- Email: [email protected]
Triarylmethine dyes: Characterization of isomers using integrated mass spectrometry
Accepted Manuscript Triarylmethine dyes: Characterization of isomers using integrated mass spectrometry Ilaria Degano, Francesca Sabatini, