JOURNAL OF MASS SPECTROMETRY

J. Mass Spectrom. 2004; 39: 1441–1449

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.728

Identification of indigoid dyes in natural organic

pigments used in historical art objects by
high-performance liquid chromatography coupled
to electrospray ionization mass spectrometry†
Maria Puchalska,1 Kasia Połeć-Pawlak,1 Irmina ZadrozP na,2 Helena Hryszko3 and
Maciej Jarosz1∗
1
Warsaw University of Technology, Faculty of Chemistry, Department of Analytical Chemistry, Noakowskiego 3, 00-664 Warsaw, Poland
2
Warsaw University of Technology, Faculty of Chemistry, Division of Organic Chemistry, Noakowskiego 3, 00-664, Warsaw Poland
3
Academy of Art in Warsaw, Faculty of Art Conservation and Restauration, Department of Chemistry, WybrzePze Kościuszkowskie 37, 00-379 Warsaw,
Poland

Received 2 June 2004; Accepted 1 September 2004

Pigments are among the most important components of historical paintings and textiles and their nature
provides the unique character of color. They can be divided into two main groups: inorganic and
organic, extracted from plants or animals. Their identification is a necessary stage in the conservation
of art objects. Reversed-phase liquid chromatography with electrospray ionization mass spectrometry
(ESI-MS) and UV/visible spectrophotometric methods were elaborated for the identification of indigoid
(indigo, indirubin, isoindigo, isoindirubin) color components of natural dyestuffs and their natural or
synthetic precursors (indican, isatin, indoxyl, 2-indolinone). ES-MS offers detection limits in the range
0.03–5.00 µg ml−1 for the color compounds examined. The method developed made it possible to identify
indigo and its isomers in genuine Indian indigo, indigo from woad and Tyrian Purple. It was applied to
the identification of natural dyes on fiber from a 19th century Japanese tapestry, ‘Cranes in the landscape’.
A procedure based on freezing and grinding of a sample before the extraction of dyes from the textile was
developed. The components of the extract obtained were identified after acidic hydrolysis as indigotin and
methylene blue. Copyright  2004 John Wiley & Sons, Ltd.

KEYWORDS: natural indigoid dyestuffs; high-performance liquid chromatography; electrospray ionization mass
spectrometry; art analysis

INTRODUCTION such as Indigofera tinctoria (Africa, Asia, East India, South

Organic colorants used in textile dying represent some of
the most fugitive materials encountered in art conservation.1
The changes brought about by fading due to light exposure
and changes in pH can dramatically affect the apprecia-
tion of a work of art. Very careful identification of coloring
agents is required with the aim of the proper reconstruc-
tion of historical art objects. Among natural products used
by ancient artists, especially the identification of indigoid
dyes is very difficult owing to the low stability and solu-
bility of the compounds.2 In addition, their structures are
extremely similar.
The blue dye indigo is one of the oldest natural dyestuffs
and has been known to since ancient times.3 – 5 Indigo has,
for centuries, been obtained from a variety of plant sources
Ł Correspondence to: Maciej Jarosz, Warsaw University of
Technology, Faculty of Chemistry, Department of Analytical
Chemistry, Noakowskiego 3, 00-664 Warsaw, Poland.
E-mail: mj@ch.pw.edu.pl
† Paper presented at the 22nd Informal Meeting on Mass
Spectrometry, Tokaj, Hungary, 2–6 May 2004.
America), Polygonum tinctorium (China, Korea) and Isatis
tinctoria (Europe). Indigo is formed in damaged leaves by
oxidation of products of hydrolysis of precursors, indican
(indoxyl-ˇ-D-glucoside) or isatan B (indoxyloketogluconate)
to indoxyl. Among the products of the reaction between
indoxyl and isatin (obtained in a side-reaction), indigotin can
be found in addition to indirubin, isoindigotin, isoindirubin,
isoindigo and indigo yellow, which are by-products of
biosynthetic indigo formation.6 The different components
create a rich blue dye widely used for textile dying. Synthetic
indigo was obtained from aniline by von Baeyer in 18787
and 20 years later all indigo was produced by the chemical
industry, which considerably decreased the price of the
dye,8 which is still used for textile dying.9 The low resistance
of the dye to expositure to light10 was the reason why
other synthetic coloring agents, e.g. Prussian Blue, available
commercially since 1704,11 and Methylene Blue, synthesized
in 1876, were also widely used.12
The purple dye Tyrian Purple, also called Royal Purple,
has been known since pre-Roman times.13 This purple dye
has been obtained from various shellfish, Murex trunculus,

Copyright  2004 John Wiley & Sons, Ltd.

