# Identification of yellow dye plant chemical markers for applications in cultural heritage using LC-MS-based metabolomics and molecular networking

Lindsay Mas-Normand, Olivier Dangles, Carole Mathe, Gérald Culioli

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## To cite this version:

Lindsay Mas-Normand, Olivier Dangles, Carole Mathe, Gérald Culioli. Identification of yellow dye plant chemical markers for applications in cultural heritage using LC-MS-based metabolomics and molecular networking. Microchemical Journal, 2025, 218, pp.115621. 10.1016/j.microc.2025.115621. hal-05442216

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Submitted on 5 Jan 2026

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# Identification of yellow dye plant chemical markers for applications in cultural heritage using LC-MS-based metabolomics and molecular networking

Lindsay Mas-Normand<sup>a</sup>, Olivier Dangles<sup>b</sup>, Carole Mathe<sup>a</sup>, Gérald Culioli<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

Metabolomics

LC-HRMS

Molecular networking

Yellow dye plants

Chemical markers

Cultural heritage

## ABSTRACT

Before the advent of synthetic dyes in the mid-19th century, dye plants were one of the main resources for coloring textiles. The analysis of specific molecules present in cultural heritage objects can help to determine the plants used to dye them and thus to better understand the context in which they have been manufactured. However, finding the botanical origin of the yellow color can be challenging. Indeed, there is a wide diversity of yellow dye plants in nature and some natural yellow compounds are present in several species, which compli- cates the identification of the producing organism. Surprisingly, metabolomics, a powerful analytical approach for comparing large sets of molecules in complex samples and detecting specific chemical markers, has so far only rarely been used for cultural heritage purposes. In this context, an LC-MS-based metabolomics approach was developed using a panel of fourteen common yellow dye plant species. Statistical analyses then revealed chemical markers for each of them and the molecules of interest were tentatively annotated using several ap- proaches, including molecular networking.

## 1. Introduction

Plants have been used for millennia and until the middle of the 19th century to dye many textiles, such as clothes, tapestries, carpets, and other objects made from leather, hair or feathers [1]. Of all the colors, yellow is the one that can be produced from the greatest diversity of species, ranging from weld to pomegranate or saffron [2]. Many flavo- noids are responsible for this color, including flavones, flavonols, iso- flavones, chalcones and aurones. Other molecules, such as carotenoids, curcuminoids, alkaloids, anthraquinones and tannins can also produce a yellow hue [1]. Determining the botanical origin of the dyes found in a cultural heritage context can provide valuable information for chemists, historians, conservators and restorers. Thus, the identification of the raw materials used to craft an object can help to determine the technique, the period and the place of manufacture, as well as the social status of the person who owned it [1-4]. The discovery of taxonomic chemical markers -defined as chemical compounds specific to certain plants or raw materials- is therefore of crucial importance for the preservation and restoration of cultural heritage objects. By identifying these chem- ical markers, scientists can accurately reconstruct ancestral recipes,

providing invaluable information on traditional techniques and know- how.

Since the 1970s, a wide range of analytical methods has been developed in the field of cultural heritage. These methods aim to obtain the most relevant information about a specific object and can be clas- sified according to their degree of intervention (low, medium, high), their usefulness, and the innovative nature they bring [5,6]. Among the methods used to characterize dyestuffs, some do not need micro- sampling, such as reflectance, fluorescence or Raman spectroscopies [7-12], while others require microsamples, such as liquid chromatog- raphy (LC) or gas chromatography (GC) [2]. Due to the chemical nature of dyeing molecules, often non-volatile and rather polar, the technique of choice among these hyphenated techniques is liquid chromatography coupled with UV-visible spectroscopy (LC-UV) and/or mass spectrom- etry (LC-MS) [13]. This analytical tool allows the separation of mole- cules of a sample and then the obtaining of characteristic UV-visible and/or mass spectra for each compound. Several LC-UV or LC-MS studies of ancient clothing or objects have been able to trace the natu- ral sources of the dyes used at the time [14-19]. For example, in Chinese yellow textiles from Dunhuang (7th-10th century) from the British

<footnote><sup>a</sup> IMBE, Aix Marseille University, Avignon University, CNRS, IRD, Avignon, France</footnote>

<footnote><sup>b</sup> SQPOV, INRAE, Avignon University, Avignon, France</footnote>

<footnote>* Corresponding author.
E-mail address: gerald.culioli@univ-avignon.fr (G. Culioli).</footnote>
Museum's collection, traces of smoketree (Cotinus coggygria; also known as young fustic), pagoda tree (Styphnolobium japonicum), Chinese cork tree (Phellodendron chinense), safflower (Carthamus tinctorius), larkspur (Delphinium semibarbatum), weld (Reseda luteola), saffron (Crocus sativus), sappanwood (Biancaea sappan) and cape jasmine (Gardenia jasminoides) have been detected. These results were consistent with the plants traditionally used in this part of the world at that time. It was also observed that the dyeing techniques were not always typical of Asia, suggesting potential exchanges of knowledge or materials along the Silk Road [20]. In yellow, orange, brown and green fibers of Polish liturgical vestments in the collections of Krakow churches (15th–17th century), the presence of weld, dyer's broom (Genista tinctoria), Persian berries (Rhamnus spp.), sawwort (Serratula tinctoria), smoketree, larkspur, bastard hemp (Datisca cannabina) and annatto (Bixa orellana) has been demonstrated. Some of these botanical species could only be identified using the MS/MS data of their extracts, demonstrating the importance of mass spectrometry for the annotation of unknown compounds [21].

However, it can be quite difficult in the field of cultural heritage chemistry to accurately determine the plants used during the manufacture of a yellow-dyed object, due to the great diversity of yellow dye plants and the many common dyestuffs present in several species as major compounds of the corresponding extracts, such as kaempferol, quercetin or luteolin [2]. In this context, the objective of the present work was to develop an analytical method dedicated to the characterization of specific chemical markers of a selection of common yellow dye plants, in order to facilitate the identification of such plants when analyzing cultural heritage objects. To do this, a metabolomics workflow involving ultra-performance liquid chromatography coupled with high-resolution mass spectrometry in tandem (UPLC-HRMS/MS), statistical analyses and the use of annotation tools, such as molecular networking, was developed.

Metabolomics, the latest branch of omics science, is emerging as an innovative and powerful analytical approach dedicated to the search for chemical markers, thanks to its ability to exhaustively analyze the whole set of metabolites (i.e., low molecular weight molecules) present in a biological sample and to handle large-scale and complex datasets [22]. A complete metabolomics workflow involves a number of steps, including sample collection, preparation and analysis, followed by data processing and chemometrics [23,24]. The most common analytical techniques used in metabolomics are nuclear magnetic resonance (NMR) and liquid chromatography, gas chromatography or capillary electrophoresis (CE) coupled with mass spectrometry (MS) [25]. Among the main applications of metabolomics are functional genomics and environmental or biological stress studies [26,27], but it is also an important tool for biomarker discovery [28]. Thus, metabolomics has been widely used in various fields, such as health, environment or food science [29], but it is still a rarely used approach both in archaeometry and in the field of natural dyes in a cultural heritage context [30]. In parallel with the development of metabolomics, gaps have appeared in the metabolite annotation stage. For this reason, original analytical tools have been created in the wake of the exponential use of metabolomics approaches, including molecular networking and predictive mass fragmentation algorithms (e.g., SIRIUS, CFM-ID...). More particularly, molecular networking is a graphical representation of untargeted mass spectrometry datasets, where structural similarities between metabolites can be visualized. This bioinformatics tool is widely used for the identification and annotation of natural products [31]. The Global Natural Products Social (GNPS) platform works on the principle that two structurally related molecules share similar ion patterns in their fragmentation spectrum (MS/MS) [32]. The MS/MS spectrum of each metabolite is compared to all the others to create a network where metabolites are represented as nodes connected to each other on the basis of their spectral similarities. Queries on spectral library databases are also available to annotate metabolites where possible.

In this context, the aim of this study was to evaluate the potential of metabolomics in the search for specific chemical markers, answering a

particular scientific question in the field of cultural heritage. For this purpose, we developed an UPLC-HRMS/MS based metabolomics approach specifically dedicated to the search for and the putative annotation of taxonomic chemical markers in plants commonly used as sources of yellow dyes. To this end, a panel of fourteen plants was selected in the light of our knowledge of their ancestral use as a yellow dye, varying the botanical families, the plant parts used or the chemical family of dye compounds (flavonoids, phenylpropanoids, anthraquinones, alkaloids...), but also considering some very closely related species (e.g., three *Rhamnus* spp. and two *Maclura* spp.). After extraction, the yellow dyes were analyzed by UPLC-HRMS/MS. Then, statistical analyses were carried out to identify potential chemical markers for each plant and their annotation was achieved using several approaches, such as molecular networking (GNPS), de novo molecular formula annotation software (SIRIUS), comparison with databases or manual annotation. Unlike previous works, the present study focused on the most discriminating metabolites for each species, not necessarily on the most abundant compounds or on colored molecules.

## 2. Experimental

### 2.1. Plant materials

The fourteen species of yellow dye plants used in this study are listed in Table 1. Dried flowers of *Carthamus tinctorius*, immature berries of *Rhamnus cathartica* and mature berries of *Rhamnus alaternus* and *Rhamnus saxatilis* were obtained from the laboratory herbarium. All other samples of dried plants were provided by Couleur Garance (Lauris, France).

### 2.2. Chemicals

Plant extraction was carried out using ultrapure water (EASYpure RF purification system; Barnstead) and HPLC-grade methanol (MeOH) (VWR Chemicals; Fontenay-sous-Bois, France).

UPLC-HRMS/MS analyses were performed using ultrapure water (Synergy UV purification system; Millipore), LC-MS-grade acetonitrile (ACN) (Carlo Erba Reagents; Val de Reuil, France) and LC-MS-grade formic acid (FA) (Fisher Chemical; Illkirch, France).

### 2.3. Sample preparation

Dried plant samples were crushed with a mill (A11 basic model; IKA). Then, 20 mg of the resulting powder obtained for each species

<caption>**Table 1**</caption>

List of the yellow dye plant species, and their corresponding part, used in this study.

