Journal of Cultural Heritage 46 (2020) 108–118

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Universal analytical method for characterization of yellow and related

natural dyes in liturgical vestments from Krakow
Katarzyna Lech
Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland

i n f o a r t i c l e A b s t r a c t

Historique de l’article : Due to the diversity of dyes, as well as the multiplicity of colorants occurring in them, their identifica-
Reçu le 17 août 2019 tion in historical objects requires the development of an effective analytical approach including also the
Accepté le 24 avril 2020 separation step. Thus, the present study was focused on high-performance liquid chromatography (with a
Disponible sur Internet le 27 June 2020
reversed phase phenyl column) coupled with spectrophotometric and tandem mass spectrometric detec-
tions with electrospray ionization (HPLC–UV–Vis–ESI MS/MS) to develop a universal analytical approach
Keywords : dedicated to a comprehensive analysis of wide range of dyes. Optimization of ion source parameters led
Natural dyes
to a total increment in intensity of peaks even up to 64%. The final method, developed using dynamic
High-performance liquid chromatography
Tandem mass spectrometry
multiple reaction monitoring mode (dMRM), includes 112 colorants in negative ion mode and/or 12 in
Method optimization positive ion mode, 62 of which are reference compounds, while 54 are compounds identified by tan-
Textile dem mass spectrometry in fibers dyed with 10 examined yellow and orange dyes (annatto, cutch, dyer’s
broom, old fustic, Osage orange, saffron, sawwort, Persian berries, weld and young fustic). The developed
method was tested for the identification of natural dyes in yellow, orange, brown and green fibers from
15th- to 17th-century textiles used in the vestments from the collections of seventeen Krakow churches.
In this way, annatto and sawwort have been found in historical samples using HPLC–UV–Vis or HPLC–ESI
MS/MS for the first time.
© 2020 L’Auteur(s). Publié par Elsevier Masson SAS. Cet article est publié en Open Access sous licence
CC BY (http://creativecommons.org/licenses/by/4.0/).

1. Introduction and the power of analytical techniques. It can be achieved using

According to application properties, natural dyes can be clas-
sified as mordant, direct and vat dyes [1,2]. Most of them contain
several or even a dozen compounds, which can occur concomitantly
in many different dyes. Chemical structures of these colorants,
thus also their properties (including polarity), may be very simi-
lar to each other or completely different. Nevertheless, due to the
presence of delocalized electrons in conjugated ␲ systems of alter-
nating single and double carbon-carbon bonds all these compounds
may absorb visible light, causing a color impression to the human
eye. These systems can be classified according to the basic skeleton
structure into flavonoids, quinochalcones, homoisoflavonoids, ben-
zophenones, anthraquinones, indigoids, phenoxazines, alkaloids,
gallotannins, apocarotenoids, curcumins, etc. [2].
Analysis of various natural colorants potentially present in
historical objects obviously requires versatile and universal ana-
lytical method based on the advanced knowledge of researcher
Adresse e-mail : klech@ch.pw.edu.pl
HPLC–UV–Vis–ESI MS/MS [3–5].
Since colorants, by definition, must have a chromophore, a
conjugated system (mainly aromatic) with delocalized ␲-electrons
that can absorb visible light, it was decided in this study to use a
column with phenyl-type stationary phase, that can retain this type
of compounds thanks to the ␲-␲ interactions occurring between
“␲-active” solutes and the stationary phase surface [6]. A phenyl-
column can be especially useful in the separation of closely related
p-electron containing species like structural isomers, which are
common among colorants. The use of phenyl-column partly also
implied the use of methanol in the mobile phase. Methanol mole-
cules, unlike acetonitrile, do not compete with solutes in their ␲-␲
interactions and do not suppress them. Retention as well as selec-
tivity are generally greater in methanol-containing mobile phases,
whereas in acetonitrile separation takes place on C18 [7].
The use of a UV-Vis detector in the analysis of colorants is
understandable. Nevertheless, a MS detection provides better sen-
sitivity to most dyes, as well as greater selectivity, especially if it
concerns tandem mass spectrometry (MS/MS) [8–13], which allows
to establish the relationships between precursor ions and product
ions. Tandem mass spectrometry can provide also structural infor-

https://doi.org/10.1016/j.culher.2020.04.011
1296-2074/© 2020 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
 K. Lech / Journal of Cultural Heritage 46 (2020) 108–118 109

mation about eluted unknown compounds, such as flavonoids or
O- and C-glycosides [14–17].
Most of analytical methods published so far and dedicated
to the simultaneous identification of dyes belonging to different
classes were carried out using HPLC–UV–Vis [18–22], HPLC–MS
[20,22], or HPLC–UV–Vis–MS/MS [10,23–25]. However, only few
of them included more than 30 compounds, or authors did not
indicate a certain number of colorants included in the method.
Separation usually was carried out in the reverse phase (RP) confi-
guration on C18 columns. Recently, an effective application of an
amide column for separation of flavonoid and tannin degrada-
tion products has been also published [26]. Since, as mentioned
above, colorants are compounds with systems of conjugated double
bonds, it seems reasonable to use columns with phenyl-type sta-
tionary phase, but, to the best of my knowledge, there is no
publication over the use of this type of columns. Moreover, nearly
all reported methods have been dedicated mostly to identifying
the basic set of flavonoids present in weld, Persian berries or
young fustic [19,20,22–25,27–29]. However, there have been no
reports which involve markers of Osage orange or cutch, especially
together.
2. Research aim
The aim of this research was to develop and optimize the univer-
sal HPLC–UV–Vis–ESI MS/MS method basing on the phenyl column
to separate and identify in a relatively short time the widest range
of colorants present in various organic dyes of natural origin. The
method includes 112 colorants in negative ion mode and/or 12 in
positive ion mode, 62 of which are reference compounds, while 54
are compounds identified by tandem mass spectrometry in fibers
dyed with 10 examined natural dyes (annatto, cutch, dyer’s broom,
old fustic, Osage orange, saffron, sawwort, Persian berries, weld and
young fustic). The developed approach is intended for the identifi-
cation of natural dyes used in historical objects, antiques and works
of art.
3. Material and methods
3.1. Apparatus
Separation and identification of the colorants was carried out
using a HPLC system coupled with two spectrophotometric detec-
tors and tandem mass spectrometric detector simultaneously
(optimized parameters of the developed method are presented in
Table 1, whereas fragmentor values for each precursor ion, the
dynamic multiple reaction monitoring (dMRM) transitions, and
collision energies (CE) are presented in Table 2. The analyses were
controlled by a MassHunter Workstation software (Agilent Tech-
nologies, USA). Spectrophotometric spectra of the colorants were
acquired from 240 to 700 nm with the use of a Lambda 20 UV–Vis
spectrophotometer (Perkin–Elmer, USA). The scan speed and the
slit width were 120 nm min−1 and 2 nm, respectively.
3.2. Chemicals and materials
Standard solutions of colorants (0.2 mg·mL−1 ) were prepared in
methanol. Only indigotin and indirubin (0.1 mg·mL−1 ) were dissol-

