Investigation of natural dyes in 15th c. documents seal threads from the Romanian Academy Library, by LC-DAD-MS (triple quadrupole)

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Case study

Investigation of natural dyes in 15th c. documents seal threads from the Romanian Academy Library, by LC-DAD-MS (triple quadrupole) Irina Petroviciu a,∗ , Florin Albu b , Ileana Cretu c , Marian Virgolici d , Andrei Medvedovici e a

National Museum of Romanian History (MNIR), 030026 Bucharest, Romania S.C. Agilrom Scientific SRL, 077190 Voluntari, Ilfov, Romania National Museum of Art of Romania (MNAR), 010063 Bucharest, Romania d “Horia Hulubei” National Research Institute for Physics and Nuclear Engineering, IRASM, 077125 Magurele, Ilfov, Romania e Department of Analytical Chemistry, Faculty of Chemistry, University of Bucharest, 050663 Bucharest, Romania b c

a r t i c l e

i n f o

Article history: Received 30 January 2017 Accepted 29 May 2017 Available online xxx Keywords: Natural dyes Liquid chromatography Mass spectrometry Documents seal threads Red

a b s t r a c t Dyes and biological sources in 40 samples from red seal threads in 38 documents issued by the Chancery of Moldavia between 1460 and 1503 were investigated by liquid chromatography with UV-Vis (DAD) and mass spectrometric (MS) detection. Lac dye (Kerria lacca Kerr), redwood type (Caesalpinia spp.) and madder (Rubia sp.), as individual dyes or in combinations, were responsible for the colour in all the dyed yarns while tannins were present in more than half of the total number of samples. The presence of major dyes, such as alizarin, purpurin, laccaic acid A and soluble redwood — a marker compound for Caesalpinia species were observed by both DAD and MS detectors while minor compounds (rubiadin, anthragallol, xanthopurpurin, munjistin, flavokermesic acid etc.) were only detected by mass spectrometry. Single stage MS detection was used in the Full Scan mode followed by data processing through Ion Extraction according to the molecular ions of compounds in the database. Tandem MS detection (MS2 ) was also achieved, through using the Product Ion Scan operating mode. Identification of dyes was made according to retention time, UV-Vis and MS data, based on information collected on standards — dyes and dyed fibers. The biological sources detected are discussed as compared with those identified in ecclesiastical embroideries from the same period, ordered by the same Prince, Stephan the Great (1457–1504). © 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction Natural dyes were the only source of color from Antiquity and until the synthetic dyes were introduced to the market, in 1856. Initially used only locally, later subject of commercialization, identification of natural dyes in historical textiles may add consistent information about the period and place an object was made. Such information is based on literature records about the natural dyes first trade, new commercial routes or regulation of use [1–3]. The interest for natural dyes in textiles from Romanian collections turned up in the identification of dyes and biological sources in the most representative local ones, dating from the 15th to the 20th century [4–9]. A special interest was given to a collection of textiles from the long reign of Stephan the Great (1457–1504), the ruler of Moldavia for almost 50 years in the second half of the 15th century [10]. Investigation of red dyes in twelve ecclesiastical

∗ Corresponding author. E-mail addresses: [email protected], [email protected] (I. Petroviciu).

textiles with donor inscriptions, from this period, preserved at Putna Monastery, showed that the combination of lac dye and madder was used for most of the objects, while kermes, the most expensive red dye at that time [11] was preferred for the more important objects, as those especially designed for Stephan’s wife, Princess Maria of Mangop [6,12,13]. A collection of documents with hanging seals emitted by the Chancery of Moldavia in the time of Stephan the Great is preserved at the Romanian Academy Library (BAR). The documents were studied from several perspectives but never as regards the colour source in the hanging seals red silk threads [14]. High performance liquid chromatography with UV-Vis detection (DAD) is the standard set up for investigation of natural dyes, since its development by Wouters, in 1985 [15–21]. In the last 15 years, mass spectrometers were increasingly exploited, in various configurations [22–28]. Their use increases the level of certitude in identifications by introducing new criteria — the molecular ion (for single stage MS detectors) and the product ion scan mode (for tandem MS instruments). MS detection provides lower detection limits for most of the dyes used in historical textiles. Such increased sensitivity is obtained by the reduction of the noise level,

http://dx.doi.org/10.1016/j.culher.2017.05.015 1296-2074/© 2017 Elsevier Masson SAS. All rights reserved.