<table><thead><tr><th>Scientific name</th><th>Common name</th><th>Family</th><th>Plant part</th></tr></thead><tbody><tr><td>Berberis vulgaris</td><td>Common barberry</td><td>Berberidaceae</td><td>Wood bark</td></tr><tr><td>Carthamus tinctorius</td><td>Safflower</td><td>Asteraceae</td><td>Flower</td></tr><tr><td>Cotinus coggygria</td><td>Smoke tree</td><td>Anacardiaceae</td><td>Heartwood</td></tr><tr><td>Datisca cannabina</td><td>False hemp</td><td>Datiscaceae</td><td>Root, branch, leaf</td></tr><tr><td>Helichrysum stoechas</td><td>Mediterranean strawflower</td><td>Asteraceae</td><td>Flower, leaf</td></tr><tr><td>Hypericum perforatum</td><td>St. John's wort</td><td>Hypericaceae</td><td>Flower, branch</td></tr><tr><td>Maclura pomifera</td><td>Osage orange</td><td>Moraceae</td><td>Heartwood</td></tr><tr><td>Maclura tinctoria</td><td>Old fustic</td><td>Moraceae</td><td>Heartwood</td></tr><tr><td>Punica granatum</td><td>Pomegranate</td><td>Lythraceae</td><td>Fruit peel</td></tr><tr><td>Reseda luteola</td><td>Weld</td><td>Resedaceae</td><td>Whole plant</td></tr><tr><td>Rhamnus alaternus</td><td>Italian buckthorn</td><td>Rhamnaceae</td><td>Berry (mature)</td></tr><tr><td>Rhamnus cathartica</td><td>Common buckthorn</td><td>Rhamnaceae</td><td>Berry (immature)</td></tr><tr><td>Rhamnus saxatilis</td><td>Dyer's buckthorn</td><td>Rhamnaceae</td><td>Berry (mature)</td></tr><tr><td>Styphnolobium japonicum</td><td>Pagoda tree</td><td>Fabaceae</td><td>Flower bud</td></tr></tbody></table>
were extracted at room temperature in triplicate in 2 mL of a mixture of MeOH/H₂O 1:1 (v/v) with an ultrasonic extractor (PEX1N model; R.E. U-S) for 10 min. Samples were centrifuged at 7000 rpm during 5 min (Frontier 5515 model; Ohaus) and the supernatants were transferred and evaporated under nitrogen at 35 °C. Finally, the dried extracts were solubilized at a final concentration of 1 mg.mL⁻¹ in MeOH/H₂O 1:1 (v/v) and filtered on PTFE syringe filters (25 mm, 0.2 µm; VWR, Radnor, PA, USA). Three experimental blank samples were extracted using the same procedure, but without any plant matrix. Moreover, quality control (QC) samples were prepared by mixing 40 µL of each plant extract and each blank sample and by dividing identically the resulting solution in ten QC samples. Additionally, an analytical blank constituted by a mixture of MeOH/H₂O 1:1 (v/v) was also prepared.

A QC was first injected ten times before starting sample analysis. Natural extracts and experimental blanks were then randomly injected and a QC was injected every five samples. The analytical blank was injected twice at the beginning and twice at the end of the injection sequence.

## 2.4. UPLC-HRMS/MS analysis

Samples were analyzed on an Acquy UPLC I-Class chromatographic system coupled to a SYNAPT G2-Si mass spectrometer (Waters), equipped with an electrospray ionization source (ESI) operating in positive and negative modes.

The separations were carried out on a Kinetex C18 column (2.1 × 100 mm, 1.7 µm, 100 Å; Phenomenex) maintained at 35 °C. The mobile phase was composed of H₂O (A) and ACN (B) with 0.1 % FA each and the flow rate was set at 0.4 mL.min⁻¹. The following gradient was applied: the eluent was maintained at 15 % B from 0 to 1 min, increased from 15 % to 100 % B from 1 to 11 min, maintained at 100 % B from 11 to 13.5 min, returned to 15 % B from 13.5 to 13.7 min and stabilized at this composition from 13.7 to 16 min. 5 µL of each sample (plant extracts, blanks and QCs) were injected for each run.

The MS source parameters were set as follows: capillary voltage 1 kV (ESI+) or 2.8 kV (ESI-), sampling cone 40 V (ESI+) or 50 V (ESI-), source offset 80 V, source temperature 120 °C, desolvation temperature 450 °C, cone gas flow 50 L.h⁻¹, desolvation gas flow 850 L.h⁻¹ and nebulizer 6.5 bar. For mass correction, a 5 mM sodium formate solution in isopropanol/H₂O 9:1 (v/v) was used to calibrate the instrument prior to the analysis. A 2 ng.µL⁻¹ leucine enkephalin solution in ACN/H₂O 1:1 (v/v) with 0.1 % FA was infused at a flow rate of 5 µL.min⁻¹ during each analysis. Acquisitions were done in Fast DDA (data-dependent acquisition) mode, both in positive and negative polarities, and the analyzer was set in the resolution mode. Ions from 50 to 1200 Da were scanned during 0.2 s. The five most intense ions with an intensity threshold greater than 5000 were fragmented in the transfer part of the TriWave, using a collision energy ramp from 10 to 40 V (m/z 50) to 40–70 V (m/z 1200). Fragment ions were scanned during 0.1 s and precursor ions were excluded for 6 s after their fragmentation. Data format was in continuum for both MS et MS/MS scans. An exclusion list was built based on data obtained with the analytical blank samples in both positive and negative modes. The thirty most intense ions observed for each mode were excluded during the entire analysis period.

## 2.5. Data processing

### 2.5.1. Data conversion and processing

Raw files were converted into "*mzML*" format using MSConvert (vers. 3.0) [33] with a 64-bit binary encoding precision and the peak picking filter (MS levels 1–2). "*Write index*", "*Use zlib compression*" and "*TPP compatibility*" options were selected. A homemade script [34] was applied to *mzML* files, in order to overcome the issue concerning *m/z* values of precursor ions of MS/MS scans in the MZmine software. The corrected *mzML* files were then imported in MZmine (vers. 2.53) [35] and cropped from 0 to 12 min with *m/z* values ranging from 50 to 1200.

The mass list of each spectrum was established in centroid mass detector from ions with a noise level above 5E4 for MS scan and 4E1 for MS/MS scan. The "*ADAP chromatogram builder*" module [36] was used with a minimum group size of 5 scans, a group intensity threshold of 1E5, a minimum highest intensity of 1.5E5 and a tolerance of *m/z* 0.005 or 10 ppm. Chromatograms deconvolution was performed with the "*Local minimum search*" algorithm set with a chromatographic threshold of 90 %, a search minimum range of 0.04 min, a minimum relative height of 0 %, a minimum absolute height of 1.5E5, a minimum ratio of 1 peak top/edge, a peak duration range from 0.01 to 1 min, a median *m/z* center calculation, a *m/z* range and a RT range for MS2 scan pairing of 0.005 Da and 0.25 min, respectively. Isotopes were grouped with a mass tolerance of *m/z* 0.005 or 10 ppm, a retention time tolerance of 0.25 min, a maximum charge of 1 and the most intense peak was chosen as representative isotope. The "*Join aligner*" module was used to align the peaks with a mass tolerance of *m/z* 0.005 or 10 ppm and a retention time tolerance of 0.25 min (weight 1/1). Missing peaks in the feature list were filled with the "*Peak finder (multithreaded)*" module with an intensity tolerance of 10 %, a mass tolerance of *m/z* 0.005 or 10 ppm and a retention time tolerance of 0.25 min. Duplicate peaks were removed using the "*New average*" filter mode, with a mass tolerance of *m/z* 0.001 or 5 ppm and a retention time tolerance of 0.1 min. The peak number ID was reset before the export of the processed data.

### 2.5.2. Statistical analysis

Statistical analyses were performed using the online platform MetaboAnalyst (vers. 6.0) [37]. The two data matrices containing *m/z* detected in positive and negative modes, respectively, were imported, normalized by "*sum*", "*square root transformed*" and "*mean centered*". As a reminder, an "*m/z* feature" (referred to as a "feature" in the following) is a chemical signal defined by its *m/z* (mass-to-charge ratio) value, retention time and peak intensity. For each matrix, a dendrogram was created using Euclidean distance measure and Ward clustering algorithm. A top 300 features heatmap was generated based on *t*-test/ANOVA method, using the same parameters as for the dendrogram. Principal component analysis (PCA) was used to observe the chemical distribution of the studied species. Then, partial least squares discriminant analyses (PLS-DA) were carried out by comparing one species to all the others, to understand which compounds (taxonomic chemical markers) were the most involved in the chemical discrimination. This procedure was repeated for each species, thus leading to fourteen PLS-DA for each ionization mode.

### 2.5.3. Construction of molecular networks

From the same LC-MS/MS datasets, molecular networks were built in ESI+ (Supporting information, MN S1) and ESI- (Supporting information, MN S2) ionization modes using the Global Natural Products Social Molecular Networking (GNPS) online platform [38]. The "*Super quick start interface*" was used and the Feature Based Molecular Networking (FBMN) analysis was chosen ( workflow version 27) [39]. The resulting data were imported into Cytoscape (vers. 3.9.1) [40] to visualize and annotate the molecular networks.

### 2.5.4. Identification and selection of taxonomic chemical markers

Three different methods were applied to identify taxonomic chemical markers for each plant. In method #1, the fifteen metabolites of each species, with the highest variable importance in projection (VIP) score obtained from the PLS-DA, were selected. In method #2, metabolites with a normalized concentration value greater than 3 on the top 300 features of the heatmap were selected. Finally, in method #3, for each species the fifteen metabolites with the greatest normalized peak intensity on the molecular networks were selected.

The results obtained from the three methods were combined and a specificity score based on molecular network intensities was determined for each molecule. This score was calculated as the peak area of the metabolite in the species divided by the sum of the peak areas of this
metabolite across all species. Chemical markers were then classified into three categories. The “Gold” category brought together metabolites obtained from the three methods and showing a specificity score greater than 90 %. The “Silver” category included compounds obtained from one of the statistical methods (method #1 or method #2; i.e., PLS-DA or heatmap) and the molecular network method (method #3) and with a specificity score greater than 80 %. Finally, the “Bronze” category gathered chemical markers obtained from one of the three methods and with a specificity score greater than 80 %.