Table 1
HPLC–UV-Vis–ESI MS conditions.

HPLC separation
Pump 1220 Infinity II LC System (Agilent Technologies, USA)
Column Zorbax SB-Phenyl, 4.6 × 150 mm, 3.5 m, 80 Å (Agilent Technologies)
Precolumn Zorbax SB-Phenyl, 4.6 × 12.5 mm,
5.0 ␮m (Agilent Technologies)
Injection volume 20 ␮L
Flow rate 0.5 · mL min−1
Eluent (A) 0.15% (v/v) formic acid in water, (B)
methanol
Gradient program Time, min %A %B
0.0 60 40
15.0 40 60
20.0 30 70
27.0 0 100
35.0 0 100
Stop time 35.0 min
Equilibrium time 8.0 min

UV-Vis detection
Detector (1) 1220 Compact Variable Wavelength Detector (Agilent Technologies, Germany), part of 1220
Infinity II LC System; (2) 1200 Variable Wavelength Detector (Agilent Technologies, Germany)
Wavelength 280, 400, 450, 480, 500, 550, 580 or 600 nm

ESI-MS detection
Detector 6460 Triple Quad LC/MS with JetStream
Technology (Agilent Technologies,
USA)
Polarity mode negative ion (NI), positive ion (PI)
Mode full scan (m/z 100–1000), product ion (MS/MS), dynamic multiple reaction monitoring (dMRM)
Ionization/probe voltage 3.0 kV (NI), 3.5 kV (PI)
Orifice voltage 135 V (full scan mode), variable
(dMRM mode; details in Table 2)
Drying gas flow 5 L·min−1
Drying gas temperature 300 ◦ C
Nebulizer pressure 45 psi
Sheath gas flow 12 L·min−1
Sheath gas temperature 380 ◦ C
Nozzle voltage 500 V
Collision energy 5 to 45 V (MS/MS mode), various
(dMRM mode; details in Table 2)
dMRM retention time width 2.2 min
110 K. Lech / Journal of Cultural Heritage 46 (2020) 108–118

Table 2
HPLC–UV–Vis–ESI MS/MS characterization of color compounds.

No. Compound name tR , min [M−H]− , m/z Frag., V Product ions, m/z (CE, V) max , nm