1442 M. Puchalska et al.
Murex brandaris, Thais haemastoma, Nucella lapillus and Pur-
pura pansa, around the Mediterranean and Atlantic coasts.14
The main coloring agent, 6,60 -dibromoindigo, does not exist
in mollusks and it is generated from precursors (chromogens)
present in the hypobranchial gland in the same way as indig-
otin in plants.15 The synthesis of 6,60 -dibromoindigo from
tyrindoxyl (equivalent of indoxyl) also give indigotin and
6,60 -dibromoindirubin, which are by-products of biosynthe-
sis from bromoisatin.16 Synthetic 6,60 -dibromoindigo was
obtained from 2-nitro-4-aminobenzaldoxime by Sachs and
Kempf in 1903.17
The main problem in the analysis of paint layers is the
very small amount of available sample. A rich matrix compo-
sition mostly creates the necessity for preliminary separation
of the components of dyes prior their identification. Vari-
ous classical procedures have been used in the past for the
identification of active color agents, e.g. acid digestion,18
spectrophotometry in the UV, visible and IR regions,10,19
micro-Raman spectroscopy,20 electron spin resonance,21 thin-
layer chromatography22 and liquid chromatography with
UV/visible detection.14,23 – 25 Mass spectrometry has been
applied to the identification of indigoid compounds using
direct inlet into different ion sources: electron ionization
(EI),26,27 laser desorption/ionization (LDI)28 and atmospheric
pressure chemical ionization (APCI).29 The present work is
the first attempt made to use electrospray ionization mass
spectrometry (ESI-MS) for the identification of indigoid com-
pounds after their separation by high-performance liquid
chromatography (HPLC). The developed method (HPLC
with parallel UV/visible diode-array detection (DAD) and
MS detection) allows the fast, easy and sensitive identifica-
tion and determination of the examined species.
EXPERIMENTAL
Apparatus
The separation and identification of indigoid dyes were
carried out using a liquid chromatographic system with
mass spectrometric detection, LC–MSD 1100 (Agilent Tech-
nologies, USA) and an LC (HP 1100, Hewlett-Packard,
Germany)/UV/VIS-DAD (G1315, HP 1100, Germany) sys-
tem. Samples were injected on to the column using a Rheo-
dyne Model 7225i injection valve with a 5 µl sample loop.
A rapid resolution column, Zorbax SB-C18 (4.6 ð 150 mm,
3.5 µm, Agilent Technologies), was used. The flow-rate was
0.6 ml min
1 and the elution program was controlled by LC
Chemstation 3D software (Hewlett-Packard, USA). The elu-
ent for liquid chromatography was degassed using a Branson
(Danbury CT, USA) Model 1210 ultrasonic bath.
Chemicals
Indoxyl-ˇ-D-glucoside (indican) was purchased from Sigma
(Sigma-Aldrich, Steinheim, Germany) and indigotin, isatin,
2-indolinone, 3-indoxyl acetate of analytical grade from
Fluka (Sigma-Aldrich, Buchs, Switzerland). Hydrochloric,
acetic and formic acid were obtained from Appli Chem
(Darmstadt, Germany), sodium carbonate, sodium hydrox-
ide, methanol, diethyl ether and dimethyl sulfoxide (DMSO)
from POCh (Gliwice, Poland) and sodium tetrahydrobo-
rate from Aldrich (Aldrich Chemical, Gillingham, USA).
Copyright  2004 John Wiley & Sons, Ltd.
All chemicals were of analytical grade. Synthetic indigo,
genuine indigo from Indigofera tinctoria, indigo from Isatis
tinctoria (woad) and Tyrian Purple from shellfish (Kremer,
Allgäu, Germany) were a gift from the National Museum
in Warsaw. Demineralized water (from a Milli-Q system
Model Millipore Elix 3; Millipore, Saint-Quentin, France)
and acetonitrile (ACN) of super gradient purity from Lab-
Scan (Lab-Scan Analytical Science, Dublin, Ireland) were
used throughout.
Synthesis of indirubin and isoindigo
Indirubin and isoindigo were synthesized following the
procedure of Russell and Kaupp21 as modified by Hoessel
et al.30
Indirubin was synthesized by reaction of 0.15 g of
isatin with 0.18 g of 3-indoxyl acetate in alkaline medium
(2.5 mM solution of sodium carbonate in 5 ml of methanol).
The reagents were stirred for 1 h under argon at room
temperature and kept under argon for the next 24 h. The
residue was then filtered and washed with methanol and
cold water until neutral pH was obtained. The product
(dark violet crystals) was dried over potassium hydroxide
in a desiccator. The yield of the synthesis was 80% and the
structure of the product was confirmed by 1 H and 13 C NMR
and elemental analysis. In NMR spectra the following signals
were found (the most characteristic, confirming the identity
of the synthesized compounds; full data concerning 1 H NMR
and 13 C NMR spectra for both indirubin and isoindigo can be
found in Supplementary Information to the paper by Hoessel
et al.30 ): 1 H NMR (DMSO-d6 , 25 ° C, υ, ppm) 11.03 (s) and 10.91
(s), NH and N0 H, respectively, and (DMSO-d6 , 90 ° C, υ, ppm):
171.51 (s), C(CO)NH, 152.56 (s), C C(NH). The total content
of carbon, hydrogen and nitrogen was found to be 72.7, 3.8,
10.6% (theoretical values: 73.3, 3.8, 10.7%). The dominant
signal in ESI-MS (registered at the apex of the HPLC peak
corresponding to the examined compound, tR D 18.1 min) at
m/z 263 was attributed to the quasi-molecular ion [M C H]C
of indirubin. The chromatographic purity of the preparation
was estimated by HPLC/DAD at 280 nm from the peak area
ratio as 85%.
In the chromatogram another peak was detected at
tR D 16.8 min corresponding to a by-product of the reaction,
indigotin. Its absorption spectrum in the UV region was
the same as that of indirubin and identification was possible
thanks to the different position of the absorption maximum in
the visible region (when the analyzed sample was relatively
large) or to the different mass spectrum, in which except
for the signal at m/z 263 also the second, characteristic for
indigotin, at m/z 132 appeared. The indigotin content in the
preparation was estimated as ca 5% using the quantified
HPLC/ESI-MS method.
Isoindigo was obtained by reaction of 0.15 g of isatin
with 0.13 g of 2-indolinone in 3 ml of glacial acetic acid
containing 50 µl of concentrated hydrochloric acid. The
reaction mixture was stirred for 3 h at 95 ° C. The residue
(dark brown crystals) was filtered, washed with methanol
and diethyl ether and dried over potassium hydroxide in a
desiccator. The yield of the reaction was 85%; the product was
examined as above. In the NMR spectra were found signals
J. Mass Spectrom. 2004; 39: 1441–1449
 HPLC/ESI-MS of natural indigoid dyestuffs 1443