### 2.5.5. Chemical markers annotation

To carry out the annotation of the selected chemical markers, several tools were used. During the construction of the molecular networks, MS/MS data of each metabolite were compared to spectral libraries on GNPS. For ions matching a database spectrum, the proposed annotation was checked on LOTUS [41], an online natural products database, and from literature data. For ions with no match, their MS spectrum was imported into SIRIUS (vers. 6.0.5) [42] and then the proposed annotation was checked as described before.

## 3. Results and discussion

To demonstrate the potential of metabolomics to authenticate specific plant-derived natural dyes in a cultural heritage context, a LC-MS-based metabolomics approach was developed and applied to a selection of fourteen common yellow dye plants. The concomitant use of HRMS and HRMS/MS data, combined with the help of various annotation tools, not only enabled the detection of specific botanical chemical markers, but also prevented the misuse of non-specific ones.

### 3.1. LC-MS metabolomics-based profiling of a selection of yellow dye plants

The preliminary step of this LC-MS-based metabolomics workflow consisted of a solid/liquid ultrasound-assisted extraction of the dried plant powders using a mixture of MeOH/H₂O, which allowed the extraction of a wide range of dye molecules. This procedure gave extraction yields (expressed as percentage of extract weight divided by dried plant weight) ranging from 3 % (M. tinctoria) to 56 % (P. granatum) (Supporting information, Fig. S1). It is worth noting that among the samples analyzed, those corresponding to heartwood and bark were those with the lowest extraction yields (less than 20 % in all cases). During the second step, the resulting plant extracts were analyzed by ESI+ and ESI- UPLC-HRMS/MS using a data-dependent acquisition (DDA) mode. The subsequent data extraction step with MZmine software allowed the detection of 1962 features (including 1666 fragmented features) in ESI+ mode, and 1690 features (including 1464 fragmented features) in ESI- mode. The final dataset matrices (ESI+ and ESI-) were separately statistically analyzed using the online MetaboAnalyst platform.

First, unsupervised PCA analyses were used to observe overall clustering effect based on chemical profiles of the samples, which also means detecting the general separation of the whole set of samples and finding hypothetical outliers (Fig. 1). For both ionization modes, more than 29 % of the total variance was explained from the two first PC axes, and 5 axes were needed to explain more than 56 % of the total variance (Supporting information, Fig. S2). In both cases, no outliers were detected and overall intra-species differentiations were less pronounced than inter-species differentiations. On the PCA score plot obtained from ESI+ data (Fig. 1a) B. vulgaris was clearly differentiated from all other species along both PC axes. This tendency could be explained by the fact that this species is one of the few among the selected plants to bio-synthesize alkaloids, more particularly isoquinoline alkaloids (e.g., berberine, magnoflorine and jatrorrhizine), which can be easily detected by LC-MS in ESI+ mode. Among the other species, on one hand, Rhamnus spp. were fairly differentiated from the others (R. cathartica

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** PCA score plots obtained from UPLC-HRMS data of the fourteen yellow dye extracts (n = 3) analyzed in (a) ESI+ and (b) ESI- modes (BV: Berberis vulgaris, CC: Cotinus coggygria, CT: Carthamus tinctorius, DC: Datisca cannabina, HP: Hypericum perforatum, HS: Helichrysum stoechas, MP: Maclura pomifera, MT: Maclura tinctoria, PG: Punica granatum, RA: Rhamnus alaternus, RC: Rhamnus cathartica, RL: Reseda luteola, RS: Rhamnus saxatilis, SJ: Styphnolobium japonicum). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

was slightly different from the two other *Rhamnus* species, *R. saxatilis* and *R. alaternus*, which appeared very similar) while, on the other hand, for *Maclura* spp., and to a lesser extent *C. coggygria*, the same trend was also observed. This tendency was more clearly observed on the PCA score plot obtained from ESI- data (Fig. 1b): the three *Rhamnus* species were clearly separated from other species along PC1 axis (and here again, *R. cathartica* was slightly differentiated from *R. saxatilis* and *R. alaternus*), while *Maclura* spp. and *C. coggygria* were differentiated from other species along PC2 axis. It should be noted that *Rhamnus* spp.
samples consisted of berries, *C. coggygria* and *Maclura* spp. samples were heartwoods, while other plant parts were extracted for other species. From a phytochemical point of view, such differences observed on the two PCA score plots could be due to the occurrence of specific chemical families in each of these particular species (e.g., xanthones from *Maclura* spp. [43], aurones from *Cotinus coggygria* [44], and anthraquinones from *Rhamnus* spp. [45]), while more common flavonoid subfamilies occurred in the vast majority of species studied herein.

Hierarchical clustering analysis (HCA) was then applied to the two dataset matrices. HCA is a descriptive statistical tool widely used to better understand multivariate datasets. Through agglomerative clustering, HCA allows identification of clusters or grouping of objects (e.g., samples or metabolites) by similarity. In the two HCA dendrograms

obtained with the ESI+ and ESI- datasets (Fig. 2), the first thing to notice was that the triplicates of each species were always grouped in the same clusters. More generally, it can be observed that *Rhamnus* spp. extracts were grouped separately from other plant extracts and, within this particular genus, extracts of *R. alaternus* and *R. saxatilis* were highly similar. Among the other species, heartwood extracts (i.e., *C. coggygria*, *M. tinctoria* and *M. pomifora*) formed a separate cluster within which the two *Maclura* species appeared to be very closely related. The main difference between the two dendrograms was due to *B. vulgaris* extracts, which in ESI+ mode (Fig. 2a) formed a specific cluster among species other than *Rhamnus* spp., whereas in ESI- mode (Fig. 2b) they were similar to species within this same group. As already argued, this difference could be due to a better detection in ESI+ mode of alkaloids characteristic of extracts from this plant. Moreover, it should also be pointed out that in both dendrograms, *S. japonicum* and *H. perforatum* on the one hand, and *C. tinctorius* and *P. granatum* on the other hand, were always found in fairly similar clusters. Lastly, even though *H. stoechas* and *C. tinctorius* belonged to two genera in the same botanical family (Asteraceae), their hydroalcoholic extracts were not very similar.

## 3.2. Detection of specific chemical markers for each yellow dye plant

As the overall chemical profiles of the species studied showed significant differences for some of them, the aim of this study was now to highlight the presence of chemical markers specific to a single species, which can later be used in a cultural heritage context as evidence of the use of a particular species for the creation of a craft object or an artwork. In order to identify such potential chemical markers, three analytical strategies were applied for each species: i) method #1: the fifteen metabolites with the highest VIP score obtained by building a PLS model pitting this particular species against all others were selected, (ii) method #2: metabolites with a normalized concentration value greater than 3 on the top 300 features of the heatmap generated based on t-test/ANOVA method were selected, and (iii) method #3: the fifteen metabolites of each species with the greatest normalized peak intensity on the molecular networks (Supporting information, MN S1-S2) were selected. Each of them had advantages and drawbacks. By comparing a species (group A) to all thirteen other species (group B) through a PLS-DA model, method #1 can lead to the identification of the most discriminating molecules, that is to say, those present in only one of the two groups. The problem that we may encounter is that a metabolite can be very abundant in only one species of group B. However, if it is absent in the other species of this group, the average area of the metabolite in this group will be close to zero and it could even be considered as a chemical marker of the species making up group A. In our case, due to the diversity of the species grouped together and the heterogeneity of their composition, PLS-DA allowed the selection of chemical markers that are major compounds of a specific plant extract, although not always specific to a single species.

On the heatmaps used with method #2, the abundance of each ion in each species was represented through a color scale. However, as the number of features was high, it was very difficult to visually exploit the data. For this reason, a maximum of 300 m/z features were chosen for each ionization mode. As the color scale was linked to the highest and lowest peak areas of the detected metabolites, reducing the number of features can impact the appearance of the heatmap. Through this method, heatmaps were used to visualize the abundance and specificity of metabolites in each species, but with the knowledge that some information would be reduced and lost, since only the 300 most relevant metabolites in each dataset were selected. Finally, by selecting through method #3 the fifteen most intense metabolites for each species in the molecular network, these chemical markers will be the most abundant compounds (and/or those that ionize best) in extracts of each species. However, they will not necessarily be species-specific, as common molecules, such as kaempferol, quercetin and rutin, are highly abundant in several plants. According to the obtained results, method #2

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** HCA dendrograms (Euclidean distance, Ward clustering) obtained from UPLC-HRMS data of the fourteen yellow dye extracts (n = 3; indexed as R1, R2 and R3) obtained in (a) ESI+ and (b) ESI- modes (BV: *Berberis vulgaris*, CC: *Cotinus coggygria*, CT: *Carthamus tinctorius*, DC: *Datisca cannabina*, HP: *Hypericum perforatum*, HS: *Helichrysum stoechas*, MP: *Maclura pomifera*, MT: *Maclura tinctoria*, PG: *Punica granatum*, RA: *Rhamnus alaternus*, RC: *Rhamnus cathartica*, RL: *Reseda luteola*, RS: *Rhamnus saxatilis*, SJ: *Styphnolobium japonicum*). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
(heatmap) selected the molecules with the highest specificity score, method #3 (molecular network) retained the most abundant compounds, while method #1 (PLS-DA) showed intermediate results. Each method provided different information and the three combined were complementary. In order to assess their robustness, chemical markers were then classified into three categories: (i) “Gold” for those that are the most specific (specificity score > 90 %) and have been highlighted by all three methods, (ii) “Silver” and (iii) “Bronze” for those that are slightly less specific (specificity score > 80 %) and have been identified by only two or one method(s), respectively. The number of chemical markers in the different categories for each species was highly variable, depending on the species and ionization mode (Supporting information, Fig. S3), and was not a crucial piece of information, due to the biases associated with the plant selection we have carried out. For example, each of *R. alaternus* and *R. saxatilis*, due to their great chemical similarity, showed only a few specific chemical markers, if any at all. For the remainder of this study, only the “Gold” chemical markers will be discussed in detail (Tables 2 and 3), but all the experimental data concerning the other two chemical markers categories are given in the chemical marker tables in Supporting information.