1 Hematein* 4.0 299 130 281 (8), 253 (15), 174 (15), 125 (15) 284, 383
2 Gallic acid* 4.1 169 90 125 (12) 273
3 Digalloylglucose 4.5 483 170 271 (25), 211 (35), 169 (35) N/A
4 Catechin* 5.2 289 130 245 (9), 123 (29), 109 (25) 280
5 Hematoxylin* 5.2 301 150 283 (17), 179 (12), 137 (21), 123 (29) 279, 397
6 Trigalloylglucose 5.3 635 170 465 (25), 313 (40), 169 (45) N/A
7 Maclurin* 5.7 261 90 151 (9), 109 (25) 317
8 Epicatechin 6.3 289 130 245 (9), 123 (29), 109 (25) N/A
9 Chlorogenic acid* 6.4 353 90 191 (8) 247, 328
10 Tetragalloylglucose (1) 6.7 787 170 617 (25), 465 (40), 169 (45) N/A
11 Tetragalloylglucose (2) 7.8 787 170 617 (25), 465 (40), 169 (45) N/A
13 Picrocrocin 9.1 375 100 179 (5) N/A
14 Fustin* 9.3 287 130 269 (5), 163 (5), 109 (21) 233, 278, 310
15 Pentagalloylglucose 9.4 939 170 787 (30), 619 (45), 617 (45) N/A
16 p-Coumaric acid (1)* 9.5 163 90 119 (12) 296, 311
17 Luteolin C-hexoside 9.7 447 170 357 (15), 327 (25), 299 (35) N/A
18 Piceatannol 10.1 243 100 225 (10), 199 (15), 175 (15) N/A
19 Dihydromorin 10.2 303 130 125 (10) N/A
20 p-Coumaric acid (2) 10.6 163 90 119 (12) 296, 311
21 Taxifolin 10.7 303 130 285 (10), 125 (15) N/A
22 Quercetin O-hexoside 10.8 463 130 301 (20), 300 (20), 271 (35), 243 (35) N/A
23 Kaempferol di-O-hexoside 10.9 609 130 284 (35), 255 (45) N/A
24 Carminic acid* 11.2 491 170 447 (14), 357 (22), 327 (22), 299 (34) 276, 310, 496
25 Eriodictyol O-glucuronide 11.3 463 130 287 (12) N/A
26 Chta (stilbenoid dimer) 11.3 487 130 377 (10), 243 (15) N/A
27 Vitexin* 11.6 431 170 311 (14), 283 (30) 270, 334
28 Quercetin O-(hex-dhex-dhex) 11.6 755 170 301 (40) N/A
29 (Epi)afzelechin-(epi)catechin dimer 11.7 561 135 425 (15), 289 (22) N/A
30 Rutin* 12.1 609 210 301 (30), 300 (38), 271 (45) 257, 358
31 Luteolin O-(hex-dhex) 12.2 593 170 285 (35) N/A
32 Methoxy-trihydroxyxanthone 12.3 273 130 258 (15), 230 (25) N/A
33 Luteolin di-O-hexoside 12.3 609 170 447 (25), 285 (45) 268, 343
34 chtB (stilbenoid dimer) 12.3 487 130 377 (10), 243 (15) N/A
35 Genistin* 12.5 431 210 269 (10), 268 (22) 261
36 Hyperoside* 12.7 463 170 300 (22), 271 (45) 257, 360
37 Luteolin/kaempferol O-(hex-dhex-dhex) 13.8 739 170 285 (40) N/A
38 Dihydrokaempferol 13.9 287 130 259 (10),125 (15) N/A
39 Myricetin* 14.1 317 130 179 (13), 151 (17), 137 (21) 254, 377
40 Luteolin 7-O-glucoside* 14.1 447 210 285 (22), 284 (33) 260, 348
41 Hesperidin* 14.8 609 170 301 (22) 284
42 Genistein O-hexoside 15.0 431 170 268 (35) N/A
43 Kaemferol O-(hex-dhex) 15.0 593 170 285 (35) N/A
44 Luteolin O-glucuronide (1) 15.0 461 130 285 (25) N/A
45 Quercitrin* 15.0 447 170 301 (14), 300 (18), 271 (42) 256, 349
46 Lawsone* 15.6 173 210 145 (20) 243, 249, 274, 332
47 O-Methyleriodictyol O-glucuronide 15.7 477 130 287 (12) N/A
48 Kaempferol 3-O-glucoside* 15.7 447 130 285 (20), 284 (30), 255 (40) 266, 350
49 Tetrahydroxyflavone 15.8 285 135 229 (20), 211 (25), 151 (20), 133 (30) N/A
50 Ellagic acid* 16.0 301 170 284 (29), 229 (21), 145 (37) 255, 366
51 Isorhamnetin 3-O-glucoside 16.1 477 130 314 (25), 285 (35), 271 (45), 243 (45) N/A
52 Apiin* 16.3 563 250 269 (34) 268, 334
53 Fisetin* 16.4 285 130 135 (17), 121 (25) 248, 319, 363
54 Luteolin O-hexoside (1) 16.5 447 170 285 (15) N/A
55 Morin* 17.0 301 130 151 (13), 149 (21), 125 (13), 107 (21) 263, 361
56 Eriodictyol 17.1 287 130 151 (10), 135 (25) N/A
57 Luteolin O-glucuronide (2) 17.1 461 130 285 (15) N/A
58 Apigenin 7-O-glucoside* 17.3 431 210 269 (18), 268 (30) 268, 332
59 Tetrahydroxyflavanone 17.4 287 130 161 (10), 125 (10) N/A
61 trans-Crocin* 17.6 975 140 651 (15), 327 (35), 283 (45) 235, 323, 432, 457
533a 140 651 (10), 327 (10)
62 Diosmin* 18.0 607 170 299 (22), 284 (45) 255, 270, 350
63 Rhamnetin O-(hex-dhex-dhex) 18.0 769 170 315 (45), 314 (45) N/A
64 Quercetin* 18.2 301 130 179 (13), 151 (13), 107 (25) 256, 371
65 Luteolin O-hexoside (2) 18.4 447 170 285 (25) N/A
66 O-methylluteolin O-glucuronide 19.1 475 130 299 (25), 285 (25), 284 (45) N/A
67 Sulfuretin* 19.9 269 130 135 (17), 133 (25) 257, 397
68 Rhamnocitrin O-(hex-dhex-dhex) 20.3 753 170 299 (35) N/A
69 trans-Crocin-3 20.8 859 100 651 (15), 489 (15), 327 (35), 283 (45) N/A
70 Datiscetin 21.1 285 100 213 (13), 151 (15), 125 (15) N/A
71 Luteolin* 21.1 285 170 133 (33) 256, 348
72 Naringenin* 21.2 271 110 151 (13), 119 (28) 288, 324 (sh)
73 Rhamnazin/ombuin O-(hex-dhex-dhex) 21.2 783 170 329 (35) N/A
74 Luteolin O-methylglucuronide 21.3 475 130 285 (15) N/A
75 Anthraflavic acid* 21.6 239 130 211 (26), 210 (30), 195 (22), 182 (42) 240, 273, 299, 346
76 Genistein* 21.6 269 170 133 (29), 132 (40), 63 (37) 261
77 Quercetin 3-methylether 21.7 315 135 300 (13), 271 (28) N/A
 K. Lech / Journal of Cultural Heritage 46 (2020) 108–118 111

Table 2 (Continued)

No. Compound name tR , min [M−H]− , m/z Frag., V Product ions, m/z (CE, V) max , nm