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through exploiting MS instruments in enhanced selectivity modes [28]. Moreover, the MS/MS configurations, such as ion trap and triple quadrupole, were proved as useful instrumentation to reveal the molecular structure of unknown dyes [29]. An analytical protocol for dyes characterization and identification by LC-DAD-MS (ion trap) was recently developed for the first time in Romania [30] and was used for the characterization of a significant number of dyes in textiles from Romanian collection [7,8,31–33]. The present work discusses the results obtained by applying an adapted version of the above-mentioned analytical protocol to the investigation of dyes in red silk threads from 38 “documents with hanging seals”, emitted by the Chancery of Moldavia in the time of Stephan the Great. 2. Materials and methods 2.1. Samples and sample preparation Forty red silk samples about 0.5 cm long (∼3-mg) from 38 documents emitted by the Chancery of Moldavia in the time of Stephan the Great (1457–1504) were available for dye analysis. Three red samples from 2 earlier documents emitted by the same Chancery, in the time of Alexander the Good (1400–1432), were also available (Table 1). For dyes extraction, samples were transferred in 1.5-mL Eppendorf tubes and 250-␮L from a mixture 37% HCl/CH3 OH/H2 O 2:1:1 (v/v/v) were added to each fiber. The tubes were placed in a heating block (Thermo Shaker TS-100, manufactured by Biosan) and kept for 10 minutes at 100 ◦ C, according to the method developed by Wouters [15]. The tubes were then moved in a vacuum desiccator (constant pressure at 50-mbar) and the solution was evaporated to dryness at room temperature. Each sample was re-dissolved in 100-␮L CH3 OH/H2 O 1:1 (v/v) mixture. After 10 minutes of centrifugation at 6000 × g, the supernatants were transferred in chromatographic vials and placed in the automatic injector tray holder. 2.2. Database

Table 1 List of documents and their dating. Documents issued in the time of Stephan the Great, dated between 1460 and 1503 are listed first, according to their inventory numbers, while below are presented the two documents from 1409 and 1425, issued in the time of Alexander the Great. Inventory number

Date

15 110 119 127 147 159 160 161 170 173 191 194a 198 200 204 231b 236 243 245c 246 265 290c 299 300 315 375 382 384 478d 480 482 502 XL/15 LXIV/7 LXXV/160 CI/66 1 CLIX/19 DCXLIII 137e 144e

1491, January 17 1499, December 6 1456, September 18 1472, June 5 1493, March 15 1466, September 11 1480 1497, February 25 1498, November 20 1493, March 7 1499 1479, March 9 1492, October 14 1497, March 10 1461, August 8 – 1468, September 24 1460, December 5 1470, May 28 1499, August 15 1472, January 25 1470, September 25 1494, February 27 1497, January 20 1490, January 14 1490, October 14 1502, March 17 1491, October 31 1479, 1487, October 8 1475, April 14 1499, November 17 1503 1488 1479 1495 1502 1500 1409, September 16 1425

a

An “in-house” developed database, relying on retention, UVVis and mass spectrometric data, was used for dyes identification. Information about dyes is based on analysis of standards. Identification of the most probable biological sources was made according to data collected on standard dyed fibers and information available in literature [1,2,15]. Retention, UV-Vis and mass spectrometric data of the dyes identified are illustrated in Table 2. 2.3. Instrumentation Samples were analysed using an Agilent 1260 LC system composed of the following modules: quaternary pump (Model G1311C), automatic injector (G1367E) and column thermostat (G1316C). The system was equipped with two detectors serially connected: a diode array detector (G4212A) and a triple quadrupole mass spectrometer (G6410B) equipped with an atmospheric pressure electrospray ion source (ESI, Model G1948B), operated under negative ion monitoring mode. 2.4. Chromatographic separation Separation was achieved on a Zorbax C18 column, 150-mm L × 4.6 i.d., 5-␮m particle size, thermostated at 40 ◦ C. The mobile phase consisted of a mixture of aqueous 0.2% (v/v) formic acid (solvent A) and methanol/acetonitrile 1:1, v/v (solvent B). Gradient elution was applied according to the following profile: at 0 min,

b c d e

Fake document, original seal. Only seal, no document. Fake document. Seal detached from document. Document issued in the time of Alexander the Good.