confirming symmetry of the molecule, corresponding to NH

and CN bonds: 1 H NMR (DMSO-d6 , 25 ° C, υ, ppm) 10.90
(s), NH and N0 H, respectively; 13 C NMR (DMSO-d6 , 90 ° C,
υ, ppm) 169.00 (s), C(CO)NH. Elemental analysis: C 73.1,
H 3.8, N 10.7% (theoretical values: 73.3, 3.8, 10.7%). In the
mass spectrum taken at the apex of the chromatographic
peak the dominant signal was registered at m/z 263 (quasi-
molecular ion [M C H]C ). The chromatographic purity of the
preparation was estimated by HPLC/DAD at 280 nm from
the peak area ratio as 95%.

Sample preparation
Standard solutions (0.1 mg ml
1 ) of indigo, isoindigo, indiru-
bin, isatin, indican, indoxyl acetate and 2-indolinone were
prepared by dissolving 5 mg of each powder in 50 ml of
DMSO. The solutions obtained were filtered over a 0.45 µm
syringe filter (Supelco, Sigma-Aldrich, Bellefonte, PA, USA).
The first five drops were discarded and only the remaining
part of the filtrate was used for the analysis. The solution
of the mixture of active agents was prepared daily by dilu-
tion of appropriate volumes of the standard solutions with
acetonitrile prior the analysis.
The solutions of the examined dyes (natural preparations)
was prepared by stirring 1 mg of each powder in 5 ml
of DMSO at room temperature until the solution was
clear. Only Tyrian Purple needed to be kept in a water-
bath at 80 ° C for 10 min. The solutions obtained were
filtered over a 0.45 µm syringe filter (the first two drops
were discarded). The solutions obtained were kept in vials
preventing light penetration to avoid decomposition of the
indigoid compounds.

Preparation of textile fiber prior analysis

A 0.5 mg amount of the fiber was frozen in liquid nitrogen,
ground in an agate mortar and transported to a conical probe.
Dyes were extracted with a mixture of 200 µl of DMSO and
10 µl of concentrated HCl for 5 min in a water-bath at 80 ° C.
The solution obtained was diluted with 200 µl of acetonitrile
and filtered over a 0.45 µm syringe filter (the first drop was
discarded).