78 Kaempferol* 22.1 285 170 239 (23), 117 (44), 93 (33) 266, 367
79 Rhamnetin O-(hex-dhex) 22.1 623 170 315 (35), 299 (45) N/A
80 Rhamnetin O-deoxyhexoside 22.6 461 170 315 (25), 314 (25), 299 (35), 271 (45) N/A
81 Luteolin methylether 22.7 299 130 284 (20) N/A
82 Isorhamnetin* 23.1 315 130 300 (16) 254, 300, 370
83 O-Methylluteolin O-methylglucuronide 23.3 489 130 474 (25), 298 (25), 283 (35), 255 (45) N/A
84 trans-Crocin-2 23.8 697 100 489 (10), 327 (15), 283 (25) N/A
85 Apigenin* 24.2 269 130 117 (27) 268, 334
86 Kermesic acid* 24.2 329 90 285 (10) 274, 308, 492
87 cis-Crocin* 24.6 975 140 651 (15), 327 (35), 283 (45) 235, 323, 432, 457
88 Rhamnetin* 24.7 315 130 300 (13), 165 (13), 121 (21) 256, 372
89 Rhamnetin O-(hex-dhex-dhex-dhex) 25.1 915 170 769 (50), 315 (38), 314 (45) N/A
90 Diosmetin* 25.3 299 130 284 (17) 252, 268, 292, 343
91 cis-Crocin-3 26.2 859 100 651 (15), 327 (35) N/A
813 100 651 (15), 327 (25)
92 Alizarin* 26.4 239 170 211 (26), 210 (30) 248, 274, 324, 429
93 cis-Crocin-2 26.7 697 100 651 (10), 327 (25), 283 (30) N/A
94 Biochanin A* 27.1 283 130 268 (17) 261, 324 (sh)
95 Rhamnocitrin 27.1 299 130 284 (15), 271 (15), 165 (15) N/A
96 Anthrarufin* 27.4 239 170 211 (26), 182 (45) 225, 252, 285, 417
97 Amentoflavone* 27.8 537 250 417 (30), 375 (30) 270, 335
98 Rhamnazin/ombuin 27.8 329 130 314 (15), 301 (15), 299 (25), 271 (35), 165 (13) N/A
99 cis-Crocin-2 28.1 651 100 327 (10) N/A
100 Genkwanin* 28.1 283 110 268 (30) 268, 335
101 Curcumin* 28.3 367 90 217 (0), 173 (8), 149 (8), 134 (28) 263, 423
102 Purpurin* 28.3 255 130 227 (22), 171 (30), 129 (38), 101 (45) 255, 290, 482
103 Acacetin* 28.4 283 130 268 (21) 269, 328
105 Rubiadin* 29.0 253 110 225 (25), 209 (22), 195 (55) 245, 278, 330, 411
107 Rhein* 29.4 283 90 239 (10), 211 (22), 183 (26) 230, 257, 431
108 Emodin* 29.5 269 170 241 (26), 225 (22) 222, 252, 266, 288, 435
109 Chrysazin* 29.8 239 170 211 (26) 223, 252, 283, 428
110 Quinizarin* 30.1 239 210 211 (18) 224,248, 278, 324, 479
111 Crocetin* 30.3 327 90 283 (0), 239 (0), 239 (4) 256, 423, 449
112 Chrysophanol* 30.8 253 170 225 (26) 225, 256, 277, 287, 429
113 Atranorin* 31.0 373 90 177 (10), 163 (14), 133 (22) N/A
114 Physcion* 31.5 283 130 268 (22), 240 (25) 254, 265, 287, 434
115 Norbixin* 31.8 379 80 335 (3), 291 (2) 455, 483
116 Bixin* 32.8 393 80 349 (3), 317 (4), 225 (3) 456, 484

No. Compound name tR , min [M+H]+ , m/z Frag., V Product ions, m/z (CE, V) max , nm

12 Isatin* 8.8 148 90 130 (15), 102 (25), 92 (20), 77 (25), 65 (30) 296, 413
13 Picrocrocin 9.1 369 100 207 (25) N/A
60 Berberine* 17.5 336 170 321 (21), 320 (29), 292 (29), 278 (44) 265, 346, 427
61 trans-Crocin* 17.6 999b 140 675 (45), 347 (45) 235, 323, 432, 457
511c 140 347 (25)
69 trans-Crocin-3 20.8 837 100 675 (35), 347 (45) N/A
87 cis-Crocin* 24.6 999b 140 675 (45), 347 (45) 235, 323, 432, 457
93 cis-Crocin-2 26.7 675 100 347 (35) N/A
99 cis-Crocin-2 28.1 675 100 347 (35) N/A
104 Indigotin* 28.7 263 90 235 (23), 219 (19), 206 (39), 132 (35), 77 (50) 291, 620#
106 Indirubin* 29.2 263 170 235 (19), 219 (23), 190 (43) 257, 550#
111 Crocetin* 30.3 329 90 311 (0), 293 (4), 275 (9), 211 (9) 256, 423, 449
116 Bixin* 32.8 395 90 377 (5), 363 (5), 145 (20) 456, 484

dhex - deoxyhexoside, hex - hexoside.

*
Data determined for standard solutions.
#
Absorption maxima determined for DMSO solutions.
a
Ion [M+2HCOO]2− ,
b
Ion [M+Na]+ ,
c
Ion [M+2Na]2+ , dhex – deoxyhexoside, hex – hexoside.

