**4. Discussion**

These results demonstrate the successful identification of many of the dyes present in samples from pre-Columbian Peruvian textiles spanning more than two millennia. This interpretation is complicated by deterioration, the use of undyed naturally colored fibers, similarity among the wide variety of plant sources, dyeing processes, and deterioration over time. The results presented above show the difficulty of positively identifying yellow colorants due to their tendency to decompose, the many possible sources from which they can be obtained, and the overlap in compositions between many of the plant sources. These challenges affect the secondary colors, as well. If yellow colorants are not identified in an orange or green sample, it is impossible to determine if yellow colorants were present to begin with, if some other method of achieving those colors was utilized, or if a naturally yellow-colored fiber was used. Mordants, dye bath temperatures, and fermentation conditions can each affect the final color obtained from various dye plants, perhaps enabling some secondary colors to be obtained without combining dyes.

This work also compares the results obtained by ambient ionization mass spectrometry with results from high-performance liquid chromatography. We consider how much sample is needed for analysis, how sensitive and selective the methods are for the dye components, and performance characteristics such as the time required for analysis and the operator skill necessary to carry out both the analyses and data interpretation. Each of the methods has capabilities and limitations that make using them together the best choice for cases where LC-MS/MS is not available, as is the case here at EMU and many other laboratories. Ideally, dyes would be identified completely non-invasively; fiber optic reflectance spectroscopy (FORS) and surface-enhanced Raman spectroscopy have been shown to be useful for some dyes, particularly the reds [20,21,42]. However, these techniques are not yet as widely available and used as are HPLC and MS. The lower limits of detection for the colorant compounds are critical for their identification by any analytical technique and are dependent upon the amounts of sample used for analyses and, more critically, how that sample is prepared for analysis.

To roughly compare the limits of detection of the ambient ionization mass spectrometry methods and HPLC-DAD, solutions of four of the colorant compounds characteristic of the dyes identified were prepared in methanol and diluted to concentrations of 1 and 10 parts per million (ppm). These compounds included luteolin, okanin, purpurin, and carminic acid. Each solution was run by DART-MS (or paper spray MS in the case of carminic acid) and by HPLC, with the chromatogram monitored at an appropriate wavelength for each compound: 350 nm for luteolin, 380 nm for okanin, 480 nm for purpurin, and 500 nm for carminic acid. Of the 1 ppm solutions, only luteolin was reliably identified with HPLC using both retention time and UV-vis spectrum; all the rest were easily detected in the 10 ppm solutions at the appropriate wavelengths.

By DART-MS, both luteolin and purpurin were readily detected in the 1 ppm solutions applied on the closed end of a capillary melting point tube, while okanin gave a large even-electron molecular ion signal at 10 ppm, placing the LOD for okanin in solution by DART-MS somewhere between 1 and 10 ppm. Interestingly, carminic acid was not detected in positive ion mode with PS-MS at either concentration, likely due to the lack of Na<sup>+</sup> ions to aid in adduct formation. This may explain why this compound was not identified in the orange-colored fibers from the Lambayeque textile (2004.1.64). The use of negative ion mode would likely remedy this problem. It is important to consider how little of the chromophore can be differentiated from background by the method of choice for small and limited samples. Further, the sample preparation method—except in the case of DART-MS, where none is necessary—strongly affects both the quantity and quality of the data obtained, with different combinations of organic solvents, chelating agents such as EDTA, and acids (formic, oxalic, HCl, etc.) yielding different results even for the same samples. Acid strength can cause changes in the overall profile of the dye as glycosidic linkages are broken to yield aglycone components [32,43], and some dye components including the chalcones are decomposed by strong acids. Solubility, particularly for indigoids, further

affects how much dye is in the solution being analyzed. How the extraction solvent(s) disrupt the binding of mordant metal ions also influences the results.