15% solvent B; from 0 to 5 min, linear increase to 25% solvent B; from 5 to 10 min, linear increase to 55% solvent B; from 10 to 16, linear increase to 100% solvent B; from min 16 to 18, constant at 100% solvent B; and step jump at 15% solvent B, with a 4 min reequilibration step. The flow rate was set at 0.8 mL/min. The injected volume was 5-␮L, from a total amount of 100-␮L resulting from the sample preparation stage. Several injections from the same solution may be performed, as described in the “Results section” below. 2.5. Detection UV-Vis spectra were acquired over the 190–640-nm range, with a resolution of 2 nm. MS detection was made in negative ion monitoring mode with the following ESI operating parameters: drying gas temperature 350 ◦ C; drying gas flow 8 L/min; pressure of the nebulizer gas 40 psi; Vcap 2500 (−). For the single stage MS mode, the triple quadrupole was using the MS2 type Scan; the data storage was set on profile and the peak width at 0.07; fragmentor 135 V; EMV 400 V; The scanning interval was between 100–600 m/z, accelerated voltage on the collision cell: 7 V; Dwell Time 500 ms. In the tandem MS working mode, product ion scan was used, with

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Table 2 Retention, UV-Visa and MS data of the dyes detected in documents seal threads from the time of Stephan the Great. Dye component

Abbreviation

Biological source

Retention

UV-Visa

[M-H]−

Product ion scan (QQQ)

Alizarin Anthragallol Ellagic acid Erythrolaccin Laccaic acid A Laccaic acid B Flavokermesic acid Munjistin Purpurin Rubiadin “Soluble redwood” Xanthopurpurin

al ag ea eryth laA laB fk mu pu ru srw xp

Madder Madder Tannins Lac dye Lac dye Lac dye Lac dye Madder Madder Madder Redwood Madder

15.1 14.1 9.8 15.8 10.3 10.3 13.5 14.0 16.2 17.4 10.5 16.0

202;248;280;430 – 254;366 – 200;226;288;492 – – – 204;256;294;480 – 258;306;336 –

239 255 301 285 536 495 313 283 255 253 243b 239

210,182 237,227,198;171 185 257,241,217,185 492,474,448,430 451,407,389 269 239 227,183,171,155 225,209,181 226;215;198;187 211;196;195

Dyes are listed in alphabetical order. a UV-Vis data is given only for the major dyes (dyes identified by DAD in the present study). b Major ion, not necessary the molecular ion.

Table 3 Results obtained by UV-Vis (at 255 nm) and mass spectrometric detection. The biological sources of dyes are also given. For dyes abbreviation see Table 2. Inventory number and sample code

Colour of fibre

Result (dyes)

DAD Documents from the time of Stephan the Great (1457–1504) Red-orange al, pu, srw 15 Red laA, al, pu, ea 110 119 Red al, pu 127 147 159 160 161 170 173 191 194 198 200 204 231 236 243 245 246 265 290 299 300 315 375 382 384 478 480 482 502 XL/15 LXIV/7 1

Red-orange Red Red Red Red Red Red-orange Red-orange Red-orange Red-orange Red-orange Red-orange Red Red-orange Red Red-orange Red Red-orange Red-orange Red-orange Red-orange Red-orange Red-orange Red-orange Red-orange Red Red-orange Red-orange Red Red Red

al, pu, srw, ea laA, al, pu al, pu laA laA, al, pu, ea laA, al, pu al, pu, srw al, pu, srw, ea al, pu, srw, ea al, pu, srw al, pu, srw, ea al, pu laA, al, pu, ea al, pu, srw laA, al, pu srw laA, al, pu al, pu, ea srw al, pu, srw al, pu, srw, ea al, pu, srw al, pu, srw, ea al, pu, srw, ea al, pu, srw laA, al, pu al, pu, srw, ea al, pu, srw laA, al, pu, ea laA, al, pu, ea laA, al, pu, srw, ea

Red-orange al, pu, srw, ea LXIV/7 2 Red laA LXXV/160 CI/66 1 Red laA, al, pu Red-orange al, pu, srw, ea CI/66 2 Red-orange al, pu, srw, ea CLIX/19 Red-orange al, pu, srw, ea DCXLIII Documents from the time of Alexander the Good (1400–1432) 137 1 Red-orange al, pu, srw, ea Pale red-orange al, pu, srw 137 2 Red al, pu, ea 144

