Optimizing HPLC–HRMS Analysis of Indigoid Dyes in Archeological Textiles: Minimizing Extraction-Induced Degradation and Identifying Transformation Markers

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript reports interesting results for the molecular characterization of indigoid compounds identified in archaeological textiles. However, several sections require substantial revision, and the reliability of the extraction procedure should be statistically assessed. The following points should be clarified and discussed:

Introduction

The aims of the paper should be stated more clearly. In particular, the authors should clarify which are the main objectives (analyze the 17 archaeological textile samples; identify new degradation compounds or markers of indigoid dyes; and/or optimize and validate an extraction method?) and the novelties. Most of the mass spectra presented are already well documented in the literature. Therefore, the acquisition of these spectra using Orbitrap MS cannot be considered a novel or significant novelty of the study.

The sentences on page 4, lines 159–165, should be moved to the Introduction,  contributing to a clearer presentation of the state of art

Page 5

The paragraph presenting the mass spectra of standard indigoid compounds does not appear to contain novel information, as these compounds and their mass spectra are already well characterized in the literature. This material could therefore be moved to the Supplementary Information.

Extraction conditions

The description of the extraction procedure is confusing and should be substantially clarified.

The extraction of chromophores from the sample is a crucial step in this type of analytical procedure, because the amount of sample available is very small,  the concentration of chromophores in the sample can be lowered by degradation of the dye itself, textile washing. Moreover, contamination from materials in dust or soil, depending on the environment where the textile was stored. All that to say that a robust and statistically evaluated extraction procedure must be used.

Extraction procedures based on a combination of hydrolysis and solvent extraction, including also the use of an ultrasonic bath, have already been reported in the literature. For instance, the authors may refer to Karapanagiotis et al. (2019), Identification of dyes in Egyptian textiles of the first millennium AD from the collection Fill-Trevisiol, Archaeological and Anthropological Sciences, 11, 2699–2710, and to other several published works.

The authors sustain as a novelty the use of ultrasound-assisted extraction for avoiding the use of high temperature. Though this is not an innovation, performing extraction in an ultrasonic bath, the temperature of the extraction solution may increase during sonication up to 60°C or even more. The temperature reached during the extraction should therefore be monitored and reported, as it may influence the extraction efficiency and potentially the degradation of the monitored compounds.

The reproducibility of the extraction procedure described in the manuscript should be demonstrated, to validate the reliability of the reported results and to assess the trend reported for indigotin degradation. The extraction procedure should be evaluated statistically using an appropriate number of replicate samples: at least analyze six samples of the same size, and of comparable composition.                                                    

Results and Discussion (from page 6 to the end)

The Results and Discussion should be reorganized to begin with the analytical results obtained for the 17 archaeological textile samples. A representative chromatogram, together with the corresponding mass spectra and relevant analytical data, should be presented and discussed in this section.  

The authors should first demonstrate the presence of indigotin and its degradation products in the archaeological samples and then discuss the significance and novelty of these findings in comparison with previously published literature.

The possible absence of other dye materials in the analyzed textiles should also be addressed, establishing their absence.  Indeed, very often in textiles tannins are present: have the authors checked for their presence?

The mass spectrum of tryptanthrin is also available in the MoNA database (https://pubchem.ncbi.nlm.nih.gov/compound/Tryptanthrin#section=LC-MS), contrary to what was reported in the manuscript.  The mass spectrum of N,N'-oxalyldianthranilic acid needs to be better interpreted.

Regarding the degradation products of indigotin, the authors may also consider citing the following reference: https://pdf.benchchem.com/80/An_In_depth_Technical_Guide_to_the_Degradation_Products_of_Indigo_Under_UV_Irradiation.pdf.

Table 1

The meaning of “identification confidence level” should be clearly defined explaining the criteria used to assign confidence level. Generally, a numerical matching score is used, for MS matching  (e.g., a match higher than 90% is considered sufficient for identification) and a positive match for retention times within 1-2%. Please, provide justification for the criterion used avoiding ++/+++.

Table 2

The notation “+- indicates that the compound was detected at the background level” is unclear and should be explained in detail specifying the analytical and statistical criteria used. In particular, the authors should provide a statistically supported criterion for determining whether a compound is present or absent in a sample. The authors should describe how the background signal was determined, and which criterion was used to distinguish a true detection from background noise.  

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The authors investigated the presence and degradation of indigo dyes and their decomposition products in medieval textiles from archaeological sources by advanced analytical techniques. The topic and the results are of interest from analytical as well as from archaeological point of view.

The manuscript describes the applied methods very clear. The authors adapted treatment methods in order to optimize the extraction of molecular components from the sensitive archaeological samples. In case of some samples, it could be shown with high probability that indigo was used for dying.  For my opinion, the work is a nice basis for future work on application HPLC and mass spectroscopy for identification of organic components in archaeological samples.

As a reader of the paper, I would wish to learn a little more about the differences between the textile samples including differences in fibres or tissues and their specific original archaeological situation. Are correlations between the specific archaeological situations of the 17 samples and the data of table 2 possible?

I like that the archaeological situation and the historical role of the sampling site are explained in the second section (“materials and methods”). I suggest to add a short statement (at the end of the discussions or in the conclusions) what the analytical results could teach about formerly human activities or the history of place. Are there any new archaeological insights possible based on the chemical data?

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been amended according to the reviewers’ suggestions. Nevertheless, some further observations are reported below.

Abstract

- Lines 34–36: These lines are not necessary and should be deleted.

Page 12, Lines 331–332

- Could the authors clarify the meaning of this sentence? Certified reference textiles containing flavonoids and tannins are not available. To investigate the absence of these compounds, the authors should extract and examine the relevant ions from the acquired chromatograms. Please clarify and comment on the approach adopted.

Page 17, Lines 426–427

- The statement reported in these lines is not correct. It is well established that acid hydrolysis at 100 °C can cause dye degradation, which is why alternative treatments have been developed. Nowadays, mild extraction procedures are commonly used for the analysis of textiles, such as extraction with a 0.1% Na₂EDTA solution in H₂O/DMF (1:1, v/v) in an ultrasonic bath at 60 °C for 1 h, as reported in https://doi.org/10.1007/s12520-021-01465-3 , or the method described in https://doi.org/10.1021/acs.analchem.8b05469 .

Since the authors employed acid hydrolysis, they should limit their statement to highlighting the role of sonication in reducing indigotin degradation. However, the potential effects of this treatment on other dyes should also be considered. What is the impact of the selected hydrolysis conditions on the stability and recovery of the other analytes? Please provide a discussion of this issue.

Page 18 and Figure 8

- It appears that the use of DMF results in a small average increase in analyte recoveries (approximately 10%) across all compounds, suggesting that no significant degradation occurs. Therefore, the comments reported in lines 452–457 appear to be contradictory.

In addition, Table S3 should report RSD values as percentages (%). The meaning of the values reported under “MeCN/DMF” is unclear and should be explained. Furthermore, the reported values do not appear to be reliable. Please verify the calculations, clarify the meaning of these values, and revise the table and corresponding discussion accordingly.

Page 19, Lines 470–472

- These lines are redundant and should be deleted.

Page 23, Lines 547–548

- These lines can be deleted.

Author Response

Please see the attached file.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

I think that the manuscript can be accepted, now.

Author Response

We thank the reviewer for the positive decision regarding the manuscript.