DART-MS, in particular, is affected by the presence of certain mordants and how the mordant and dye bind to either cellulose or proteinaceous fibers, at least for some dyes such as logwood [39] with transition metal mordants such as Fe, Sn, and Cu. A recent study of mordants in Paracas textiles with X-ray fluorescence spectroscopy [7] showed that Fe and Cu may indeed have been used as mordants in Paracas textiles, which would likely influence the signal intensity observed in the DART-MS results. The binding of dye colorants with cotton fibers appears to be quite strong, as DART-MS spectra show reliably stronger signals from dyes applied to wool and other animal hair yarn compared to those dyed on cotton [25,31]. Nearly all of the fibers investigated herein were camelid wool; two of the Paracas samples, three of the Chancay yarn balls, and three samples, including both warp threads from the Lambayeque textile, were cotton, as identified visually with optical and/or scanning electron microscopy or with attenuated total reflectance Fouriertransform infrared spectroscopy (ATR-FTIR). Two of the Paracas samples yielded FTIR spectra consistent with the presence of both cellulose and protein, which is consistent with previous reports of some of the yarns containing a mixture of fibers [27,44].

The primary advantage of ambient ionization mass spectrometry methods is the ability to characterize molecules with little or no sample preparation and very short analysis times on the order of a few seconds. However, collecting data is not the same as interpreting data, and making sense of the results takes expertise and, ideally, another method such as HPLC-DAD for confirmation. Knowing what does and does not ionize by DART-MS (e.g., glycosides such as carminic acid) and under what conditions (e.g., benzoisoquinoline alkaloids such as sanguinarine forming ions in positive mode, but not in negative) requires analysis of known materials such as the database of Peruvian plant dyes, as in this case. Comparing ambient ionization mass spectrometry results directly, even with multivariate analysis, may provide more insight into dye sources that would be difficult or even impossible otherwise. The application of these methods to art and archaeological materials is in its infancy, and much work remains.

#### **5. Conclusions**

Here, we have shown the characterization of the dyes present in both the primary and secondary colors found in ancient Peruvian textiles from multiple cultural periods over a span of nearly 1800 years, including the Paracas Necropolis, the Nazca, the Wari, the Chancay, and the Lambayeque. The dyes were identified by both direct analysis in real time time-of-flight mass spectrometry (DART-MS) and paper spray MS, and these results were compared to ones obtained from extraction and separation with high-performance liquid chromatography (HPLC) with ultraviolet-visible diode array detection (DAD). The ambient ionization MS methods were simple and fast: DART-MS required no sample preparation at all, and paper spray results were obtained in a few seconds of analysis time once the samples were extracted into an appropriate solvent system (30:1 methanol:HCl). In general, the ambient ionization MS results compared well with the more traditional HPLC-DAD analyses, which provided the advantage of separation and identification of isomeric species (e.g., indigotin and indirubin). Analysis with DART-MS yielded the general classes of dyes, either through rapid identification of marker compounds (e.g., indigotin/indirubin, purpurin, luteolin, etc.) or the marked absence of such (as was the case with carminic acid). Many of the possible yellow dyes have overlapping compositions, making their identification difficult regardless of analytical approach. Oxidative decomposition, either due to age or light exposure, further complicates the conclusive identification of yellow dyes in the Peruvian textiles. Chalcone biomarkers characteristic of *Bidens* were observed in some of the samples by use of DART-MS; these findings were confirmed with HPLC-DAD. The speed and simplicity of ambient ionization mass spectrometry holds significant promise for the identification of textile dyes using only small samples, though much work

remains to understand the breadth and depth of possible sources of dyes available to the artisans of the ancient Andes.