RESULTS AND DISCUSSION

The aim of this research was to develop an efficient and
robust LC method enhanced by the potential of ESI-MS

Figure 1. Chromatogram (SIM) of the mixture of standard

compounds obtained under optimum separation conditions
described in Table 2 and UV/visible and ESI mass spectra (TIC)
for selected peaks: (1) indican (5.0 µg ml
1 , tR D 4.2 min);
(2) isatin (5.0 µg ml
1 , tR D 7.7 min); (3) 2-indolinone
(2.0 µg ml
1 , tR D 8.5 min); (4) indoxyl acetate (1.0 µg ml
1 )
and co-eluted isoindigo (2.0 µg ml
1 , tR D 13.1 min),
UV/visible spectrum shown in the inset of the second mass
spectrum; (5) indigotin (1.0 µg ml
1 , tR D 16.8 min);
(6) indirubin (1.0 µg ml
1 , tR D 18.1 min), UV/visible and ESI
mass spectra shown in the middle of spectra set. The first peak
on the chromatogram (not numbered) refers to the void volume
of the column and was identified as DMSO. Bold solid line,
chromatogram of the standards mixture; thin solid line, blank.

Copyright  2004 John Wiley & Sons, Ltd. J. Mass Spectrom. 2004; 39: 1441–1449
1444 M. Puchalska et al.

Table 1. Characteristics of selected indigoid compounds and their precursors

max (nm)
Class Compound Structure (DMSO) [M C H]C

Indigoid coloring agents Indigo, indigotin (blue) 617 263

Indirubin (red) 540 263

Isoindirubin (red) 552 263

Isoindigo (brown) 365 263

490

6,60 -Dibromoindigo (purple) 585 421

597

Indigoid precursors Indican 218, 280 296

Isatan 218, 280 310

(continued overleaf )

Copyright  2004 John Wiley & Sons, Ltd. J. Mass Spectrom. 2004; 39: 1441–1449
 HPLC/ESI-MS of natural indigoid dyestuffs 1445

Table 1. (Continued)
max (nm)
Class Compound Structure (DMSO) [M C H]C

Indoxyl acetate 280 176

2-Indolinone — 134

Isatin 416 148

detection for the characterization of the different indigoid

2
pigment components (Table 1). The identification of these
components, their precursors and degradation products
allows the characterization of the sources and preparation 1.5
methods of the samples examined. The developed method a
Absorbance

should also be useful in analysis of art objects such as ancient

textiles. 1

b
HPLC/ESI-MS method for the identification of the 103 0.5 c
main components of indigoid dyes 40
Under the developed optimum conditions (Table 2), both
Relative detector response, cps

0
detection modes could and even have to be applied. Some 200 400 600
30
of the examined compounds are colorless in the visible Wavelength, nm
region (indican, 2-indolinone) and MS is necessary for their a
identification; also, co-eluted indoxyl acetate and isoindigo 20
can be differentiated exclusively by ESI-MS. On the other b
hand, UV/visible detection is useful for the identification of
isatin and 2-indolinone, as their chromatographic peaks are 10
not very well separated but their absorption spectra differ
substantially (a solution of 2-indolinone is transparent in the c
visible region). 0
0 5 10 15 20 25
The chromatogram of the mixture of standard com- Time, min
pounds (Fig. 1) consists of six peaks and the last three are
identified as indigoid compounds. Figure 2. Degradation of indigotin to isatin caused by daylight.
The reagents used for indirubin and isoindigo synthesis Chromatograms (HPLC/ESI-MS, SIM) and absorption spectra
(potential precursors), except for indoxyl acetate, are eluted (in the inset) of (a) fresh solution, (b) solution after 7 days and
in the first 10 min of the chromatographic separation, with (c) solution after 30 days.
indican as the first, isatin the second and 2-indolinone the
third peak. In the mass spectrum of colorless indican (chro-
matographic peak 1) few signals appear. The relatively weak The mass spectrum obtained for chromatographic peak
peak at m/z 296 can be attributed to [M C H]C and the strong 2 contains a strong signal at m/z 148 and a weak signal at
peak at m/z 318 to [M C Na]C . The sodium ions are naturally m/z 170 related to isatin quasi molecular ions [M C H]C and
present in indican preparations extracted from plants and [M C Na]C . The identification of isatin can be confirmed by
purified by Fluka. The instability of the indican molecular ion UV/visible detection thanks to the characteristic absorption
in the electrospray source is highlighted by the appearance maximum at 416 nm. This possibility is of special importance,
of a dominant signal at m/z 134 (parent ion with eliminated because 2-indolinone (peak 3) eluted on the tail of isatin peak
glucoside group). The fragment ion at m/z 106 originates is colorless. Its mass spectrum consists of the base signal at
from the ion at m/z 134 by elimination of a CO group. m/z 134 corresponding to the molecular ion [M C H]C and a

Copyright  2004 John Wiley & Sons, Ltd. J. Mass Spectrom. 2004; 39: 1441–1449
1446 M. Puchalska et al.