Result (biological sources of dyes, common names) MS al, pu, ru, mu, xp, srw laA, laB, fk, eryth, al, pu, ru, mu, xp, ag, ea al, pu, ru, mu, xp, ag, srw, laA(−), laB(−), fk, eryth al, pu, ru, mu, xp, ag, ea, srw laA, laB, fk, eryth, al, pu, ru, mu, xp, ag al, pu, ru, mu, ag, ea laA, laB, fk, eryth laA, laB, fk, eryth, al, pu, ru, mu, xp, ag, ea(−) laA, laB, fk, eyth, al, pu, ru, mu, xp, ag, ea(−) al, pu, ru, mu, xp, ag, srw, ea(−) al, pu, ru, mu, xp, ag, srw, ea al, pu, ru, mu, xp, ag, srw, ea al, pu, ru, mu, xp, ag, srw al, pu, ru, mu, xp, ag, srw, ea al, pu, ru, mu, xp, ag, ea laA, laB, fk, eryth, al, pu, ru, xp, ag, ea al, pu, ru, mu, xp, ag, srw laA, laB, fk, eryth, al, pu, ru srw laA, laB, fk, eryth, al, pu, ru, xp al, pu, ru, mu, xp, ag, ea srw al, pu, ru, xp, srw al, pu, ru, mu, xp, ag, srw, ea al, pu, ru, srw al, pu, ru, mu, xp, ag, srw, ea(−) al, pu, ru, mu, xp, ag, srw, ea al, pu, ru, mu, xp, ag, srw laA, laB, fk, eryth, al, pu, ru, mu, xp, ag al, pu, ru, mu, xp, ag, srw, ea al, pu, 14, ru, mu, xp, ag, srw laA, laB, fk, eryth, al, pu, ru, mu, xp, ag, ea laA, laB, fk, eryth, al, pu, ru, mu, xp, ag, ea laA, laB, fk, eryth, al, pu, ru, mu, xp, ag, srw, ea(−) al, pu, ru, mu, xp, ag, srw, ea laA, laB, fk, eryth laA, laB, fk, eryth, al, pu, ru, mu, xp, ag al, pu, ru, mu, xp, ag, srw, ea al, pu, ru, mu, xp, ag, srw, ea al, pu, ru, mu, xp, ag, srw, ea

Madder and redwood type Lac dye, madder and tannins Madder, redwood type and traces of lac dye Madder, redwood type and tannins Lac dye and madder Madder and traces of tannins Lac dye Lac dye, madder and traces of tannins Lac dye, madder and traces of tannins Madder, redwood type and traces of tannins Madder, redwood type and traces of tannins Madder, redwood type and tannins Madder and redwood type Madder, redwood type and tannins Madder and traces of tannins Lac dye, madder and tannins Madder and redwood type Lac dye and madder Redwood type Lac dye and madder Madder and tannins Redwood type Madder and redwood type Madder, redwood type and tannins Madder and redwood type Madder, redwood type and traces of tannins Madder, redwood type and tannins Madder and redwood type Lac dye and madder Madder, redwood type and tannins Madder and redwood type Lac dye, madder and tannins Lac dye, madder and traces of tannins Lac dye, madder, redwood type and traces of tannins Madder, redwood type and tannins Lac dye Lac dye and madder Madder, redwood type and tannins Madder, redwood type and tannins Madder, redwood type and tannins

al, pu, ru, mu, xp, ag, srw, ea al, pu, ru, mu, xp, ag, srw al, pu, ru, mu, xp, ag, ea

Madder, redwood type and tannins Madder and redwood type Madder and tannins

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the following operating parameters: start mass 50 m/z; end mass 600 m/z; MS2 Step size 0.1 m/z; scan time 100 ms; collision energy 20 V.

2.6. Data processing The Agilent MassHunter Quantitative Analysis B.06.00 software was used for controlling the chromatographic system and for data acquisition/processing. The analysis procedure follows the methodology described in detail in an earlier publication, where an ion trap mass spectrometer was used instead of the triple quadrupole [30]. Each sample was first analysed with a single stage MS detection and Full Scan operating mode. The resulted data was processed by extracting chromatograms, according to the molecular ions of the dyes in the database. The major compounds were identified according to their retention, UV-Vis and mass spectrometric data while the minor ones (associated in the biological source with the major dyes detected) only based on retention and mass spectrometric data. An additional injection was made with tandem MS detection in Product Ion Scan operating mode, to confirm the dyes presence, if needed. In such situations, each compound was unambiguously identified based on its MS2 spectrum, through comparison with data collected on standards [30].