ved in dimethyl sulfoxide (DMSO). Detailed list of reagents and dyes
is presented in Supplementary Material, Section 1.
The 89 silk fibers (yellow, orange, brown and green) were taken
from 15th- to 17th-century textiles used in the vestments belon-
ging to the collections of seventeen Krakow churches (all samples
are depicted in [47]).
3.3. Dyeing and extraction of fibers
The mordant dyes were applied to the wool pre-treated with
alum, while the direct dyes were applied directly to the wool
without any initial pre-treatment. Extraction of brown and green
fibers was processed twice, using consecutively two extraction
methods, with DMSO and with acidic-methanol solution, whereas
yellow and orange fibers were extracted only using the second pro-
cedure. Schemes of dyeing and extraction methods are presented
in Figs. 1 and 2, respectively.
4. Results and discussion
The reported research had three stages. Firstly, standard solu-
tions of colorants were used for HPLC–UV–Vis–ESI MS/MS method
development. Then, the method was expanded on markers (com-
mercially unavailable) found in extracts of wool dyed with 10
112 K. Lech / Journal of Cultural Heritage 46 (2020) 108–118
Fig. 1. Scheme of the fiber dyeing method.
Fig. 2. Scheme of extraction method.
yellow, orange and brown natural dyes; the markers were determi-
ned using MS/MS data. Finally, the developed approach was used to
identify colorants in yellow, orange, brown and green fibers taken
from 15th- to 17th-century textiles.
4.1. Method development
The method was developed basing on a phenyl column and
the mixture of 33 colorant standards (compounds are listed in
Supplementary Material, Figure S-1). The variety and multiplicity
of natural colorants required the use of gradient elution to achieve
their good separation in a reasonable time (between 25 and 35 min).
Considering the type of column as well as my previous nonpubli-
shed studies conducted on the influence of the pH modifiers on
the intensity of the MS signal, a standard mobile phase consis-
ting of 0.15% of formic acid in water (A) and methanol (B) was
used. Detection was carried out using a UV-Vis detector at 280,
450 and 600 nm and a mass spectrometry detector in the selective
ion monitoring mode (SIM) of negative ionization. At first, a recti-
linear scouting gradient of A and B components (15:85 to 0:100)
performed for 30 min was applied, but that did not enable the elu-
tion of all non-polar compounds. Moreover, only one compound
(gallic acid) eluted before 14 min, thus the gradient needed to be
significantly improved. The initial content of organic solvent in the
mobile phase was increased in the next few runs, as well as its rise
in time at different points of the analysis was changed several times
to achieve the best separation.
None of the tested conditions was able to achieve elution of
the entire set of compounds with proper resolution. The best per-
formance was obtained by using the 35-min method (gradient is
showed in Table 1). The first compound was eluted at 4.1 min, and
the peaks of further compounds were fairly evenly distributed in
the chromatogram (Supplementary Material, Section 2). They were
sharp and symmetrical. Only myricetin and luteolin 7-O-glucoside
as well as purpurin and curcumin fully co-eluted with each other.
Similar retention times characterized also carminic acid and genis-
tin, luteolin and genistein, rhamnetin and apigenin, ellagic acid and
fisetin, as well as ellagic acid and apigenin 7-O-glucoside. Further
modification of the gradient, even its extension, did not signifi-
cantly improve separation of these compounds. Nevertheless, the
high selectivity of MS/MS detector in the MRM mode enabled full
discrimination of the mixture components, especially since all iso-
baric compounds were well separated and their retention times
were stable. Thus, these separation conditions were considered to
be sufficient to start optimization of MS detection.
The ion source conditions and the MS/MS parameters were opti-
mized using the same mixture of standards as described above.
Gas flow, nebulizer pressure, sheath gas flow that correspond to
sheath gas temperature, and capillary voltage were tested at seve-
ral levels in SIM mode (details are presented in Supplementary
Material, Section 23). The final values of ESI parameters are defined
in Table 1. However, regardless of the conditions, two compounds
(bixin and crocetin) remained almost undetected by MS.
Nevertheless, the crucial parameters affecting the intensity of
the MS signal were fragmentor (orifice voltage), optimal values
of which were very much compound-dependent, just like the CE
values for each MRM transition. Both these parameters were deter-
mined individually for each standard solution using MassHunter
Optimizer software. Each sample (made up in 0.15% formic acid
in water/methanol, 3:7, v/v) was introduced directly into the ESI
MS/MS using flow injection analysis. Considering structures of the
colorants, literature [30–33], and my previous nonpublished stu-
dies, small acids, flavonoids and anthraquinones were examined
in negative ion mode, berberine, indigoids and their derivatives
in the positive mode, whereas apocarotenoids in both modes. In
total, MRM conditions for 61 standards were optimized (results are
shown in Table 2).
4.2. Fragmentation of color compounds
The MS/MS experiments provided precise information about
fragmentation pathways of the colorants, which are similar inside
the same group of compounds. On this basis the compounds were
classified into six groups: flavonoids, O- and C-glycosides, anthra-
quinones and indigoids, acids and others, which did not fit to any of
 K. Lech / Journal of Cultural Heritage 46 (2020) 108–118 113

these group. Fragmentation pathways and the origin of individual

product ions are specified in Supplementary Material, Section 4.

4.3. Analysis of dye colorants

The HPLC–UV–Vis–ESI MS method developed with the aid of

commercially available standards has been extended with addi-
tional data achieved based on the analysis of selected dyes and
identification of the compounds contained therein with the sup-
port of the acquired MS/MS spectra and literature data. Extracts of
eight yellow (weld, dyer’s broom, sawwort, Persian berries, old fus-
tic, young fustic, annatto and saffron), one orange (Osage orange)
and one brown (cutch) dyes were examined. Full scan MS analysis
and the subsequent fragmentation of the predominant pseudo-
molecular ions were used to obtain information about molecular
weights of the colorants and the sugar moieties, as well as structure
of unglycosylated compounds or aglycones.

4.3.1. Weld and dyer’s broom

Presence of luteolin (71), apigenin (85), diosmetin (90), and
luteolin 7-O-glucoside (40) was confirmed in weld extract by com-
parison with the standards (Fig. 3a). In addition, the identities of
two luteolin O-hexosides (54 and 65) and luteolin di-O-hexoside
(33) were established on the basis of [M−H]− ions (at m/z 447
and 609), losses of 162 Da corresponded to hexoside moiety
([M−H−Hex]− and [M−H−2Hex]− ) and characteristic ion at m/z
133 that is luteolin fingerprint produced via retro-Diels–Alder
(RDA) reaction ([1,3 B−H]− ).
The same compounds, except luteolin di-O-hexosides, were
identified in dyer’s broom extract (Fig. 3b). Furthermore, geni-
stein (76), genistin (35) and vitexin (27) were also found in
the extract. Remaining compounds were characterized based on
MS/MS spectra. One of them was luteolin-C-hexoside (17) with
pseudo-molecular ion at m/z 447 and fragmentation pathway typi-
cal for C-glycosides (ions at m/z 357, 327 and 299 corresponded
to [0,3 X−H]− , [0,2 X−H]− ) and [0,1 X−H]− , respectively) as well as
genistein O-hexoside (42) with precursor ion at m/z 431 and
[M−H−Hex]•− at m/z 268. The next compound identified in dyer’s
broom was luteolin methyl ether (81, [M−H]− at m/z 299), probably
substituted with methyl group at the C-3 position. It was confirmed
by the loss of methyl radical ([M−H−CH3 ]•− at m/z 284) as well as
by the by the characteristic [1,3 B−H]•− ion at m/z 148 formed via
RDA reaction.

