*2.3. Methods: HPLC-DAD*

### 2.3.1. Sample Preparation (HPLC and PS-MS)

Indigoids in the blue and purple samples were initially extracted following an adaptation of the procedure [15] used by Degano and Colombini. Roughly 1–2 mm of yarn was treated with 50 μL of dimethylsufoxide (DMSO) at 60 ◦C for 60 min in a sonicator bath, resulting in Solution I. Originally, the anthraquinone colorants were extracted from red yarns using a 1:1 (*v/v*) mixture of dimethylformamide (DMF) with 0.1% Na2EDTA in water, as reported elsewhere [32]. This mixture purportedly preserves the glycosides and results in a more accurate representation of all the compounds present in a dyed yarn. A 50 μL portion of the EDTA/DMF solution was added to a similar size fragment of yarn in a microcentrifuge tube, which was then sonicated at 60 ◦C for 60 min to yield Solution II.

A third protocol [33] was later determined to be more reliable, wherein yarn fragments were extracted with 50–100 μL of 30:1 (*v/v*) methanol:HCl solution, hereafter referred to as Solution III. Fibers were then sonicated for 60 min at room temperature to assist extraction. While the EDTA/DMF extractant did likely preserve the glycosides, the chromatograms for the textile yarns were of low quality, possibly due to a problem with the detector controller board that was identified after a portion of the analyses were completed. As the project spanned several years and numerous operators, the methanol/HCl extract was selected as it appeared to be more reliable for the identification of the characteristic colorants in these samples by HPLC and PS-MS.