3. Results and discussion Lac dye (Kerria lacca Kerr), redwood type (Caesalpinia spp.) and madder (Rubia tinctorum L.) — as individual dyes or in combinations, were responsible for the colour in all the 43 samples analysed, 40 from the time of Stephan the Great and 3 from Alexander the Good (Table 3). Tannins were also present in more than half of the total number of samples. 3.1. Lac dye Lac dye was detected in 15 samples, based on the presence of laccaic acid A, laccaic acid B, flavokermesic acid (also called laccaic acid D) and erythrolaccin (Fig. 1). Amongst all the lac dye components, only laccaic acid A was identified by UV-Vis data at 255 nm (Table 2), correlated with retention. Its presence was confirmed by the molecular ion (m/z = 536 [M-H]− ) as well as by the ion formed by its decarboxylation (m/z = 492 [M-H-44]− ) in the Full Scan–Ion Extracted Chromatogram (FS-IEC), as described in Section 2.6. Identification of laccaic acid B by MS detection was possible for all the samples where laccaic acid A was present. Its detection was based on the presence of the molecular ion (m/z = 495, [M-H]− ) as well as by the ion formed by its decarboxylation (m/z = 451 [M-H-44]− ) in the FS-IECs. The presence of laccaic acid B is confirmed in the MS2 spectrum through the ion at m/z = 495, as compared with data

Fig. 1. Illustrative chromatograms supporting identification of dyes (laccaic acid A, laccaic acid B, flavokermesic acid, also called laccaic acid D and erythrolaccin) in lac dye (Kerria lacca Kerr) in a sample taken from document 160, dated 1480. A. Overlaid chromatograms resulting from UV detection and single stage MS in full scan (FS) mode. B. Overlaid ion extracted chromatograms (IECs) according to molecular ions m/z values obtained for the target compounds. C. Overlaid chromatograms for tandem MS (product ion scan mode, precursor ions are the molecular ones). D. Product ion mass spectra for each of the dyes identified.

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3.2. Redwood type

Fig. 2. Fragmentation mechanism proposed for erythrolaccin in lac dye (Kerria lacca Kerr).

available in literature [24]. The low amount of laccaic acid B as compared with laccaic acid A was explained by Wouters who observed that laccaic acid B is vulnerable to the acid hydrolysis extraction procedure [34]. The presence of flavokermesic acid, a minor compound in lac dye, was revealed by its molecular ion (m/z = 313, [M-H]− ) and the ion formed through decarboxylation in the ESI source (m/z = 269, [M-H-44]− ), as revealed in the FS-IECs. Due to its reduced sensitivity, the UV-Vis detection failed to identify flavokermesic acid (a low amount of the analyte exists in the insects, and even less preserved in acid hydrolysed extracts, as compared to laccaic acid A). Wouters and Verhecken showed that, during the standard extraction method based on acid hydrolysis (also used in our experiments), only 51% laccaic acid A and 3% flavokermesic acid are not degraded [34,35]. Erythrolaccin was also identified in all the samples where lac dye was present. Its detection was based on the presence of the molecular ion (m/z = 285, [M-H]− ) in the FS-IEC correlated with the retention time, as compared with data collected on a standard lac dye dyed fiber. The product ion mass spectra of erythrolaccin (m/z = 285) is presented in Fig. 1 and a possible fragmentation mechanism is suggested in Fig. 2. Detection of erythrolaccin in samples from textiles in Romanian collections should not surprise, this yellow dye component in lac dye being already observed in fibers from 15th c. liturgical embroideries preserved at Putna Monastery (Romania) [12]. The presence of erythrolaccin suggests that dyes were extracted from the biological source in an alkaline medium [36]. From the 15 samples in which lac dye was identified, in 2 samples (documents 160 and LXXV/160) it was used as individual dye source, in 2 (documents 119 and LXIV/7) in a dyeing combination with madder and redwood type, while in 11 other samples together with madder sources only. Tannins were also detected in 7 samples. In all these cases, lac dye and madder were used together. All the samples where lac dye was detected were described as red.

