2.3.2. HPLC-ESI-Q-ToF System

HPLC-ESI-Q-ToF MS analyses were carried out using a 1200 Infinity HPLC, coupled with a Jet Stream ESI interface with a Quadrupole-Time of Flight tandem mass spectrometer 6530 Infinity Q-ToF detector (all modules were Agilent Technologies, Santa Clara, CA, USA). The ESI operating conditions were: drying gas (N2, purity >98%): 350 ◦C and 10 L/min; capillary voltage 4.5 KV; nebulizer gas 35 psig; sheath gas (N2, purity >98%): 375 ◦C and 11 L/min. The nozzle, skimmer, and octapole were set at 1000 V, 65 V, and 750 V, respectively. High resolution MS and MS/MS spectra were acquired in negative mode in the range 100–1000 *m*/*z* with a scan rate of 1.04 spectra/sec, employing the AutoMS/MS acquisition mode (1 precursor per cycle, CID voltage for tandem mass spectra 30 V, collision gas N2, purity 99.999%, FWHM (Full Width Half Maximum) of quadrupole mass bandpass used during MS/MS precursor isolation 4 *m*/*z*). MassHunter® Workstation Software (B.04.00) was used to carry out mass spectrometer control, data acquisition, and data analysis. The mass spectrometer was calibrated daily using Agilent tuning mix HP0321. An Agilent Zorbax Extend-C18 column (2.1 × 30 mm, particle size 1.8 μm) with precolumn Extend-C18 column (2.1 × 12.5 mm, particle size 1.8 μm) was used for the chromatographic separation. The injection volume was 20 μL and the flow rate was 0.2 mL/min.

The two eluent solutions were: A: trifluoroacetic acid (FA, 0.1% *v*/*v*) in bi-distilled water; B: FA (0.1% *v*/*v*) in acetonitrile (ACN, HPLC grade). The elution program started at 15% B, held for 1 min, then a linear gradient to 50% B was applied in 5 min, then to 70% B in 2 min and to 90%B in 7 min; 100% was reached after further 5 min; re-equilibration time took 5 min.

### **3. Results and Discussion**

HPLC/DAD and HPLC-ESI-Q-ToF MS analyses were performed in order to determine the dyestuff used in the Viking Age textiles from the Gokstad ship's grave.

The chromatograms obtained from the extracts after DMSO extraction did not show any relevant peaks. The extracts obtained after EDTA/DMF procedure gave satisfactory results for sample S1, providing the highest peaks in HPLC-ESI-Q-ToF MS analysis. Only the harsh extraction based on hydrolysis followed by extraction in ethylacetate provided results for sample S2, S3, and S4, and was also successfully applied to S1.

The three samples S1, S3, and S4 showed (Figures 2, 4 and 5) a very similar composition, consisting of the presence of anthraquinones typical of madder-type dyestuffs.

In detail, Figure 2 shows the Extracted Ion Chromatograms for the HPLC-ESI-Q-ToF MS chromatograms of the EDTA/DMF extract of sample S1 from the ship's tent, where anthragallol, alizarin, munjistin, purpurin, and pseudopurpurin were detected. The detected anthraquinones are typical of a madder-dye source. The overall molecular profile was not typical of a specific dye source, although the highest content in alizarin respect to the other components may point to *Rubia tinctorum* as the original raw material. The good results obtained by the mild extraction might suggest that a relatively high amount of dye survived in the sample, or that a different recipe was used to obtain the desired color, with respect to the other samples. For comparison, the inset presents the results obtained by HPLC-DAD (shown at 254 nm) relative to sample S1 after hydrolysis followed by extraction in ethylacetate, which provided the highest peaks in the UV-Vis range.

**Figure 2.** EIC HPLC-ESI-Q-ToF MS chromatograms of the EDTA/DMF extract of sample S1, acquired in negative mode; where Anth is anthragallol (EIC—*m*/*z* 255.029), Ali is alizarin (EIC—*m*/*z* 239.031), Munj is munjistin (EIC—*m*/*z* 283.002), Pur is purpurin (EIC—*m*/*z* 255.029), and PsPur is pseudopurpurin (EIC—*m*/*z* 299.019). In the inset, the HPLC-DAD chromatogram at 254 nm relative to sample S1 extract after HCl/MeOH + AcOEt is presented for comparison.

The second sample collected from the ship's tent (S2, Figure 3), characterized by a yellow/white color at the time of analysis, showed the presence of traces of alizarin and purpurin (only detected by HPLC-ESI-Q-ToF MS, thanks to its superior sensitivity with respect to HPLC-DAD), pointing again to the use of a madder-based dye.

Samples S3 (Figure 4) and S4 (Figure 5), collected from the embroidery, contained the anthraquinones anthragallol, alizarin, purpurin, and pseudopurpurin; rubiadin was only present in S3.

**Figure 3.** EIC HPLC-ESI-Q-ToF MS chromatograms of the extract of sample S2 extract after HCl/MeOH + AcOEt, acquired in negative mode; where Ali is alizarin (EIC—*m*/*z* 239.031), Pur is purpurin (EIC—*m*/*z* 255.029).

**Figure 4.** EIC HPLC-ESI-Q-ToF MS chromatograms of the extract of sample S3 extract after HCl/MeOH + AcOEt, acquired in negative mode; where 4-OH-ba is 4-hydroxybenzoic acid (EIC—*m*/*z* 137.024), Anth is anthragallol (EIC—*m*/*z* 255.029), Ali is alizarin (EIC—*m*/*z* 239.031), Munj is munjistin (EIC—*m*/*z* 283.002), Pur is purpurin (EIC—*m*/*z* 255.029), PsPur is pseudopurpurin (EIC—*m*/*z* 299.019), Rubiadin (EIC—*m*/*z* 253.051). In the inset, the corresponding HPLC-DAD chromatogram at 254 nm is presented for comparison.

**Figure 5.** EIC HPLC-ESI-Q-ToF MS chromatograms of the extract of sample S4 extract after HCl/MeOH + AcOEt, acquired in negative mode; where 4-OH-ba is 4-hydroxybenzoic acid (EIC—*m*/*z* 137.024), Anth is anthragallol (EIC—*m*/*z* 255.029), Ali is alizarin (EIC—*m*/*z* 239.031), Pur is purpurin (EIC—*m*/*z* 255.029), PsPur is pseudopurpurin (EIC—*m*/*z* 299.019). In the inset, the corresponding HPLC-DAD chromatogram at 254 nm is presented for comparison.

In addition, the chromatograms feature 4-hydroxybenzoic acid, most probably due to degradation processes involving the fabric (silk) [34]. Peaks attributed to further unknown phenols based on their early retention times and maxima of absorption around 275 nm in the DAD spectra were also detected as highlighted in the insets in Figures 4 and 5, but were not confirmed by HPLC-ESI-Q-ToF MS analysis.

Table 2 summarizes the results obtained by both analytical systems (HPLC-DAD, HPLC-ESI-Q-ToF); it includes detected markers and identified dyes (associated with the presence of these compounds).


**Table 2.** Molecular markers detected along with identified materials in the analyzed extracts.