4.3.2. Persian berries

Chromatograms acquired for extract of Persian berries (Fig. 3c)
showed four peaks corresponding to unglycosylated compounds,
that is quercetin (64), rhamnetin (88), luteolin (71) and emodin
(108). Moreover, another twelve peaks corresponded to glycosides.
After comparison with standards three of them were found to be
hyperoside (36), hesperidin (41), and quercitrin (45), whereas the
rest was identified based on two MS/MS data sets. The first one,
acquired for glycosides (Supplementary Material, Figures S-5 and S-
6), led to establish types of glycans. The lost fragment of 146 Da was
identified to be a single deoxyhexose moiety, while 308 or 454 Da
corresponded to deoxyhexose and hexose or two deoxyhexoses and
one hexose linked glycosidically, respectively. Moreover, the same
type of spectra but acquired at higher collision energies and MS/MS
spectra of aglycones released by acid hydrolysis of O-glycosidic
bonds using hydrochloric acid were the basis of aglycone identifica-
tion. In consequence, two new O-methylated flavonols were found
Fig. 3. Chromatograms of (a) weld, (b) dyer’s broom, (c) Persian berries, (d) cutch,
at tR 27.1 and 27.8 min. Their identities were confirmed on the basis
(e) Osage orange, (f) old fustic, (g) young fustic, (h) sawwort and (i) annatto extracts
of pseudo-molecular ions (at m/z 299 and 329, respectively), as well acquired by UV-Vis detector; peak numbers are decoded in Table 2.
as characteristic fragmentation pathways. Apart from ions corres-
ponding to the loss of one or two • CH3 radicals, the MS/MS spectra
of both compounds showed also ions at m/z 165 and 121, that were
114 K. Lech / Journal of Cultural Heritage 46 (2020) 108–118

formed via RDA reaction ([1,3 A−H]− ) and further loss of CO2 , res-
pectively. The results indicate both compounds were substituted
with one methoxy group at the A ring (like rhamnetin), whereas the
second compound had also another methoxy substituent localized
at the rings B. Thus, flavonols were identified to be rhamnocitrin
(95) and rhamnazin/ombuin (98), respectively.

4.3.3. Cutch
UV chromatogram of cutch extract acquired at 280 nm sho-
wed two significant peaks (Fig. 3d). The first one was identified
to be catechin (4) by comparison to the standard. MS/MS spec-
tra of the second compound (tR 6.3 min) were identical to these
of catechin (Supplementary Material, Figures S-7), thus this com-
pound was identified to be epicatechin (8), the isomer of catechin;
it was compatible with available literature [34,35]. Moreover, very
intense peak corresponding to quercetin (64) and few smaller peaks
were observed at 400 nm. One of them matched to kaempferol (78),
whereas the rest was identified based on their MS/MS spectra.
The compound eluted at tR 21.7 min was identified to be quer-
cetin O-methyl ether on the basis of [M−H]− ion at m/z 315 and
the loss of 15 Da corresponded to •CH3 radical (Supplementary
Material, Figures S-7). It was identified to be quercetin 3-O-mthyl
ether (77), the compound that has been previously isolated from
Acacia catechu Willd. [36].
The MS/MS spectra of the next compound (tR 15.8, [M−H]−
at m/z 285) were in a high m/z region very similar to spectra of
kaempferol (for example in ions at m/z 239, 229, 227, 211 and 185),
but they showed also some differences, especially below m/z 200
(Fig. 4a). Ions at m/z 133 and 151 were formed by C-ring clea-
vage of bonds 1 and 3; it was the evidence for presence of two
hydroxyl groups at the A-ring and one hydroxyl group at the B-ring
(assuming we were dealing with flavonol). Thus, the compound
was identified to be tetrahydroxyflavone (49), probably substituted
with hydroxy groups at position C-3, -5, -7 and -3 .
The identities of taxifolin (21) was established by comparing
MS/MS data with those available in the literature [35,37], i.e. based
on the [M−H]− ion at m/z 303, the loss of H2 O (m/z 285) and pre-
sence of intense ion at m/z 125 (Fig. 4b). Dihydrokaempferol (38,
[M−H]− at m/z 287), also called aromadedrin, was identified in the
analogous way. Apart from the signal at m/z 259 corresponding to
the loss of CO molecule (Fig. 4c), the ion at m/z 125 was present
[38], thus, signal at m/z 125, which corresponds to the anion of
phloroglucinol, is a result of heterocyclic ring fission (HRF) and can
be a diagnostic fragment ion for C-5 and C-7 dihydroxy-substituted
flavanonols.
The MS/MS spectra acquired for ion [M−H]− at m/z 561 of the
last peak (tR 11.7 min) showed five product ions (Supplementary
Material, Figures S-7) characteristic for fragmentation pathway of
oligomeric proanthocyanidin [39,40]. Ion at m/z 435 was formed
by heterocyclic C-ring fission (HRFC ), ions at m/z 425 and 407 were
a result of RDAC reaction within the same ring and further loss of
H2 O, respectively, whereas ions at m/z 289 and 273 were formed by
quinone methide fission (QMCD ) of the interflavan bond (between
C- and D-ring). Thus, the examined compound was identified to be Fig. 4. MS/MS spectra of (a) tetrahydroxyflavone, (b) taxifolin, (c) dihydrokaempfe-
B-type dimer of (epi)afzelechin and (epi)catechin (29). rol, (d) dihydromorin, (e) tetrahydroxyflavanone, (f) methoxy-trihydroxyxanthone
and (g) piceatannol acquired in negative ion mode.