Redwood type is the name of the soluble dyes from the species of Caesalpinia. The main dye component in these species is brazilein which is formed from the oxidation of a precursor called brazilin, a neoflavonoid derivative [37]. In historical textiles, brazilein is broken down while another component could be seen in the chromatograms [16,37]. Detection of redwood type is thus based on the presence of this marker compound, called “soluble redwood, srw” [16] or “type C” [38]. It can be observed in the UV chromatogram at 255 nm, while experiments performed on standards showed that it could be also detected in the FS-IEC, based on a major ion of m/z = 243 [26,33]. The presence of “srw” may be confirmed by the presence of the signal at m/z = 243 in the MS2 spectrum, as compared with data collected on a standard redwood type dyed fiber (Table 2). In the present set of samples, “soluble redwood (srw)” was observed in 26 items out of the total of 43, which suggests the use of redwood type sources. In all but one case, the presence of “srw” was observed by both detectors. Redwood type was detected as individual dye source in two instances (documents 245 and 290), with lac dye and madder in 2 samples and with madder type sources in other 22 samples. An example to illustrate the detection of redwood type in a dyeing combination with madder, in the presence of tannins, in a red sample from document CI 66 is presented in Fig. 3. All samples of redwood type have red-orange hue, exception made by those also containing lac dye. The two documents where redwood type was detected as individual dye source (documents 245 and 290) are mentioned in the archives as “fakes from the time of Stephan the Great” (documents from his time, not issued by the Chancery of Moldavia). The threads exhibit a dull red-orange hue. Literature mentions that fake documents may be revealed by thoughtful observations, such as “low quality parchment, seals aspect, seal threads colour etc.” [39], which were also noticed for the two documents under discussion: “brown-red translucent waxy [parchment], without surface grinding treatment for writing” [40]. Detection of redwood type, a light fugitive (low quality) dye, as individual source, exclusively in these two documents (out of a series of 40 investigated), comes to support the classification of the two objects as “fakes from the time of Stephan the Great”. 3.3. Madder Madder (Rubia tinctorum L.) is identified in historical textiles by the two main dye components, alizarin and purpurin, while large amounts of rubiadin would point to the use of wild madder (Rubia peregrina L.) [41–43]. In the present set of samples, alizarin and purpurin were identified in 39 samples (all but 4 samples), by UV detection at 255 nm. Their presence was also confirmed by the mass spectrometric data, where the molecular ions of alizarin (m/z = 239 [M-H]− ) and purpurin (m/z = 255 [M-H]− ) were observed in the FS-IECs and also supported by their fragmentation patterns, as compared with data collected on standards (Table 2). Munjistin (m/z = 283 [M-H]− and m/z = 239 [M-H-44]− ) and rubiadin (m/z = 253 [M-H]− ) were also identified by means of their MS2 spectra (Table 2). Two other anthraquinones, showing absolute retention at minutes 14.4 (m/z = 255, major ion, probably [M-H]− ) and 16.02 (m/z = 239, major ion, probably [M-H]− ), respectively, were also observed in the FS-IECs of the samples where alizarin and purpurin were present. According to the relative retention in the chromatogram with respect to the two main dyes, and also considering the mass to charge ratios of the major ions in the MS2 spectra, the two unknown compounds were tentatively attributed to anthragallol and xanthopurpurin. Mass spectrometric data available in literature [28] and that collected on standards

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Fig. 3. Illustrative chromatograms supporting identification of dyes in redwood type, Caesalpinia spp. (“srw”) and madder, Rubia tinctorum L. (alizarin, purpurin, anthragallol, munjistin, xanthopurpurin and rubiadin) in a sample taken from document CI 66, dated 1495. Ellagic acid from tannins is also present. A. Overlaid chromatograms resulting from UV detection and single stage MS in full scan (FS) mode. B. Overlaid ion extracted chromatograms (IECs) according to molecular ions m/z values obtained for the target compounds. C. Overlaid chromatograms for tandem MS (product ion scan mode, precursor ions are the molecular ones). D. Product ion mass spectra for each of the dyes identified.