**Supplementary Materials:** The Supplementary Tables and Figures are available online at https: //www.mdpi.com/article/10.3390/heritage4030091/s1. Figure S1, (a) Tapestry fragment with fish and snake designs (MCCM accession number 2002.1.100), made of camelid fiber; attributed to the Nazca, Early Intermediate Period. (b–d) Samples from (a). Figure S2, (a) Mantle fragment with crossloopknit stitched embroidered border of hummingbird motifs (MCCM accession number 2002.1.3), made of camelid fiber; attributed to the Nazca, Early Intermediate Period. (b–d) Samples from (a). Figure S3, (a) Fragments of a mantle border (MCCM accession number 2002.40.4 A-C), made of camelid fiber; attributed to the Nazca, Early Intermediate Period. (b–c) Samples from (a). Figure S4, (a) Tie-dye textile fragment (MCCM accession number 2002.1.148), made of camelid and cotton fibers; Wari related, Middle Horizon. (b–h) Samples from (a). Figure S5, (a) Tie-dye textile fragment (MCCM accession number 2002.1.1), made of camelid fiber; Wari related, Middle Horizon. (b–h) Samples from (a). Figure S6, (a) Single interlocked tapestry administrator's tunic (MCCM accession number 2002.1.16), made from cotton and camelid fibers, Wari Middle Horizon. (b–g) Samples from (a). Figure S7, (a) Brocade textile fragment with winged staff-bearer figures in headdresses (MCCM accession number 2002.1.83), made from cotton and camelid fibers, Wari Middle Horizon. (b–c) Samples from (a). Figure S8, (a) Woven band (MCCM accession number 2003.40.5), no additional information available, attributed to Wari. (b–f) Samples from (a). Figure S9, (a) Weaver's work basket yarns (MCCM accession number 2002.1.126 A-U), cotton and camelid fibers, Late Intermediate Period Chancay. (b–h) Samples from (a). Figure S10, (a) Red tasseled tunic fragments (MCCM accession number 2004.64.1), cotton and camelid fibers, Late Intermediate Period Pacatnamú. (b–k) Samples from (a), brown warp shown in Figure 2b. Figure S11, Paracas Necropolis yarn fragment samples collected by Anne Paul in 1985 from the Museo Nacional de Arqueología, Anthropología, e Historiadel Perú (MNAAHP). (a) Orange yarn from mummy bundle textile designated 310-58c 02912; (b) yellow yarn from mummy bundle textile designated Cave #5 12-5236 0093, cloth (oldest). Figure S12, ParacasNecropolis yarn fragment samples collected by Anne Paul in 1985 from the Museo Nacional de Arqueología, Anthropología, e Historiadel Perú (MNAAHP), mummy bundle 382. (a) Specimen 10, subspecimen 05904, green yarn from mantle; (b) specimen 45, subspecimen 02763, orange loose threads from skirt; Specimen 48, subspecimen 01017, (c) skirt fringe blue and (d) black/purple (continued next page). Figure S12, continued. Specimen 49, subspecimen (23808) 03174, (e) orange from skirt ground cloth; Specimen 54 subspecimen 02846, (f) gold embroidery threads; (g) blue fibers from turban ground cloth; (h) green threads from turban. Figure S13, continued. Specimen 68, subspecimen02929, (i) poncho purple braid and (j) poncho border ground cloth green fibers; Specimen 72, subspecimen 02519, (k) black skirt fringe and (l) red skirt embroidery thread. Figure S14, Paracas Necropolis yarn fragment samples collected by Anne Paul in 1985 from the Museo Nacional de Arqueología, Anthropología, e Historiadel Perú (MNAAHP), mummy bundle 421. (a) Specimen 39, subspecimen 03083, red loose fibers from mantle; (b) specimen 132, subspecimen 03096, brown mantle fibers from box. Figure S14, Paracas Necropolis yarn fragment samples collected by Anne Paul in 1985 from the Museo Nacional de Arqueología, Anthropología, e Historiadel Perú (MNAAHP), mummy bundle 421. (a) Specimen 39, subspecimen 03083, red loose fibers from mantle; (b) specimen 132, subspecimen 03096, brown mantle fibers from box. Figure S16, Example chromatogram at 500 nm (a) for a red sample from 2002.1.1, yarn from tie-dye Wari textile, indicative of a plant red consistent with Relbunium. UV-vis spectrum for the major peak identified as purpurin (b) detected at 10.4 min. DART mass spectra in negative ion mode (c) and positive ion mode (d) of the same sample. Figure S17, Example chromatogram at 495 nm (a) for a red sample from 2004.61.1, yarn from Lambayeque tasseled tunic, indicative of insect red consistent with cochineal. UV-vis spectrum for the major peak compared with that of carminic acid standard (b) detected at 6.6 min. Paper spray mass spectrum (c) in positive ion mode of the same sample extracted in methanol:HCl. Figure S18, UV-vis spectra for peaks observed in the chromatogram shown in Figure 1 in the main text. Spectrum of peak at 7.8 min in the extract from the gold embroidery thread from Paracasturban 382-54 02896 (a), compared to the spectrum of the okaninstandard (b). Spectrum of peak at 8.6 min in the extract from the gold embroidery thread from Paracasturban 382-54 02896 (c), compared to the spectrum of the buteinstandard (d). Figure S19, Example chromatogram at 606 nm (a) for a purple sample from 382-68 02929, yarn from poncho braid, indicative of both Relbunium and indigo. UV-vis spectra for the major peaks (b) show that purpurin (λmax~480 nm) and indigotin (λmax at 606 nm) co-elute under the chromatographic conditions used; only a trace of indirubin (c) was observed, in part because it does not absorb strongly at 606 nm. DART mass spectrum in negative ion mode (d) of the same sample. Figure S20, Example chromatograms at 350 nm (a) and 606 nm (b) for a green sample from 382-68 02929, yarn from poncho border ground cloth, indicative of indigo and an as-yet undetermined but unique yellow. The UV-vis spectrum for the peak at 10.6 min (b, inset) is consistent with that of indigotin. The other peaks observed at 350 nm gave similar spectra (c–f), which did not correlate with any of the standards or reference dyes. Figure S21, Example chromatograms at 350 nm (a) and 450 nm (b) for an orange sample from 310-56c 02912, yarn from a decorative border from a Paracas Necropolis period textile. The UV-vis spectrum for the peak at 10.5 min (b, inset) is consistent with that of purpurin from Relbunium. No major peaks were observed at 350 nm and none could be identified as any of the known yellow components. No signal consistent with any of the yellow colorants was observed above 1% relative abundance by DART-MS; the negative ion spectrum (c) shows only purpurin and caffeic acid. Figure S22, Example chromatogram at 375 nm (a) for the deteriorated brown fibers from 421-132 03096 (brown). The chromatogram from the gold yarn consistent with Bidens or Coreopsis is shown in gray for comparison. A trace of butein may be present around 8.6 min, but the poor signal-to-noise ratio limits the identification. Several signals consistent with yellow colorants from Bidens or Coreopsis were observed both by negative ion DART-MS (b) and paper spray mass spectrometry (c); no glycosides were detected, likely due to the sample extraction in methanol:HCl. Table S1, Detailed results from analysis of yarns from ancient Peruvian textiles from the Michael C. Carlos Museum collections. Table S2, Detailed results from analysis of yarns from Paracas Necropolis samples collected in 1985 by Anne Paul at the MNAAHP in Lima, Peru.