### 3.3. Putative annotation of chemical markers from a selection of yellow dye plants and comparison with those previously described in literature

In this part, “Gold” chemical markers detected in ESI+ and ESI-modes (Tables 2 and 3, respectively) for each plant were putatively annotated (see part 2.5.5) and compared with compounds commonly defined as specific constituents of the same species in previous studies dealing with dyeing methodologies and/or specifically dedicated to the analysis of cultural heritage objects (Table 4).

For *B. vulgaris*, not surprisingly, many isoquinoline-type alkaloids (i. e., jatrorrhizine, magnoflorine, berbamine, berbamunine as “Gold” chemical markers and berberine as a “Silver” chemical marker) were identified in wood bark hydroalcoholic extracts, mainly in ESI+ mode, as strong markers of this species (Tables 2 and 3; Supporting information, Table S1). This result was clearly consistent with previous studies, which have shown that hydroalcoholic extracts from various plant parts of *B. vulgaris*, and more widely from all *Berberis* spp., contained berberine as a major constituent, as well as other alkaloids present in lesser quantities, such as magnoflorine and jatrorrhizine [46,47]. Nevertheless, such alkaloids were not limited to this genus and, particularly in Asia and the Americas, plants of various botanical origins containing these compounds have been used since ancient times. In these contexts, which differ from that of this study, it is often the ratios between these different compounds that provide answers as to the botanical origin of dyes extracted from various objects of heritage interest [48–53]. In the field of dyeing and/or cultural heritage, berberine, berbamine, jatrorrhizine, and magnoflorine have been already defined as chemical markers of *B. vulgaris*, while berbamunine has never been described as such. Finally, a number of chemical markers already described in this species (e.g., demethyleneberberine, oxyacanthine, tetrahydrojatrorrhizine) were not detected as “Gold” chemical markers in this study (Table 4).

The hydroalcoholic extracts of flowers of *C. tinctorius* are known for their rich chemodiversity, which encompasses chemical families as varied as flavonoids, including the unusual quinochalcone subfamily, polyacetylenes, phenylpropanoids and alkaloids [54]. Here, two quinochalcones (anhydrosafflor yellow B and hydroxysafflor yellow B/C) and two other unidentified compounds were defined as “Gold” chemical markers of a flower extract of this species (Tables 2 and 3; Supporting information, Table S2). These two quinochalcones have been already described as characteristic of the use of *C. tinctorius* as a dyeing agent [55]. However, a wide variety of common quinochalcones in safflower extracts, such as the red pigment carthamin or safflor yellow A and their derivatives, were not detected as “Gold” chemical markers in this study (Table 4). This could be due to the low amounts naturally present in this

specific plant sample (due to the cultivar and/or environmental factors) or because of their instability.

In the case of *C. coggygria*, the “Gold” chemical markers of the heartwood hydroalcoholic extracts were fustin (a dihydroflavonol also called dihydrofisetin) and one of its isomers, which were detected in both ionization modes, together with two other compounds only detected in ESI- mode and putatively annotated as a gallotannin and a condensed tannin showing MS data compatible with those of a procyanidin trimer (Tables 2 and 3; Supporting information, Table S3). These results were consistent with the fact that this plant has already been reported to produce a wide range of phenolic compounds, including hydrolysable tannins (mainly gallotannins), phenolic acids, numerous classes of simple flavonoids (aglycones and glycosides), as well as other types of mixed (e.g., flavonolignans) or more complex (e.g., condensed tannins) phenolic derivatives. More precisely, the flavonoids produced by this plant belong to various subfamilies, among which chalcones (e. g., butein) and aurones (e.g., sulfuretin) are characteristic metabolites of its extracts [44]. Interestingly, fustin, which has already been used as a chemical identifier of this species, was the only simple flavonoid defined here as a “Gold” chemical marker, whereas other flavonoids, such as sulfuretin, fisetin, butin and butein, often cited as such were not (Table 4). Nevertheless, it can be specified that sulfuretin and disulfuretin were detected as “Silver” chemical markers.

Hydroalcoholic extracts from *D. cannabina* are known for their high flavonoid content, mainly flavonols (e.g., galangin, datiscetin) often present as rutinosides (e.g., galanginoside, cannabin), and for the occurrence of triterpenes [56,57]. It can also be noted that the only other species of the genus *Datisca*, *D. glomerata*, is known, like many plants in the Cucurbitaceae family, to biosynthesize glycosylated triterpenes of the cucurbitacin type [58]. However, no such compounds have been described in *D. cannabina* to date. In this study, three “Gold” chemical markers of *D. cannabina* were annotated as glycosylated flavonols: cannabin in ESI+ mode, and galanginoside and datinoside in ESI- mode. Two other “Gold” chemical markers were partially annotated as a triterpenoid (ESI+) and a dihydrochalcone glycoside (ESI-), while an unidentified one was detected in ESI- (Tables 2 and 3; Supporting information, Table S4). The two flavonols galangin et datiscetin, typically considered as good chemical markers of this species (Table 4), were only detected as “Silver” chemical markers.

*H. stoechas* belongs to a genus whose members give hydroalcoholic extracts particularly rich in phenylpropanoids, pyrones, acylphloroglucinol derivatives and flavonoids [59]. In this case, such molecules, namely two phenylpropanoid derivatives (caffeoylquinic and dicafeoylquinic acids), two flavonoids (3,5-dihydroxy-6,7,8-trimethoxyflavone and quercetagitrin) and five acylphloroglucinol derivatives, were identified as “Gold” chemical markers (Tables 2 and 3; Supporting information, Table S5). Interestingly, most of them are detected in both positive and negative modes. Even if the use of *Helichrysum* spp. as dyeing plants is described in the literature [60], very few studies were aimed at characterizing molecules specific to plants belonging to this genus in heritage objects (Table 4), and the chemical markers described here are being used for the first time in this specific context.

*H. perforatum* hydroalcoholic extracts are mainly composed by various classes of phenolic compounds, including prenylated phloroglucinols (e.g., hyperforin and adhyperforin), flavonoids (e.g., flavonols, such as kaempferol, quercetin and rutin; flavones, such as luteolin), anthraquinone derivatives (e.g., hypericin and pseudohypericin) and phenolic acid derivatives [61]. According to these data, metabolomics allowed here to highlight, as “Gold” chemical markers, a biflavonoid (biapigenin) and a hyperforin derivative in ESI+, and a quercetin glycoside in ESI- (Tables 2 and 3; Supporting information, Table S6). *Hypericum* spp., and more particularly *H. perforatum*, are well-known as flavonol-containing dyeing plants [62] and their presence is supposed to be chemically characterized by quercetin, quercetin glycosides and xanthones, which is consistent with our findings [63] (Table 4). However, the capacity of phloroglucinol derivatives as chemical markers in
<table><caption>**Table 2**