Table 2. Optimal HPLC/UV/MS conditions of the separation and detection of the examined indigoid dyes

Method Conditions

HPLC separation Column Zorbax SB-C18 (150 ð 4.6 mm, 3.5 µm)
Eluent A, ACN; B, 0.15% formic acid
Gradient program 3 min 20% A, 8 min 50% A, 10 min 55% A, 22 min 55% A
Additional steps for Tyrian Purple 35 min 100% A, 45 min 100% A
Flow-rate 0.6 ml min
1
Injection volume 5 µl
UV/visible detection Instrument HP 1100 LC
Wavelength 280, 610 nm
Dwell time 10 ms
ESI-MS detection Instrument LC-MSD 1100 (Agilent Technologies)
Ionization voltage 4000 V (positive ion mode)
Orifice voltage 90 V
Nebulizing gas flow and temperature 10 l min
1 , 300 ° C
Observed mass range (TIC) m/z 50–500
Observed selected ions (SIM) m/z 134, 148, 176, 205, 263, 296, 338, 340, 418, 420, 422
Acquisition step 0.2
Dwell time 50 ms

Table 3. Parameters of HPLC/ESI-MS methods for the determination of the examined indigoid colorants (calculated for
concentrations of the compounds in injected samples)

Parameter Indigotin Isoindigo Indirubin Isatin

Selected ion, m/z 263 263 263 148

Calibration graph,a y D ax C b:
a 598367 341891 319965 80950
SDa 10607 6943 6789 907
b 16205 430 47560 27036
SDb 5983 6558 15643 7783
Linearity range (µg ml
1 ) 0.03–4.20 0.03–4.00 0.03–5.00 0.45–18.20
R2 n D 6 0.999 0.998 0.999 0.993
SDŁb 10710 13291 28190 13800
Detection limitc (µg ml
1 ) 0.01 0.01 0.01 0.15
Quantification limit (µg ml
1 ) 0.03 0.03 0.03 0.45

a
y, Peak area (relative units); x, concentration of the compound in the injected sample (µg ml
1 ); SDa and SDb , standard deviations of
a and b, respectively.
b
SDŁ , mean standard deviation of regression.
c
Detection limit: DL D 3SDbl /a, where SDbl is the standard deviation calculated using blanks.

weak signal at m/z 106 (molecular ion with eliminated CO
group).
ESI-MS detection is necessary when indoxyl (acetate)
used for the synthesis of indigorubin has to be identified,
because of its co-elution with isoindigo (tR D 13.1 min) and
the lack of characteristic absorption maxima in its spectrum
in the visible range. The mass spectrum of indoxyl (acetate)
consists of two signals: the base signal at m/z 134 attributed
to the ion [M C H]C (the ion of indoxyl acetate at m/z 176 is
not registered) and a weaker signal at m/z 106 (fragment ion
formed by CO elimination).
Isoindigo eluted in peak 4 can be simply identified
because, in contrast to indoxyl acetate, its UV/visible
spectrum is obtained (absorption maxima at 365 and 490 nm).
Also, the mass spectrum is quite different: a quasi-molecular
ion [M C H]C is registered at m/z 263 and the next two signals
at m/z 235 and 219 are created by loss of CO, [MH
CO]C ,
or CONH2 [MH
CONH2 ]C .
Indigotin and indirubin (peaks 5 and 6) can be easily
differentiated thanks to their different retention times of
16.8 and 18.1 min, respectively. Their absorption spectra
also differ substantially in the visible region; indigotin
absorbs at 617 nm and indirubin at 540 nm. In the ESI
mass spectrum of indigotin, the doubly charged quasi
molecular ion [M C 2H]2C registered at m/z 132 creates
a unique chance for unequivocal identification of this
compound, as it is not observed in the spectra of the
other substances examined. The fragment ion at m/z
206 is formed by the elimination of two CO groups,
[MH
CO
HCO]C .