**Author Contributions:** Funding acquisition, R.S. and R.A.A.; Investigation, J.C.A., S.M., B.W. and R.A.A.; Methodology, R.A.A.; Project administration, R.S. and R.A.A.; Resources, K.A.d.M. and K.J.; Validation, R.A.A.; Writing—original draft, J.C.A., R.S. and R.A.A.; Writing—review & editing, J.C.A., S.M., B.W., K.A.d.M., K.J., R.S. and R.A.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Andrew W. Mellon Foundation, Grant ID# G-1806-05955. The APC was funded in part by the EMU College of Arts and Sciences Dean's Faculty Professional Development Award program.

**Data Availability Statement:** Data not found in the paper and Supplementary Materials can be requested from the corresponding author.

**Acknowledgments:** The authors acknowledge a number of undergraduate students who contributed to this work, including Clara Gonzales from Georgia State University; Bennet Dunstan and Christin Neiman from the EMU Chemistry Department; and Chelsea Van Buskirk, Ethan Burke, and Jenna Zoerman from the Fall 2019 Analytical Instrumentation curriculum-based undergraduate research experience at EMU. Support for graduate student authors was provided by the EMU College of Arts and Sciences Dean's Faculty Professional Development Award Program, the James H. Brickley Faculty Professional Development and Innovation Award, EMU Faculty Research Fellowships, the EMU Chemistry Department and Sellers Fund, and an EMU Graduate School Research Support Award to J.C.A. The AccuTOF DART mass spectrometer was purchased through NSF MRI-R2 award #0959621, and the HPLC-DAD and FTIR instruments used for this project were purchased with funding from the Kresge Foundation. SEM-EDS analyses were made possible by JEOL USA through the loan of a JSM-7000 Neoscope in August–September 2019.

**Conflicts of Interest:** The authors declare no conflict of interest.

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