List of “Gold” chemical markers for each plant species detected in ESI+ mode.</caption><thead><tr><th rowspan="2">Species</th><th rowspan="2">Putative annotation*</th><th rowspan="2">Molecular formula</th><th rowspan="2">m/z values</th><th rowspan="2">RT (min)</th><th colspan="4">Annotation</th><th rowspan="2">References</th></tr><tr><th>GNPS</th><th>Sirius</th><th>Lotus</th></tr></thead><tbody><tr><td rowspan="2">B. vulgaris</td><td>Magnoflorine<br>(in-source fragment ion)</td><td>C<sub>20</sub>H<sub>24</sub>NO<sub>4</sub><sup>+</sup><br>(C<sub>18</sub>H<sub>16</sub>O<sub>4</sub>)</td><td>342.1720 [M]<sup>+</sup><br>(297.1116 [M+H]<sup>+</sup>)</td><td>1.28</td><td>X</td><td>✓</td><td>✓</td><td>[70]</td></tr><tr><td>Berbamine/Oxyacanthine</td><td>C<sub>37</sub>H<sub>40</sub>N<sub>2</sub>O<sub>6</sub></td><td>305.1531 [M+2H]<sup>2+</sup></td><td>2.26</td><td>X</td><td>✓</td><td>✓</td><td>[70,71]</td></tr><tr><td rowspan="2">C. tinctorius</td><td>Jatrorrhizine/Columbamine</td><td>C<sub>20</sub>H<sub>20</sub>NO<sub>4</sub><sup>+</sup></td><td>338.1430 [M]<sup>+</sup></td><td>3.00</td><td>✓</td><td>✓</td><td>✓</td><td>[70]</td></tr><tr><td>Unidentified m/z 539<br>(+)-Fustin/(-)-Fustin<br>(in-source fragment ion)</td><td>C<sub>24</sub>H<sub>26</sub>O<sub>14</sub><br>(C<sub>15</sub>H<sub>12</sub>O<sub>6</sub><br>(C<sub>14</sub>H<sub>10</sub>O<sub>4</sub>)</td><td>539.1377 [M+H]<sup>+</sup><br>Not detected<br>(243.0644 [M+H]<sup>+</sup>)<br>289.0701 [M+H]<sup>+</sup></td><td>0.54</td><td>X</td><td>X</td><td>X</td><td>-</td></tr><tr><td rowspan="2">C. coggygria</td><td>(+)-Fustin/(-)-Fustin<br>(in-source fragment ion 1)</td><td>C<sub>18</sub>H<sub>12</sub>O<sub>6</sub><br>(C<sub>14</sub>H<sub>10</sub>O<sub>4</sub>)</td><td>(243.0645 [M+H]<sup>+</sup>)</td><td rowspan="2">2.18</td><td rowspan="2">✓</td><td rowspan="2">✓</td><td rowspan="2">✓</td><td rowspan="2">[72]</td></tr><tr><td>(in-source fragment ion 2)<br>(in-source fragment ion 3)</td><td>(C<sub>13</sub>H<sub>10</sub>O<sub>3</sub>)<br>(C<sub>7</sub>H<sub>4</sub>O<sub>3</sub>)</td><td>(215.0703 [M+H]<sup>+</sup>)<br>(137.0232 [M+H]<sup>+</sup>)<br>593.1871 [M+H]<sup>+</sup></td></tr><tr><td rowspan="2">D. cannabina</td><td>Cannabin<br>(in-source fragment ion)</td><td>C<sub>28</sub>H<sub>32</sub>O<sub>14</sub><br>(C<sub>16</sub>H<sub>12</sub>O<sub>5</sub>)</td><td>481.2945 [M+H]<sup>+</sup></td><td rowspan="2">3.75</td><td rowspan="2">✓</td><td rowspan="2">✓</td><td rowspan="2">✓</td><td rowspan="2">[73]</td></tr><tr><td>Unidentified triterpenoid</td><td>C<sub>30</sub>H<sub>40</sub>O<sub>5</sub></td><td>481.0973 [M+H]<sup>+</sup></td></tr><tr><td rowspan="2">H. stoechas</td><td>Dicaffeoylquinic acid</td><td>C<sub>25</sub>H<sub>24</sub>O<sub>12</sub></td><td>499.1240 [M-H<sub>2</sub>O+H]<sup>+</sup></td><td>2.82</td><td>✓</td><td>✓</td><td>✓</td><td>[76,77]</td></tr><tr><td>3,5-dihydroxy-6,7,8-trimethoxyflavone</td><td>C<sub>18</sub>H<sub>16</sub>O<sub>7</sub></td><td>345.0972 [M+H]<sup>+</sup></td><td>6.76</td><td>✓</td><td>✓</td><td>✓</td><td>[78]</td></tr><tr><td rowspan="2">H. perforatum</td><td>Acylphloroglucinol derivative “D”</td><td>C<sub>26</sub>H<sub>32</sub>O<sub>7</sub></td><td>457.2225 [M+H]<sup>+</sup></td><td>8.99</td><td>X</td><td>✓</td><td>✓</td><td>[59,74,79]</td></tr><tr><td>I3,I8-biapigenin/Amentoflavone</td><td>C<sub>36</sub>H<sub>18</sub>O<sub>10</sub></td><td>539.0978 [M+H]<sup>+</sup></td><td>4.52</td><td>(✓)</td><td>✓</td><td>✓</td><td>[80]</td></tr><tr><td rowspan="2">M. pomifera</td><td>33-deoxy-33-hydroperoxyfurohyperforin</td><td>C<sub>35</sub>H<sub>52</sub>O<sub>6</sub></td><td>591.3666 [M+Na]<sup>+</sup></td><td>9.61</td><td>X</td><td>✓</td><td>X</td><td>[81,82]</td></tr><tr><td>Methoxyxanthone</td><td>C<sub>14</sub>H<sub>10</sub>O<sub>6</sub></td><td>275.0549 [M+H]<sup>+</sup></td><td>2.08</td><td>X</td><td>✓</td><td>X</td><td>-</td></tr><tr><td rowspan="2">Lespedezaflavanone D/3-(3,4-dihydroxyphenyl)-5,7-dihydroxy-6,8-bis(3-methylbut-2-enyl)chroman-4-one<br>(in-source fragment ion)</td><td rowspan="2">Lespedezaflavanone D/3-(3,4-dihydroxyphenyl)-5,7-dihydroxy-6,8-bis(3-methylbut-2-enyl)chroman-4-one<br>(in-source fragment ion)</td><td>C<sub>25</sub>H<sub>28</sub>O<sub>6</sub><br>(C<sub>21</sub>H<sub>20</sub>O<sub>6</sub>)</td><td>425.1958 [M+H]<sup>+</sup></td><td>5.81</td><td>X</td><td>✓</td><td>✓</td><td>[43,83]</td></tr><tr><td>(369.1328 [M+H]<sup>+</sup>)</td><td>397.1640 [M+H]<sup>+</sup></td><td>6.33</td><td>✓</td><td>✓</td><td>✓</td><td>[43]</td></tr><tr><td rowspan="2">M. tinctoria</td><td>Prenylated xanthone</td><td>C<sub>23</sub>H<sub>24</sub>O<sub>6</sub></td><td>425.1959 [M+H]<sup>+</sup></td><td>7.17</td><td>X</td><td>✓</td><td>✓</td><td>[43,83]</td></tr><tr><td>Lespedezaflavanone D/3-(3,4-dihydroxyphenyl)-5,7-dihydroxy-6,8-bis(3-methylbut-2-enyl)chroman-4-one<br>(in-source fragment ion)</td><td>C<sub>25</sub>H<sub>28</sub>O<sub>6</sub><br>(C<sub>21</sub>H<sub>20</sub>O<sub>6</sub>)</td><td>(369.1334 [M+H]<sup>+</sup>)</td><td>1123.0296 [M+K]<sup>+</sup></td><td>0.54</td><td>X</td><td>✓</td><td>✓</td><td>[84,85]</td></tr><tr><td rowspan="2">P. granatum</td><td>Punicalagin isomer “A”</td><td>C<sub>48</sub>H<sub>28</sub>O<sub>30</sub></td><td>1067.0623 [M-H<sub>2</sub>O+H]<sup>+</sup></td><td>0.74</td><td>X</td><td>✓</td><td>✓</td><td>[84,85]</td></tr><tr><td>Punicalagin isomer “B”<br>(in-source fragment ion)</td><td>C<sub>48</sub>H<sub>28</sub>O<sub>30</sub><br>(C<sub>34</sub>H<sub>22</sub>O<sub>22</sub>)</td><td>(765.0567 [M-H<sub>2</sub>O+H]<sup>+</sup>)</td><td>435.0562 [M+H]<sup>+</sup></td><td>2.08</td><td>(✓)</td><td>✓</td><td>✓</td><td>[84]</td></tr><tr><td rowspan="2">Ellagic acid<br>(in-source fragment ion)</td><td>Ellagic acid-O-pentoside<br>(in-source fragment ion)</td><td>C<sub>19</sub>H<sub>14</sub>O<sub>12</sub><br>(C<sub>14</sub>H<sub>6</sub>O<sub>8</sub>)</td><td>303.0129 [M+H]<sup>+</sup></td><td>303.0133 [M+H]<sup>+</sup></td><td>2.26</td><td>✓</td><td>✓</td><td>✓</td><td>[84,85]</td></tr><tr><td>Unidentified m/z 511<br>(in-source fragment ion)</td><td>-</td><td>511.3400<br>(453.3361)</td><td>5.93</td><td>X</td><td>X</td><td>X</td><td>-</td><td>-</td></tr><tr><td>R. luteola</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>R. alaternus</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>R. cathartica</td><td>Unidentified m/z 176</td><td>C<sub>7</sub>H<sub>13</sub>NO<sub>4</sub></td><td>176.0918 [M+H]<sup>+</sup></td><td>0.52</td><td>X</td><td>X</td><td>X</td><td>-</td><td>-</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 2 (continued)**</caption><thead><tr><th rowspan="2">Species</th><th rowspan="2">Putative annotation*</th><th rowspan="2">Molecular formula</th><th rowspan="2">m/z values</th><th rowspan="2">RT (min)</th><th colspan="4">Annotation</th><th rowspan="2">References</th></tr><tr><th>GNPS</th><th>Sirius</th><th>Lotus</th></tr></thead><tbody><tr><td rowspan="2">R. saxatilis</td><td>Quercetin-3-O-rhamninoside</td><td>C<sub>33</sub>H<sub>40</sub>O<sub>20</sub></td><td>757.2183<br/>[M+H]<sup>+</sup></td><td>2.16</td><td>✓</td><td>✓</td><td>X</td><td>[68,86]</td></tr><tr><td>Rhamnetin-3-O-rhamninoside</td><td>C<sub>34</sub>H<sub>42</sub>O<sub>20</sub></td><td>771.2342<br/>[M+H]<sup>+</sup></td><td>3.12</td><td>✓</td><td>✓</td><td>✓</td><td>[68,86]</td></tr><tr><td rowspan="2">S. japonicum</td><td>–</td><td>–</td><td>–</td><td>–</td><td>–</td><td>–</td><td>–</td><td>–</td></tr><tr><td>Narcissin</td><td>C<sub>28</sub>H<sub>32</sub>O<sub>16</sub></td><td>625.1767<br/>[M+H]<sup>+</sup></td><td>2.59</td><td>✓</td><td>✓</td><td>✓</td><td>[87]</td></tr><tr><td rowspan="2">S. japonicum</td><td>Isorhamnetin</td><td>C<sub>16</sub>H<sub>12</sub>O<sub>7</sub></td><td>317.0651<br/>[M+H]<sup>+</sup></td><td>4.44</td><td>✓</td><td>✓</td><td>✓</td><td>[88,89]</td></tr><tr><td>Soyasaponin gamma-G</td><td>C<sub>48</sub>H<sub>74</sub>O<sub>17</sub></td><td>923.4990<br/>[M+H]<sup>+</sup></td><td>5.96</td><td>X</td><td>✓</td><td>X</td><td>[90,91]</td></tr></tbody></table>

RT: Retention time.

X Not identified, (✓) Other isomer identified, ✓ Right isomer identified.