Copyright  2004 John Wiley & Sons, Ltd. J. Mass Spectrom. 2004; 39: 1441–1449
 HPLC/ESI-MS of natural indigoid dyestuffs 1447

The developed HPLC/ESI-MS method can also be used

for quantitative studies. Parameters of the methods suitable 103
for the determination of selected indigotin dyes and isatin
are presented in Table 3.
The stability of indigotin, indirubin and isoindigo 100
solutions in DMSO was examined. They were exposed to c
daylight (indirect sunlight filtered by glass) for 30 days
at room temperature and their UV/visible spectra were 80

Relative detector response, cps

1
recorded every day (when changes were observed, also
chromatograms with ESI-MS detection were registered). It
was found that solutions of indirubin and isoindigo are 60
stable relative to indigotin and the period of exposure. In
the case of indigotin the process was completed by 50% after
7 days; after 30 days, in the examined solution only isatin 0
0 10 20
(degradation product) could be found (Fig. 2). 40
Isatin, the detected degradation product, eluted at
4.2 min. The absorption maximum of the compound a
separated by HPLC was situated at 416 nm and in its mass 20
spectrum the base peak was registered at m/z 134. The above b
observation indicates that the identification of a large per-
c
centage of isatin in a dye or pigment solution in DMSO
can prove the former presence of indigotin, degraded by 0
5 10 15 20 25
exposure to daylight. Time, min

Identification of components of natural and Figure 3. Chromatograms (HPLC/ESI-MS, SIM) of three

synthetic dyes preparations of indigo dyes: (a) synthetic (5 µg ml
1 );
Three indigo preparations, synthetic, obtained from Isatis (b) extracted from Indigofera tinctoria (20 µg ml
1 );
tinctoria (woad) and obtained from Indigofera tinctoria, were (c) extracted from Isatis tinctoria (20 µg ml
1 ). Inset, magnified
analyzed by the developed method. The chromatograms part of chromatogram (c).
obtained are presented in Fig. 3. It was found that in each
dye the main coloring agent is indigotin, but its content is
103 103
different. It was determined using quantified HPLC/ESI-MS
method as 72.9% (w/w) in synthetic indigo, 11.9% (w/w) in
indigo obtained from Indigofera tinctoria and 1.8% (w/w) in 20 SIM 2.0
indigo from Isatis tinctoria. As can be seen on the magnified
Relative detector response, cps

Relative detector response, cps

part of chromatogram of the last product, indigo from Isatis
tinctoria also contains a small amount of indirubin (ca 0.1%).
15 1.5
Unfortunately, such results indicate that for recognition of
the origins of indigotin dyes another method could also be 421
required, e.g. based on the identification of other substances
present in the examined samples. 10 1.0
The second natural dye consisting of indigoid com- 205
pounds analyzed by the developed HPLC/ESI-MS method
was Tyrian Purple, extracted from the mollusk Purpura lapil-
5 0.5
lus. The chromatogram reconstructed for the ion at m/z
263
263 shown in Fig. 4 consists of three major peaks. The first,
registered at 13.1 min, corresponds to isoindigo, the sec-
ond, at 16.8 min, was identified as indigotin and the third, 0
0 5 10 15 20 25 30
at 18.1 min, was related to indirubin. The chromatogram Time, min
reconstructed for the ions at m/z 419, 421 and 423 (reflecting
the isotopic pattern of the pseudomolecular ion of 6,60 - Figure 4. Chromatogram (HPLC/ESI-MS, SIM) of the extract
dibromoindigo with two bromine atoms, [M C H]C ) consists from Tyrian Purple (500 µg ml
1 ) obtained under the
of one peak at 28.1 min and proves the presence of this experimental conditions given in the Table 2 and
compound in the examined preparation. This result is con- chromatograms reconstructed for ions at m/z 263, 205 and
firmed by the chromatogram reconstructed for the ion at 421.
m/z 205 (fragment ion [M C H
2Br
2CO]C formed by the
elimination of bromine and CO from the 6,60 -dibromoindigo using the quantified HPLC/ESI-MS method, the following
molecule). In addition to the peak at tR D 28.1 min, also percentages of active components were found: 9.2% (w/w)
two matrix-originated unidentified peaks are present in this of indigotin, 14.4% (w/w) of isoindigo and 4.7% (w/w)
chromatogram. In the examined Tyrian Purple preparation of indirubin. The content of 6,60 -dibromoindigotin could

Copyright  2004 John Wiley & Sons, Ltd. J. Mass Spectrom. 2004; 39: 1441–1449
1448 M. Puchalska et al.