4.3.4. Osage orange and old fustic

Chromatograms of Osage orange extract showed several peaks
(Fig. 3e). Four of them were recognized to be dihydrokaempferol
(38), morin (55), naringenin (72), and kaempferol (78). Other com-
pounds observed in the chromatogram were identified based on
MS/MS spectra.
The first compound eluted at tR 10.2 min showed pseudo-
molecular ion at m/z 303 and MS/MS spectrum similar to the one
of taxifolin, except fragments at m/z 177, 217 and 285 that were
much less intense, and the ion at m/z 125 (formed by HRF) that was
the main signal on the spectrum (Fig. 4d). It was identified to be
dihydromorin (19).
MS/MS spectra of the next peak (tR 17.4 min, [M−H]− at m/z 287)
showed one main product ion at m/z 125 and another one, much
less intense, at m/z 161 (Fig. 4e), both formed by HRF. Moreover,
signals produced via RDA (m/z 135 [1,3 A−H]− and 151 [1,3 B−H]− )
were also observed, as well as signal corresponding to the loss of
 K. Lech / Journal of Cultural Heritage 46 (2020) 108–118
H2 O (m/z 269), but their intensities were very low. Thus, this com-
pound was identified to be tetrahydroxyflavanone (59) (with two
hydroxy groups substituted at C-5 and C-7 positions of the A-ring).
According to literature [41,42], Maclura pomifera Raf. Schneid.
besides flavonoids contains also xanthones. One of them, methoxy-
trihydroxyxanthone (32), eluted at 12.3 min. MS/MS spectra of its
pseudo-molecular ion (m/z 273) showed the main signal (m/z 258)
formed by the loss of • CH3 , which is characteristic decomposition
of methoxy group (Fig. 4f). Further fragment ions corresponded to
subsequent losses of CO molecules were observed at m/z 230, 202
and 174. Ion at m/z 107 formed via RDA reaction proved that this
xanthone was substituted by two hydroxy groups at the A-ring,
thus, a methoxy group and the third hydroxy group were linked to
the B-ring.
The extract of old fustic contained almost the same colo-
rants as the extract of Osage orange, including dihydromorin (19),
dihydrokaempferol (38), tetrahydroxyflavone (49), morin (55),
tetrahydroxyflavanone (59), and kaempferol (78), nevertheless,
xanthanone was present only in trace amount and naringenin was
missing (Fig. 3f). Furthermore, the most intense peak at 280 nm (tR
10.1 min) corresponded to piceatannol (18). It was identified on the
basis of [M−H]− ion at m/z 243 and its MS/MS spectrum (Fig. 4g),
that was identical with the one reported in the literature [43]. The
main product ions at m/z 225, 199 and 175 corresponded to the
loss of H2 O, C2 H2 O, CO2 and C3 O2 , respectively, whereas ions at
m/z 201 and 159 were formed by two sequential losses of C2 H2 O
[44]. Unequivocal identification of this as well as attributed other
compounds would require thorough characterization with the aid
of complementary techniques, such as nuclear magnetic resonance
(NMR).
Moreover, also two stilbenoid dimers (26 and 34, [M−H]− at m/z
487) were identified. Their MS/MS spectra were almost identical
with the ones of piceatannol, except two additional signals at m/z
109 and 377 corresponding to the anion of benzenediol and to the
loss of benzenediol moiety, respectively (Supplementary Material,
Figure S-8).
4.3.5. Other yellow dyes
Extracts of young fustic and annatto showed a small number
of colorants. Chromatograms of young fustic showed only sulfure-
tin (67) and fustin (14) (Fig. 3g), while in the annatto extract only
bixin (116) and small peak of norbixin (115) were found (Fig. 3i).
All these compounds were identified by their comparison with the
standards.
Colorants observed in sawwort extract (Fig. 3h) (such
as eriodictyol (56), eriodictyol O-glucuronide (25), O-
methyleriodictyol O-glucuronide (47), two isomers of luteolin
O-glucuronide (44 and 57), O-methylluteolin O-glucuronide
(66), luteolin O-methylglucuronide (74), and O-methylluteolin
O-methylglucuronide (83)) and saffron (cis- and/or trans-crocin
(87 and 61), crocin-3 (91 and 69), crocin-2 (93), and kaempferol
di-O-hexoside (23)) were identified based on previously conducted
research, which has been published elsewhere [8,45]. Crocins and
picrocrocin (13) were included into the method in both, negative
and positive ion modes.
4.4. Analysis of historical samples
The developed HPLC–UV–Vis–ESI MS/MS method was used to
analyses yellow, orange, brown and green fibers taken from 15th-
to 17th-century textiles being a part of Catholic liturgical vest-
ments. The 75 DMSO extracts and 89 methanol/water/formic acid
extracts were tested in positive or in both ion modes, respectively.
All samples, identified colorants and dyes are listed in Ref. [47].
Weld (Reseda luteola L.) was identified in 31, whereas dyer’s
broom (Genista tinctoria L.) was found in 36 yellow and green fibers.
115
Fig. 5. Chromatograms acquired for methanolic extracts of yellow sample (textile
No. 231) by MS detector in (a) negative and (b) positive MRM modes, and by (c)
UV-Vis detector at 280 and 450 nm; peak numbers are decoded in Table 2.
In most samples both these dyes were used with other dyes (Fig. 5).
In green yearns, they were accompanied mostly by indigo or woad,
as it was evidenced by the presence of indigotin (104) and indiru-
bin (106). Nevertheless, in one sample (No. 164) a green hue was
obtained using dyer’s broom individually, probably by the use of
proper mordant, maybe copper salt.
Furthermore, the green fibers were also dyed with Persian ber-
ries (the genus Rhamnus L.) or sawwort (Serratula tinctoria L.). These
dyes, along with indigo/woad, were identified (Fig. 6) in three
and seven extracts, respectively. Although the literature refers that
sawwort has been used mainly for mixed colors, including green
[1,2], this is the first time that it has been identified in historical
samples.
Another flavonoid dye identified in extracts obtained from yel-
low and brown samples was young fustic (Cotinus coggygria Scop.).
This yellow dye was used both individually and in mixtures with
other dyes (Fig. 5).
In addition, chromatographic profiles obtained for seven green
samples, mostly taken from Persian or Turkish textiles, did not cor-
respond to any of the previously tested yellow dyes. Nevertheless,
their chromatographic profiles indicated three different dyes. The
first one (unknown yellow flavonoid dye I), found in three samples
(No. 329, No. 330 and No. 332) was relatively simple (Fig. 7a); only
hyperoside (36), kaempferol 3-O-glucoside (48), and a small signal
of rutin (30) were observed. Nevertheless, another intense peak was
found at tR 16.1 min. Fragmentation of its [M−H]− ion at m/z 477 led
to ions at m/z 314, and further to 285, 271 and 243. Since chroma-
togram of an extract hydrolysed with hydrochloric acid apart from
kaempferol and quercetin contained also isorhamnetin ([M−H]−