<table><caption>**Table 3**

List of “Gold” chemical markers for each plant species detected in ESI- mode.</caption><thead><tr><th rowspan="2">Species</th><th rowspan="2">Putative annotation*</th><th rowspan="2">Molecular formula</th><th rowspan="2">m/z values</th><th rowspan="2">RT (min)</th><th colspan="4">Annotation</th><th rowspan="2">References</th></tr><tr><th>GNPS</th><th>Sirius</th><th>Lotus</th></tr></thead><tbody><tr><td rowspan="2">B. vulgaris</td><td>Berbamunine</td><td>C<sub>36</sub>H<sub>40</sub>N<sub>2</sub>O<sub>6</sub></td><td>595.2800 [M-H]<sup>-</sup></td><td>1.82</td><td>X</td><td>✓</td><td>✓</td><td>[92,93]</td></tr><tr><td>Hydroxysafflor yellow B/C</td><td>C<sub>27</sub>H<sub>32</sub>O<sub>16</sub></td><td>611.1602 [M-H]<sup>-</sup></td><td>1.20</td><td>X</td><td>✓</td><td>X</td><td>[94]</td></tr><tr><td rowspan="3">C. tinctorius</td><td>Anhydro)safflor yellow B</td><td>C<sub>48</sub>H<sub>52</sub>O<sub>26</sub></td><td>1043.2683 [M-H]<sup>-</sup></td><td>2.48</td><td>X</td><td>✓</td><td>✓</td><td>[95]</td></tr><tr><td>Unidentified m/z 329</td><td>C<sub>18</sub>H<sub>34</sub>O<sub>5</sub></td><td>329.2310 [M-H]<sup>-</sup></td><td>4.15</td><td>X</td><td>X</td><td>X</td><td>-</td></tr><tr><td>(+)-Fustin/(−)-Fustin</td><td>C<sub>15</sub>H<sub>12</sub>O<sub>6</sub></td><td>287.0541 [M-H]<sup>-</sup></td><td>1.69</td><td>✓</td><td>✓</td><td>✓</td><td>[72]</td></tr><tr><td rowspan="2">C. coggygria</td><td>(+)-Fustin/(−)-Fustin</td><td>C<sub>15</sub>H<sub>12</sub>O<sub>6</sub></td><td>575.1196 [2M-H]<sup>-</sup></td><td>2.16</td><td>✓</td><td>✓</td><td>✓</td><td>[72]</td></tr><tr><td>(in-source fragment ion)</td><td>(C<sub>14</sub>H<sub>12</sub>O<sub>5</sub>)</td><td>(259.0596 [M-H]<sup>-</sup>)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="7">D. cannabina</td><td>Pentagalloylglucose</td><td>C<sub>41</sub>H<sub>32</sub>O<sub>26</sub></td><td>939.1114 [M-H]<sup>-</sup></td><td>2.60</td><td>✓</td><td>✓</td><td>✓</td><td>[96,97]</td></tr><tr><td>Procyanidin trimer</td><td>C<sub>45</sub>H<sub>38</sub>O<sub>16</sub></td><td>833.2092 [M-H]<sup>-</sup></td><td>2.77</td><td>X</td><td>✓</td><td>X</td><td>[98]</td></tr><tr><td>Galanginoside</td><td>C<sub>27</sub>H<sub>30</sub>O<sub>14</sub></td><td>577.1563 [M-H]<sup>-</sup></td><td>2.88</td><td>(✓)</td><td>✓</td><td>✓</td><td>[73]</td></tr><tr><td>Dihydrochalcone glycoside</td><td>C<sub>21</sub>H<sub>24</sub>O<sub>9</sub></td><td>419.1330 [M-H]<sup>-</sup></td><td>3.13</td><td>X</td><td>✓</td><td>X</td><td>-</td></tr><tr><td>Datinoside</td><td>C<sub>28</sub>H<sub>32</sub>O<sub>15</sub></td><td>607.1660 [M-H]<sup>-</sup></td><td>3.36</td><td>X</td><td>X</td><td>X</td><td>[99]</td></tr><tr><td>Unidentified m/z 765</td><td>C<sub>39</sub>H<sub>58</sub>O<sub>15</sub></td><td>765.3703 [M-H]<sup>-</sup></td><td>4.36</td><td>X</td><td>X</td><td>X</td><td>-</td></tr><tr><td>Caffeoylquinic acid</td><td>C<sub>16</sub>H<sub>18</sub>O<sub>9</sub></td><td>707.1819 [2M-H]<sup>-</sup></td><td>0.92</td><td>✓</td><td>✓</td><td>✓</td><td>[76,77]</td></tr><tr><td rowspan="2">H. perforatum</td><td>Quercetatrin</td><td>C<sub>21</sub>H<sub>20</sub>O<sub>13</sub></td><td>479.0818 [M-H]<sup>-</sup></td><td>1.74</td><td>(✓)</td><td>✓</td><td>✓</td><td>[74,75]</td></tr><tr><td>Dicaffeoylquinic acid</td><td>C<sub>25</sub>H<sub>24</sub>O<sub>12</sub></td><td>1031.2467 [2M-H]<sup>-</sup></td><td>2.86</td><td>✓</td><td>✓</td><td>✓</td><td>[76,77]</td></tr><tr><td rowspan="2">H. stoechas</td><td>Acylphloroglucinol derivative “A”</td><td>C<sub>21</sub>H<sub>24</sub>O<sub>7</sub></td><td>387.1435 [M-H]<sup>-</sup></td><td>7.01</td><td>X</td><td>(✓)</td><td>(✓)</td><td>[59,100]</td></tr><tr><td>Acylphloroglucinol derivative “B”/“C”</td><td>C<sub>24</sub>H<sub>30</sub>O<sub>7</sub></td><td>429.1903 [M-H]<sup>-</sup></td><td>8.72</td><td>X</td><td>✓</td><td>X</td><td>[59]</td></tr><tr><td rowspan="2">M. pomifera</td><td>Acylphloroglucinol derivative “D”</td><td>C<sub>26</sub>H<sub>32</sub>O<sub>7</sub></td><td>455.2067 [M-H]<sup>-</sup></td><td>8.97</td><td>X</td><td>✓</td><td>✓</td><td>[59,79]</td></tr><tr><td>Acylphloroglucinol derivative “E”</td><td>C<sub>28</sub>H<sub>36</sub>O<sub>7</sub></td><td>483.2373 [M-H]<sup>-</sup></td><td>10.18</td><td>X</td><td>✓</td><td>X</td><td>[59]</td></tr><tr><td rowspan="2">M. tinctoria</td><td>Acylphloroglucinol derivative “F”</td><td>C<sub>30</sub>H<sub>40</sub>O<sub>7</sub></td><td>511.2694 [M-H]<sup>-</sup></td><td>11.08</td><td>X</td><td>✓</td><td>X</td><td>[59]</td></tr><tr><td>Isoquercetin/Hyperoside</td><td>C<sub>21</sub>H<sub>20</sub>O<sub>12</sub></td><td>927.1852 [2M-H]<sup>-</sup></td><td>2.33</td><td>(✓)</td><td>✓</td><td>✓</td><td>[80]</td></tr><tr><td rowspan="2">P. granatum</td><td>O-methylated flavonoid</td><td>C<sub>16</sub>H<sub>14</sub>O<sub>7</sub></td><td>317.0646 [M-H]<sup>-</sup></td><td>1.71</td><td>X</td><td>✓</td><td>X</td><td>-</td></tr><tr><td>Oxyresveratrol</td><td>C<sub>14</sub>H<sub>12</sub>O<sub>4</sub></td><td>243.0649 [M-H]<sup>-</sup></td><td>2.71</td><td>X</td><td>✓</td><td>✓</td><td>[101,102]</td></tr><tr><td rowspan="3">R. luteola</td><td>Oxyresveratrol derivative</td><td>C<sub>36</sub>H<sub>40</sub>O<sub>20</sub></td><td>791.1973 [M-H]<sup>-</sup></td><td>3.32</td><td>X</td><td>✓</td><td>X</td><td>-</td></tr><tr><td>Unidentified m/z 329</td><td>C<sub>19</sub>H<sub>22</sub>O<sub>5</sub></td><td>329.1380 [M-H]<sup>-</sup></td><td>3.41</td><td>X</td><td>X</td><td>X</td><td>-</td></tr><tr><td>Prenylated flavonoid</td><td>C<sub>20</sub>H<sub>22</sub>O<sub>7</sub></td><td>373.1300 [M-H]<sup>-</sup></td><td>3.83</td><td>X</td><td>✓</td><td>X</td><td>-</td></tr><tr><td rowspan="4">R. alaternus</td><td>Unidentified m/z 457</td><td>C<sub>25</sub>H<sub>30</sub>O<sub>8</sub></td><td>457.1856 [M-H]<sup>-</sup></td><td>5.16</td><td>X</td><td>✓</td><td>X</td><td>-</td></tr><tr><td>Prenylated xanthone</td><td>C<sub>23</sub>H<sub>26</sub>O<sub>7</sub></td><td>413.1593 [M-H]<sup>-</sup></td><td>6.29</td><td>X</td><td>✓</td><td>X</td><td>-</td></tr><tr><td>Prenylated dihydroflavonol</td><td>C<sub>25</sub>H<sub>28</sub>O<sub>7</sub></td><td>439.1747 [M-H]<sup>-</sup></td><td>6.54</td><td>X</td><td>✓</td><td>X</td><td>-</td></tr><tr><td>Prenylated xanthone</td><td>C<sub>23</sub>H<sub>26</sub>O<sub>6</sub></td><td>397.1642 [M-H]<sup>-</sup></td><td>6.71</td><td>X</td><td>✓</td><td>X</td><td>-</td></tr><tr><td rowspan="2">R. cathartica</td><td>Unidentified m/z 487</td><td>C<sub>30</sub>H<sub>48</sub>O<sub>5</sub></td><td>487.3415 [M-H]<sup>-</sup></td><td>6.33</td><td>X</td><td>X</td><td>X</td><td>-</td></tr><tr><td>Unidentified m/z 341</td><td>C<sub>16</sub>H<sub>22</sub>O<sub>8</sub></td><td>341.1223 [M-H]<sup>-</sup></td><td>2.26</td><td>X</td><td>X</td><td>X</td><td>-</td></tr><tr><td rowspan="2">R. saxatilis</td><td>Luteolin-7-O-glucoside</td><td>C<sub>21</sub>H<sub>20</sub>O<sub>11</sub></td><td>895.1945 [2M-H]<sup>-</sup></td><td>2.41</td><td>✓</td><td>✓</td><td>✓</td><td>[103]</td></tr><tr><td>Chrysoeriol</td><td>C<sub>16</sub>H<sub>12</sub>O<sub>6</sub></td><td>299.0547 [M-H]<sup>-</sup></td><td>4.37</td><td>✓</td><td>✓</td><td>X</td><td>[103]</td></tr><tr><td>R. alaternus</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>R. cathartica</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>R. saxatilis</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td rowspan="4">S. japonicum</td><td>Soyasaponin III</td><td>C<sub>42</sub>H<sub>68</sub>O<sub>14</sub></td><td>795.4530 [M-H]<sup>-</sup></td><td>5.42</td><td>X</td><td>✓</td><td>✓</td><td>[89]</td></tr><tr><td>Azukisaponin I</td><td>C<sub>42</sub>H<sub>68</sub>O<sub>13</sub></td><td>779.4579 [M-H]<sup>-</sup></td><td>5.79</td><td>X</td><td>✓</td><td>✓</td><td>[89]</td></tr><tr><td>Soyasaponin</td><td>C<sub>34</sub>H<sub>86</sub>O<sub>21</sub></td><td>1051.5488 [M-H<sub>2</sub>O-H]<sup>-</sup></td><td>5.87</td><td>X</td><td>✓</td><td>X</td><td>-</td></tr><tr><td>Soyasaponin gamma-G</td><td>C<sub>48</sub>H<sub>74</sub>O<sub>17</sub></td><td>921.4864 [M-H]<sup>-</sup></td><td>5.96</td><td>X</td><td>✓</td><td>X</td><td>[90,91]</td></tr><tr><td>R. luteola</td><td>Phaseoside IV</td><td>C<sub>48</sub>H<sub>76</sub>O<sub>17</sub></td><td>905.4901 [M-H<sub>2</sub>O-H]<sup>-</sup></td><td>6.25</td><td>X</td><td>✓</td><td>X</td><td>[89]</td></tr></tbody></table>

RT: Retention time.