not been quantified because of the lack of a standard. The detection allowed the twin chromatogram to be obtained. In
complexity of the Tyrian Purple composition indicates the the mass spectrum obtained in the apex of the peak registered
natural origin of the dye and the ratio of indigoid compounds at 10.2 min one signal was found at m/z 284. According to the
present shows good agreement with the characteristics of the literature data, as the most probable the basic dye Methylene
dye extracted from Purpura lapillus. Blue (a thiazine cationic dye first obtained at the end of the
19th century, used for silk and cotton dying) was proposed;
Identification of color agents on a fiber from a its molecular mass is 284, which agrees with the mass of the
Japanese tapestry molecular ion obtained in the spectrum. Internal standard
The extract of blue fiber taken from a Japanese tapestry addition of Methylene Blue confirmed the identification of
(19th century) was first analyzed by the HPLC/UV/visible the compound from its retention time. The mass spectrum
method. The chromatogram shown in Fig. 5 reconstructed of the compound eluted in the second peak clearly proved
for 610 nm consists of two peaks. The second peak at a its identity (indigotin). The mass ratio of these two dyes,
retention time 16.8 min was undoubtedly identified from its Methylene Blue : indigotin, was estimated by HPLC/ESI-MS
retention time as indigotin, but the first peak at 10.2 min (SIM mode) from the peak area ratio as 3 : 1, because their
was of unknown origin. The parallel analysis with ESI-MS quantification was not possible.

CONCLUSIONS
1.5
Relative detector response, mV

1
The coupling of reversed-phase liquid chromatography with
UV/visible detection significantly improves the selectivity
1.0 of the spectrophotometric method used alone for the
identification of isomeric indigoid compounds. Coupling
2
of HPLC with ESI-MS also allows this detection technique to
0.5 be used for the analysis of dye solutions in DMSO, because
in the chromatographic step color agents are separated from
this solvent, which dramatically affects the ionization of the
0 examined dyes. The developed method, which is cheap and
0 5 10 15 20
fast, can serve as an efficient tool for screening hydrophobic
Time, min indigoid dyes in different art objects, offering confirmative
the identification of blue coloring matters.
284.0

100 1
[M]+
CH3 CH3
80 Acknowledgement
N S N+ The authors thank to Ms ElPzbieta Rosłoniec to the Laboratory of the
H2C CH2
60 National Museum, Warsaw, Poland, for the gift of natural dyes.
N
40
REFERENCES
20
1. ZadroPzna I, Połeć-Pawlak K, Głuch I, Ackacha MA, Mojski M,
Witowska-Jarosz J, Jarosz M. Old master paintings—a fruitful
0
field of activity for analysts: targets, methods, outlook. J. Sep. Sci.
100 200 300 400 2003; 26: 996.
m/z 2. Halpine SM. An improved dye and lake pigment analysis
method for high-performance liquid chromatography and
diode-array detector. Stud. Conserv. 1996; 41: 76.
100
263.0

O H
3. Ensley BD, Ratzkin BJ, Osslund TD, Simon MJ, Wackett LP,
N [M+H]+ 2 Gibson DT. Expression of naphthalene oxidation genes in
[MH-CO]+
235.0

80
N Escherichia coli results in the biosynthesis of indigo. Science 1983;
[MH-CONH2]+ 222: 67.
60 H O [MH-CO-HCO]+ 4. Fitzhugh W. Artist’s Pigments, a Handbook of Their History and
219.0

[M+2H]2+ Characteristics, vol 3. Oxford University Press: Oxford, 1997.

132.0

40 5. Seldes A, Burucúa JE, Maier MS, Abad G, Jáuregui A,

206.0

Siracusano G. Blue pigments in South American Painting

190.0

20 (1610–1780). J. Am. Inst. Conser. 1999; 38: 100.

6. Maugarda T, Enauda E, Choisyb P, Legoya MD. Identification
0 of an indigo precursor from leaves of Isatis tinctoria (woad).
Phytochemistry 2001; 58: 897.
100 200 300 400
7. Von Baeyer A. Synthese des Isoatins und Indigblaus. Chem. Ber.
m/z 1878; 11: 1228.
8. Fairbanks VF. Blue gods, blue oil, and blue people. Mayo Clin.
Figure 5. Chromatogram (HPLC/UV/visible) of the extract of
Proc. 1994; 69: 889.
blue fiber from the tapestry and ESI mass spectra of the 9. Son YA, Hong JP, Kim TK. An approach to the dying of polyester
compounds eluted in the chromatographic peaks: fiber using indigo and its extended wash fastness properties.
(1) Methylene Blue; (2) indigotin. Dyes Pigments 2004; 61: 263.