at m/z 315), the compound eluting at 16.1 min was identified to be
isorhamnetin O-hexoside (51); the identity was confirmed at a later
stage by comparison with the standard. The depicted composition
was consistent with the one of larkspar (Delphinium semibarbatum
Bien. ex Boiss.) presented in the literature [46].
The chromatographic profile of the second dye (the unknown
yellow flavonoid dye II) found in one sample (D-31), was charac-
terized by presence of luteolin (71), luteolin 7-O-glucoside (40),
luteolin O-glucuronide (44), and luteolin C-hexoside (17) (Fig. 7b).
Since the composition was partly like the one of sawwort, it was
determined to be a luteolin-based and sawwort-like dye.
The third profile was more diverse, but in-depth analysis of the
results showed two yellow dyes that have been used, the unknow
yellow flavonoid dye II (already identified in the sample D-31), and
116 K. Lech / Journal of Cultural Heritage 46 (2020) 108–118
Fig. 6. MRM chromatograms acquired in negative and positive MRM modes for green samples: (a) No. 172 dyed with sawwort and indigo and (b) No. 328 dyed with Persian
berries and indigo; peak numbers are decoded in Table 2.
Fig. 7. MRM chromatograms acquired in negative ion mode for methanolic extracts
of samples: (a) No. 329, (b) D-31, and (c) No. 320; peak numbers are decoded in
Table 2.
another dye (the unknow yellow flavonoid dye III), that has not
been used individually in any sample. This combination (Fig. 7c)
was found in three fibers (two from textile No. 299 and one from
No. 320). In addition to luteolin (71), luteolin 7-O-glucoside (40),
luteolin O-glucuronide (44), and luteolin C-hexoside (17) assigned
to unknow yellow flavonoid dye I, the chromatograms showed
also distinct peaks corresponding to rutin (30), hyperoside (36),
and ellagic acid (50), as well as to four unknown compounds elu-
ting at tR 17.6 min (U1, 577→ 269, 239), 22.5 min (U2, 621→ 313,
298, 283), 23.8 min (U3, 591→ 283, 268) and 27.1 min (U4, 299
→ 284, 256, 165, 151, 139, 133, 124). Results suggested that the
last compound was probably methoxytrihydroxyflavone (U4), per-
haps luteolin methyl ether. However, MS/MS spectrum of ion at
m/z 285 (Fig. 8a) acquired for the peak X (21.1 min) showed that
together with luteolin also another isobaric compound was eluted
at that time. The new compound was much easier to fragment. Its
spectrum achieved after removal of luteolin signals (Fig. 8b) cor-
Fig. 8. (a) MS/MS spectra of compounds eluting in the peak X from the extract of
sample No. 320 and luteolin, and (b) MS/MS spectrum generated by subtracting
spectra of peak X and luteolin.
responded to reported datiscetin (70) spectra [20,31]. According to
literature datiscetin is the main colorant of only one dye, bastard
hemp (Datisca cannabina L.), but complete chromatographic profile
of this dye, especially using with a mild extraction method, has not
been characterized so far. Nevertheless, despite the lack of data to
compare with, the obtained results led to the assumption that the
flavonoid yellow dye III is bastard hemp.
Annatto (Bixa orellana L.) was identified in four samples (No.
196, No. 231, No. 271 and F-11/3), always together with ano-
ther dye (mainly with brazilwood), or in three-dye mixture (i.e.,
annatto/dyer’s broom/young fustic). The use of annatto was confir-
med by the presence of bixin (116) noted both in positive ion mode
and at 400, 450 and/or 480 nm (Fig. 5). Since bixin, like other apo-
carotenoids, is very difficult to extract from a fiber, observed peaks
were low intensity, but clear enough to prove the use of annatto.
Apart from natural dyes also synthetic dyes were found in six
fibers (three red, one blue, one green and one purple). They were
used for secondary dyeing to change the color or to improve the
appearance of textiles.
 K. Lech / Journal of Cultural Heritage 46 (2020) 108–118
5. Conclusion
HPLC–UV–Vis–ESI MS/MS approach for analysis of various
colorants (flavonoids, quinochalcones, benzophenones, alkaloids,
gallotannins, apocarotenoids and curcumins) was developed and
optimized to identify the compounds in historical objects. To the
best of my knowledge, it is the first approach dedicated to iden-
tification of natural dyes in historical objects that includes such a
number of compounds.
At the beginning a phenyl column and 0.15% formic acid in
water-methanol eluent were used to develop a 35-min separation
method of 32 standards. The method was extended while keeping
the same analysis time; finally, it included 62 colorant standards
and 54 markers from 10 natural dyes. Some of the dyes have never
been examined for textiles. A phenyl column enabled good sepa-
ration of positional isomers and homologues, thus, it was great
to separate compounds present in particular dyes. Further opti-
mization of MS ion source and dMRM parameters led to improve
detection response.
The developed method was used to identify natural dyes in
89 yellow, orange, brown and green fibers taken from liturgical
vestments from the 15th to the 17th centuries and belonging to
collections of seventeen Krakow churches. Several dyes have been
identified, including indigo, weld, dyer’s broom, Persian berries,
and young fustic, as well as sawwort that has never been identified
in any ancient textiles so far. Although the developed approach sho-
wed low MS response to non-glycosylated apocarotenoids, it made
possible to identify annatto in the historical samples using MS/MS
detection for the first time. Moreover, HPLC–UV–Vis–ESI MS/MS
technique was proven to be a useful tool in recognizing of unk-
nown dyes in historical samples by acquiring the additional MS/MS
spectra, which were the basis for the identification of larkspur and
bastard hemp.
Developed method is continuously improved and extended with
new colorants, that are studied, what will be published soon.
Conflict of interest
None declared.
Acknowledgements
The financial support by the statutory fund of the Faculty of
Chemistry Warsaw University of Technology, Poland, and by the
Ministry of Science and Higher Education, Poland, under the project
number NPRH3/H11/82/2014 is gratefully acknowledged.
The author would like to thank Perlan Technologies Polska
(Warsaw, Poland) and Tomasz Gromulski for the opportunity
to conduct research using 1220 Infinity II LC Systems (Agilent
Technologies). The author would like to express their gratitude
to Dr. Natalia Krupa (Pontifical University of John Paul II, Kra-
kow, Poland), Dr. Ioannis Karapanagiotis (“Ormylia” Art Diagnosis
Centre, Greece), and to the management of the Botanical Garden
of Polish Academy of Sciences (Powsin, Poland) for kindly donating
samples and chemical standards.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.culher.2020.04.011.
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