X Not identified, (✓) Other isomer identified, ✓ Right isomer identified.

<footnote>* Annotation based on spectral databases and/or literature data.</footnote>

<footnote>* Annotation based on spectral databases and/or literature data.</footnote>
<caption>**Table 4**</caption>

Comparison of “Gold” chemical markers highlighted in this work for the selection of 14 yellow dye plants and taxonomic chemical markers described in literature for the same plant species in studies dealing with applications in the fields of dyeing and/or cultural heritage (non-exhaustive list).

<table><thead><tr><th>Plant species</th><th>“Gold” chemical markers</th><th>Chemical markers in literature</th><th>References</th></tr></thead><tbody><tr><td><i>B. vulgaris</i></td><td>Berbamine/Oxyacanthine, Berbamunine, Jatrorrhizine/ Columbamine, Magnoflorine</td><td>Berbamine, Berberine, Berberine derivative, Chrysin derivative, Demethyleneberberine, Jatrorrhizine, Magnoflorine, Oxyacanthine, Phenolic acid, Salicylic acid, Syringic acid, Tetrahydrojatrorrhizine, Unidentified m/z 379, Vanillic acid</td><td>[104-106]</td></tr><tr><td><i>C. tinctorius</i></td><td>Anhydrosafflor yellow B, Hydroxysafflor yellow B/C, Unidentified m/z 329 + m/z 539</td><td>4-Hydroxybenzaldehyde, 4-Hydroxybenzoic acid, 6-Hydroxykaempferol-3-O-β-D-glucoside, Anhydrosafflor yellow B, Apigenin, Carthamin, Carthamin degradation products m/z 449 + m/z 477, Cartormin, Ct0-Ct6, CtX-CtZ, Deglucosyl hydroxysafflor yellow A, Hydroxysafflor yellow A, Hydroxysafflor yellow B/C, Hyperoside, Kaempferol, Kaempferol-3-O-glucoside, Kaempferol-0-deoxyhexosyl-hexoside, p-Coumaric acid, p-Hydroxystyrene, Quercetin, Quercetin-0-hexoside, Quinochalcone, Rutin, Safflomin A, Safflomin C, Safflor yellow A, Unidentified m/z 582 + m/z 613, Y1-Y2</td><td>[16,20,55,107-110]</td></tr><tr><td><i>C. coggygria</i></td><td>(+)-Fustin, (-)-Fustin, Pentagalloylglucose, Procyanidin trimer</td><td>4',7-dihydroxyflavanol, Butein, Butin, Fisetin, Fisetin xyloside, Fustin, Isoliquiritigenin, Liquiritigenin, Myricetin, Naringenin, Quercetin, Sulfuretin, Sulfuretin glucoside, Taxifolin</td><td>[3,20,111,112]</td></tr><tr><td><i>D. cannabina</i></td><td>Cannabin, Datinoside, Dihydrochalcone glycoside, Galanginoside, Unidentified triterpenoid, Unidentified m/z 765</td><td>Datiscetin, Datiscetin-3-O-rutinoside, Galangin</td><td>[113,114]</td></tr><tr><td><i>H. stoechas</i></td><td>3,5-dihydroxy-6,7,8-trimethoxyflavone, Acylphloroglucinol derivatives “A” + “B”/“C” + “D” + “E” + “F”, Caffeoylquinic acid, Dicaffeoylquinic acid, Quercetagitrin</td><td>–</td><td>–</td></tr><tr><td><i>H. perforatum</i></td><td>33-Deoxy-33-hydroperoxy-furohyperforin, 13,II8-Biapigenin/Amentoflavone, Isoquercetin/Hyperoside</td><td>Quercetin</td><td>[115]</td></tr><tr><td><i>M. pomifera</i></td><td>Gallic acid derivative, Methoxyxanthone, O-methylated flavonoid, Oxyresveratrol, Oxyresveratrol derivative</td><td>Alvaxanthone, Dihydrokaempferol, Dihydromorin, Kaempferol, Macluraxanthone, Methoxytrihydroxyxanthone, Morin, Naringenin, Osajaxanthone, Osajin, Pomiferin, Tetrahydroxyflavanone</td><td>[21,116-119]</td></tr><tr><td><i>M. tinctoria</i></td><td>Lespedezaflavanone D/3-(3,4-dihydroxyphenyl)-5,7-dihydroxy-6,8-bis(3-methylbut-2-enyl)chroman-4-one, Prenylated dihydroflavonol, Prenylated flavonoid, Prenylated xanthone, Unidentified m/z 329 + m/z 457</td><td>2,4,6-Trihydroxybenzoic acid, 2,4-Dihydroxybenzoic acid, 4-Hydroxy-benzoic acid, Dihydrokaempferol, Dihydromorin, Kaempferol, Maclurin, Morin, Piceatannol, Stilbenoid dimer (ChtA-ChtB), Tetrahydroxyflavanone, Tetrahydroxyflavone</td><td>[21,120]</td></tr><tr><td><i>P. granatum</i></td><td>Ellagic acid, Ellagic acid-O-pentoside, Punicalagin isomer “A” + “B”, Unidentified m/z 487 + m/z 511</td><td>Ellagic acid, Ellagic acid-rhamnoside, Ellagic acid-xylopyranoside, Ellagitanins, Galloy-HHDP-hexoside, Malic acid, Punicalin, Punicalagin A, Punicalagin B</td><td>[121,122]</td></tr><tr><td><i>R. luteola</i></td><td>Chrysoeriol, Luteolin-7-O-glucoside, Unidentified m/z 341</td><td>Apigenin, Apigenin-6,8-di-C-glucoside, Apigenin-7-O-glucoside, Chrysoeriol, Chrysoeriol glycoside, Diosmetin, Luteolin, Luteolin-3',7-di-O-glucoside, Luteolin-4',7-di-O-glucoside, Luteolin-3'-O-glucoside, Luteolin-4'-O-glucoside, Luteolin-7-O-glucoside, Rutin</td><td>[14,20,21]</td></tr><tr><td><i>R. alaternus</i></td><td>–</td><td>Kaempferol, Kaempferol-3-O-rhamninoside, Kaempferol-4'-O-rhamninoside, Quercetin, Quercetin-3-O-rhamninoside, Quercetin-4'-O-rhamninoside, Rhamnazin-3-O-rhamninoside, Rhamnetin-3-O-rhamninoside, Rhamnocitrin, Rhamnocitrin-3-O-rhamninoside, Rhamnocitrin-4'-O-rhamninoside</td><td>[68]</td></tr><tr><td><i>R. cathartica</i></td><td>Quercetin-3-O-rhamninoside, Rhamnetin-3-O-rhamninoside, Unidentified m/z 176</td><td>Emodin, Frangulin, Isorhamnetin, Isorhamnetin-Rhamnose-Glucose, Kaempferide, Kaempferol, Kaempferol-3-O-rhamninoside, Kaempferol-4'-O-rhamninoside, Kaempferol-Glucose, Quercetin, Quercetin-3-arabinoside, Quercetin-3-O-rhamninoside, Rhamnazin, Rhamnazin-3-O-rhamninoside, Rhamnetin, Rhamnetin-3-O-rhamninoside, Rhamnetin/Isorhamnetin-Rhamnose, Rhamnocitrin, Rhamnocitrin-3-O-rhamninoside, Rhamnocitrin-4'-O-rhamninoside, Rutin</td><td>[62,68,123-125]</td></tr><tr><td><i>R. saxatilis</i></td><td>–</td><td>Emodin, Isorhamnetin, Isorhamnetin-3-O-rhamninoside, Kaempferol, Kaempferol-3-O-rhamninoside, Kaempferol-3-O-acetyl-rhamninoside, Quercetin, Quercetin-3-O-glucoside, Quercetin-3-O-rhamninoside, Quercitrin, Rhamnazin, Rhamnazin-3-O-acetyl-rhamninoside, Rhamnazin-3-O-rhamninoside, Rhamnetin, Rhamnetin-3-O-rhamninoside, Rhamnetin-3-O-rhamninoside, Rhamnetin-0-rutinoside, Rhamnocitrin, Rhamnocitrin-3-O-acetyl-rhamninoside, Rhamnocitrin-3-O-rhamninoside, Rutin isomer</td><td>[20,68]</td></tr><tr><td><i>S. japonicum</i></td><td>Azukisaponin I, Isorhamnetin, Narcissin, Soyasaponin, Soyasaponin III, Soyasaponin gamma-G, Phaseoside IV</td><td>Dimethylquercetin-3-O-rutinoside, Isorhamnetin, Isorhamnetin-3-O-rutinoside, Kaempferol, Kaempferol-3-O-rutinoside, Quercetin, Quercetin-3-O-glucoside, Rutin</td><td>[20,126]</td></tr></tbody></table>

ancient samples seems to be much more limited because of their instability to light and dioxygen [64].

Hydroalcoholic extracts of heartwood of *Maclura* spp. usually contain flavonoids (mainly flavones, flavanones, chalcones, isoflavones) as well as their glycosylated and prenylated derivatives, but also xanthones and simple phenolic acids [43,65]. When both species *M. pomifera* and *M. tinctoria* were compared to all other species, several flavonoids and phenolics compounds, such as morin, dihydromorin, quercimeritrin, maclurin and macluraxanthone, were found as

characteristic chemical markers of their extracts (Supporting information, Table S9). Several of these compounds have already been used as chemomarkers for *Maclura* spp. in previous studies in the field of cultural heritage chemistry (Table 4). Nevertheless, despite their taxonomic proximity, these two *Maclura* species also presented their own chemical markers in this metabolomics approach. In the case of *M. pomifera*, a methoxylated xanthone and a gallic acid derivative in ESI+, and an O-methylated flavonoid, oxyresveratrol and one of its derivative in ESI-were defined as “Gold” chemical markers of this species. For *M. tinctoria*,
a greater number of “Gold” chemical markers were detected, including two prenylated flavonoids and a prenylated xanthone in ESI+, and two prenylated xanthones, two prenylated flavonoids, and two unidentified compounds (with m/z 329 and 457, respectively) in ESI- (Tables 2 and 3; Supporting information, Tables S7 and S8). These results were in line with previous chemical studies in the field of cultural heritage, which have already used prenylated flavonoids and prenylated xanthones as characteristic compounds of these two species (Table 4).

In addition to simple phenolic acids and a great variety of flavonoids, hydroalcoholic extracts of pomegranate (P. granatum) peels are characterized by high contents of tannins (mainly hydrolysable tannins, including gallotannins and especially ellagitannins) [66]. Ellagic acid and specific ellagitannins (e.g., punicalagins, punicalins) are particularly abundant and characteristic of P. granatum peels extracts. Accordingly, punicalagins A and B, ellagic acid and one of its derivatives together with two unidentified features were defined as “Gold” chemical markers of P. granatum using the present approach (Tables 2 and 3; Supporting information, Table S10). These findings were consistent with previous reports in the field of cultural heritage (Table 4).

Weld (R. luteola) was one of the first yellow dye plant to be analyzed in depth by chemists at the end of the 19th century for identifying the dyeing molecules of its extracts. R. luteola hydroalcoholic extracts are composed mainly by three flavones, luteolin, apigenin and chrysoeriol, and their di- and mono-glycosylated derivatives [14,67]. Luteolin-7-O-glucoside, chrysoeriol and an unidentified compound were identified as “Gold” chemical markers of this species (Tables 2 and 3; Supporting information, Table S11). It is interesting to note that despite their presence in significant quantities, luteolin and apigenin only appear as silver chemical markers because of their lesser specificity. In fact, luteolin and apigenin are much more common flavones in nature than chrysoeriol. This is in line with numerous studies already carried out on this species (Table 4), which conclude that R. luteola was used to dye a sample only if these two compounds are present together with chysoeriol in a sample [67].

Hydroalcoholic extracts of *Rhamnus* spp. berries contain flavonols, such as quercetin, kaempferol, rhamnetin, rhamnazin or rhamnocitrin, as well as their 3- or 4'-O-glycosides (rhamnoside and isorhamnoside) in higher proportions [68]. Anthraquinones, mainly emodin, and anthocyanins are also present in such extracts [69]. Considering the three *Rhamnus* species studied here as a whole, rhamnocitrin, and kaempferol rhamninosides were highlighted through the present metabolomics approach as strong chemical markers of this genus both in ESI+ and ESI-(Supporting information, Table S15). These results are in line with the bibliography, as these compounds have already been used as chemical markers for these three plant species in the field of cultural heritage (Table 4). However, when the three species were taken separately, no “Gold” chemical markers can be detected in R. *saxatilis* and R. *alaternus* extracts because of their strong chemical similarity. For R. *cathartica* extracts, which are slightly different from those of the other two species, quercetin and rhamnetin 3-O-rhamninosides and an unidentified compound (m/z 176) were defined as chemical markers in ESI+ for this species (Tables 2 and 3; Supporting information, Tables S12-S14). These results showed that in this case it is very difficult to discriminate between closely related species (same botanical genus) within a large panel of plants. In addition, berry maturity seems to play an important role in the chemical discrimination of the three species, since berries of R. *saxatilis* and R. *alaternus* used in this study were mature while those of R. *cathartica* were immature. However, at the genus level, flavonol rhamninosides appear to be good taxonomic markers, in line with the literature in this field (Table 4).

Flower buds from *Styphnolobium japonicum*, previously known as *Sophora japonica*, mainly contain flavonoids (e.g., kaempferol, quercetin, isorhamnetin and their glycosides), triterpenoids (mainly as saponins, e.g., kaikasaponins, soyasaponins), and various other compounds (e.g., alkaloids, maltol glycosides and putrescine derivatives) in their hydroalcoholic extracts. Using the chemical marker

selection method developed in this study, two flavonoid derivatives were defined as “Gold” chemical markers in ESI+, and five triterpene saponins in ESI- (one of them being detected in both ionization modes) (Tables 2 and 3; Supporting information, Table S16). It is interesting to note that, for possible use in the field of heritage, these chemical markers appear to be much more specific than those used until now, such as quercetin, kaempferol, isorhamnetin and their corresponding glycosides (such as rutin) (Table 4).

In this study, it is interesting to note that kaempferol and quercetin were found in seven of the fourteen plant species studied. Yet these molecules are often used as chemical markers in the identification of dye plants. That is why it is essential to define more robust taxonomic chemical markers for each specific dye plant. However, the best way to confirm the use of a particular plant species in the manufacture of a heritage object is to assess the occurrence of a set of specific molecules, rather than just one or two. It is important to consider that the molecules identified as chemical markers in this study depend not only on the choice of the plant panel selected, i.e., their number and taxonomic diversity (genus and family), but also on the harvest location and season, the maturity stage, or the part of the plant that has been selected for extraction. In addition, extraction conditions (i.e., technique, solvents and duration) and MS analysis conditions (ionization parameters and detector sensitivity) may also drastically influence the detection of certain compounds and thus their selection as taxonomic markers. Furthermore, some molecules presented as chemical markers here may be degraded over the lifetime of an ancient object, to the point that they would no longer be detected during analysis. This potential degradation may be caused by the dyeing parameters (pH, temperature and duration) used during their original manufacture, the object's conservation conditions (exposure to sunlight and humidity), and the dye extraction method. In a heritage context, dye extraction is often different from that described here, and can lead to degradation of certain compounds [127]. In fact, most textiles undergo a mordanting step during dyeing, to enhance dye fixation. A metal cation (aluminum, copper...) is added to form a complex between the natural fibers and the colored molecules [1]. During extraction, an acid medium (hydrochloric, formic, trifluoroacetic acid...) is used to disrupt this complex. However, it can degrade certain molecules, such as glycosylated flavonoids, whose osidic bonds are hydrolyzed with concomitant release of the corresponding aglycones.

## 4. Conclusion

The study of ancient objects helps us to better understand the historical and geographical context of their manufacture, as well as the producing techniques that have been used. The yellow color of these objects can be produced from a wide variety of dye plants. Many yellow dye molecules (e.g., flavones and flavonols) are common in Nature, making it difficult to identify their botanical origin. In this paper, we presented a metabolomics workflow involving UPLC-HRMS/MS, statistical analyses and molecular networking, in order to determine specific chemical markers of a selection of common yellow dye plants. Two ionization modes (ESI+ and ESI-) were used to detect the greatest possible number of chemical families in the hydroalcoholic extracts of these plants. For plant discrimination, three different and complementary selection methods (PLS-DA, heatmap, molecular network) were combined in order to select only the most robust chemical markers for each plant species. Thus, the “Gold” chemical markers selected in this study were those that were the most specific (specificity score > 90 %) and were retained each time when each of the three selection methods was applied. Molecular networking and in silico MS/MS fragmentation have made it possible to putatively annotate at different levels a large number of these chemical markers. All in all, this study enabled us to identify new markers for certain plants, to confirm others alone or in combination with others, or, on the contrary, to eliminate some of them. In the future, these chemical markers could be useful for characterizing
yellow plant dyes in heritage objects through targeted LC-MS analyses. However, further studies are needed to validate that these molecules remain stable well after their extraction, but also during the dyeing process and after they have been complexed to a fiber, considering in particular the type of mordant used and the long periods of time during which they are exposed to harsh physical and chemical conditions (exposure to light, oxidation...). Thus, while this work has shown that metabolomics is an analytical tool of choice in cultural heritage chemistry, it could be used routinely in the future to answer scientific questions specific to this field, e. g., the study of the degradation of chemical markers in the context of the original dyeing and/or conservation of a heritage object, or in the extraction step carried out prior to the analysis itself.

### CRediT authorship contribution statement

**Lindsay Mas-Normand:** Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. **Olivier Dangles:** Writing – review & editing, Supervision, Funding acquisition, Conceptualization. **Carole Mathe:** Writing – review & editing, Supervision, Funding acquisition, Conceptualization. **Gérald Culioli:** Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Funding acquisition, Data curation, Conceptualization.

### Funding

The PhD grant of L. Mas-Normand was financially supported by the IMPLANTEUS "Sustainable Mediterranean Agriculture for Health" Graduate School of Avignon University funded from the French National Research Agency as part of the France 2023 investment programme for the future, under reference ANR-18-EURE-0009. One part of the LC-MS analyses was granted by the TERSYS federative research structure (Avignon University, France).

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

The authors are grateful to Dr. R. Lugan, Dr. O. Chevallier, V. Kuentz, and S. Raynal for their help during the acquisition of LC-MS profiles on the "Metaboscope" platform (Avignon University, France), to C. Joliet and D. Saouli (IMBE, Avignon University, France) for their help with extraction and sample preparation, to Dr. E. Garayev (IMBE, Aix-Marseille University, France) for the script used to correct the *m/z* values of precursor ions of MS/MS scans, and to L. Camoin ("Couleur Garance", Lauris, France) for the selection of dye plants and the helpful discussions.

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.microc.2025.115621.

### Data availability

Raw data for UPLC-HRMS/MS analyses obtained in ESI+ and ESI-modes were deposited on the MassIVE platform under the IDs MSV000098899.

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