Copyright  2004 John Wiley & Sons, Ltd. J. Mass Spectrom. 2004; 39: 1441–1449

10. Vautier M, Guillard C, Herrmann J-M. Photocatalytic degrada-
tion of dyes in water: case study of indigo and of indigo carmine.
J. Catal. 2001; 201: 46.
11. Zollinger H. Color Chemistry: Synthesis, Properties and Applications
of Organic Dyes and Pigments. VCH: Weinheim, 1987.
12. Mertz V, Weith W. Mitteilungen aus dem Universitäts-
Laboratorium in Zürich; Veber Derivate des Dimethylalanins.
Ber. Dtsch. Chem. Gese. 1877; 10: 760.
13. McGovern PE, Michel RH. Royal Purple dye: tracing the
chemical origins of the industry. Anal. Chem. 1985; 57: 1514A.
14. Cooksey CJ. Tyrian Purple: 6,60 -dibromoindigo and related
compounds. Molecules 2001; 6: 736.
15. Wouters J, Verhecken A. Composition of Murex dyes. J. Soc.
Dyers Colour. 1992; 108: 404.
16. Michel RH, Lazar J, McGovern PE. The chemical composition
of the indigoid dyes derived from the hypobranchial glandural
secretions of Murex mollusks. J. Soc. Dyers Colour. 1992; 108: 145.
17. Sachs F, Kempf R. Uber p-halogen-o-nitrobenzaldehyde. Ber.
Dtsch. Chem. Ges. 1903; 36: 3299.
18. Cordy A, Yeh K. Blue dye identification on cellulosic fibers:
indigo, logwood, and Prussian blue. J. Am. Instit. Conserv. 1984;
24: 33.
19. Miliani C, Romani A, Favaro G. A spectrophotometric and
fluorimetric study of some anthraquinoid and indigoid colorants
used in artistic paintings. Spectrochim. Acta, Part A 1998; 54: 581.
20. Vandenabeele P, Moens L. Micro-Raman spectroscopy of
natural and synthetic indigo samples. Analyst 2003; 128: 187.
21. Russell GA, Kaupp G. Oxidation of carbanions. IV. Oxidation of
indoxyl to indigo in basic solution. J. Am. Chem. Soc. 1969; 91:
3851.
22. Kokubun T, Edmonds J, John P. Indoxyl derivatives in woad in
relation to medieval indigo production. Phytochemistry 1998; 49:
79.
Copyright  2004 John Wiley & Sons, Ltd.
HPLC/ESI-MS of natural indigoid dyestuffs 1449
23. Wouters J, Rosario-Chirinos N. Dye analysis of pre-Columbian
Peruvian textiles with high-performance liquid chromatography
and diode array detection. J. Am. Inst. Conserv. 1992; 31:
237.
24. Maugard T, Enaud E, Choisy P, Legoy MD. Identification of
an indigo precursor from leaves of Isatis tinctoria (woad).
Phytochemistry 2001; 58: 897.
25. Orska-Gawryś J, Surowiec I, Kehl J, Rejniak H, Urbaniak-
Walczak K, Trojanowicz M. Identification of natural dyes in
archeological Coptic textiles by liquid chromatography with
diode array detection. J. Chromatogr. A 2003; 989: 239.
26. Clark RJH, Cooksey CJ. Bromoindirubins: the synthesis and
properties of minor components of Tyrian Purple and the
composition from Nucella lapillus. J. Soc. Dyers Colour. 1997;
113: 316.
27. McGovern PE, Lazar J, Michel RH. Caveats on the analysis of
indigoid dyes by mass spectrometry. J. Soc. Dyers Colour. 1991;
107: 280.
28. Wyplosz N. Laser Desorption Mass Spectrometric Studies of Artists’
Organic Pigments. University of Amsterdam: Amsterdam, 2003
(MOLART report ISBN 90-77209-01-6).
29. Szostek B, Orska-Gawryś J, Surowiec I, Trojanowicz M.
Investigation of natural dyes occuring in historical Coptic textiles
by high-performance liquid chromatography with UV–Vis and
mass spectrometric detection. J. Chromatogr. A 2003; 1012: 179.
30. Hoessel R, Leclerc S, Endicott JA, Nobel ME, Lawrie A, Tun-
nah P, Leost M, Damiens E, Marie D, Marko D, Niedeberger E,
Tang W, Eisenbrand G, Meijer L. Indirubin, the active con-
stituent of a Chinese antileukaemia medicine, inhibits cyclin-
dependent kinases. Nature Cell Biol. 1999; 1: 60.
J. Mass Spectrom. 2004; 39: 1441–1449