fully support the identity of these compounds (Fig. 2). Detection of alizarin and purpurin as main dyes and rubiadin, munjistin, anthragallol and xanthopurpurin as minor components indicate the use of madder (Rubia tinctorum L.). In 4 out of the 39 samples where Rubia tinctorum L. was identified (documents 159,204,265,144), it was used as an individual dye source. In all the samples, tannins were also present, as suggested through the detection of ellagic acid (see also section Tannins). Madder was detected in a dyeing combination with lac dye in 8 cases, tannins being observed in 2 samples (documents 161 and XL/15). In 24 other samples, madder was identified in a dyeing combination with redwood type, tannins being determined in about half of them. From the 2 samples (documents 119 and LXIV/7 1) where the 3 biological sources — lac dye, redwood type and madder — were detected together, traces of tannins were only observed in one case (LXIV/7 1). Except for those samples where madder was

used in a dyeing combination with lac dye, all the samples where madder was detected have a red-orange hue. 3.4. Tannins Tannins are substances of vegetable origin, which are mainly used to process animal hides and skins to leather [2]. Due to their properties, they also played an important role in dyeing. When used in combination with iron salts they give the fibers a brown or black hue. They may be also used for weighting silk and as mordants in wool, silk and cotton dyeing. Identification of tannins is based on the detection of ellagic acid, exhibiting a polyphenolic structure [16]. In the set of samples under discussion, ellagic acid was present in the UV chromatogram obtained with UV detection at 255 nm in 18 out of the total number of 43 samples, which suggests the

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use of tannins. The identification of ellagic acid was confirmed by the molecular ion (m/z = 301 [M-H]− ), observed in the FS-IECs. In 6 other samples, ellagic acid was detected only through mass spectrometry, which suggest that only traces of tannins were used. Tannins were absent in the samples where individual dyes were used, in those where the combination of lac dye and madder was identified (with an exception) and in some of the samples where madder and redwood type were detected together. 3.5. Chronology and comparison with results on religious embroideries from the time of Stephan the Great Results are in perfect correlation with earlier ones reported for samples in documents seal threads preserved in other Romanian museums, where madder with redwood type or lac dye, in the presence of tannins were detected [44]. No correlation between the dyes detected and the date was observed for documents emitted by Stephan the Great within an interval of 43 years (1460–1503). When comparing with dyes confirmed in the 2 documents emitted by Alexander the Good (in 1409 and 1425), no difference between the sources preferred by the two princes was noticed. The same biological sources — lac dye, madder and redwood type — were used in the ecclesiastical pieces and the documents seal threads. However, more valuable insect dyes, such as Polish carmine scale, kermes or Armenian carmine scale were especially kept for the extremely valuable religious embroideries. The sources were always correlated with the dyed fiber function: expensive insect dyes for the warp which is visible to the viewer and redwood type, a dye with poor light fastness, for the weft, which is invisible to the observer. In original documents seal threads emitted by Stephan the Great, redwood type was never used as individual dye source, but always in combination with madder and/or lac dye, to achieve a red hue, with good light fastness. 4. Conclusions Liquid chromatography with serially coupled UV-Vis and mass spectrometric detection systems (LC-DAD-MS or MS/MS) was shown to be a valuable tool in dyes characterization and identification. The association of the information provided by each detector is helpful to distinguish between the major and minor dyes and facilitates a clear attribution of the biological source/sources used. The tandem MS detection approach achieved through space delayed quadrupole mass analyzers enables unequivocal identification of each dye in complex matrices, due to its high sensitivity and selectivity. Lac dye, redwood type and madder as individual dyes or in combinations, were responsible for the colour in all the 43 samples analysed, 40 from the time of Stephan the Great (1457–1504) and 3 from Alexander the Good (1400–1432). Redwood type, a dye with poor lightfastness, was detected in the seal threads of 2 documents known to be fakes from the time of Stephan the Great. Keeping in mind that lac dye is a biological source of Oriental origin, with a rare use in Western Europe, it is worth to emphasize its frequent detection in this geographical area. Dyes and biological sources in a large number of dated documents, issued within a period of less than 50 years by the same Chancery, were identified. This set of results serve as an ideal landmark for the use of the respective sources and may be further used to fit unknown items in place and time. Acknowledgements The authors express their gratitude to dr. Gabriela Dumitrescu, Oana Lucia Dimitriu and Liana Nast˘as¸elu from the Romanian

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Academy Library for providing access to their collections and for sharing their knowledge on documents.

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Please cite this article in press as: I. Petroviciu, et al., Investigation of natural dyes in 15th c. documents seal threads from the Romanian Academy Library, by LC-DAD-MS (triple quadrupole), Journal of Cultural Heritage (2017), http://dx.doi.org/10.1016/j.culher.2017.05.015