigotin and indirubin, or alizarin and xanthopurpurin, based solely on molecular mass. Yellow colorants are particularly difficult to differentiate with DART-MS alone, as insource collision-induced dissociation, useful for pure substances, yields complex results for mixtures [25]. DART-MS and another "ambient" ionization method, paper spray, [26] have shown potential efficacy for characterizing a wide variety of dyes in reference collections [9], though HPLC with MS/MS detection is necessary for identifying true unknowns.

The work reported herein stems from projects carried out over several years, with a focus on the primary colors of red, blue, and yellow as well as the secondary colors of purple, green, and orange in dyed textiles from several pre-European contact civilizations of South America, ranging in time from 800 BCE to 1460 CE. The sampled objects are either from the Paracas Necropolis or have been attributed stylistically to the Nazca, Wari, and Chancay, all of which rose and fell throughout that period within the geographic area that is present-day Peru. Here, we compare the results of two approaches—ambient ionization mass spectrometry and high-performance liquid chromatography—for the identification of these dyes in yarns from ancient Peruvian textiles. We compare the capabilities and limitations of these approaches in terms of (1) the amount of sample necessary, (2) the limits of detection, (3) the selectivity, (4) the speed of analysis, and (5) the skill required of the analyst. This study serves as a step in the process of validating the use of ambient ionization mass spectrometry for characterizing dye colorants in ancient textiles.

#### **2. Materials and Methods**
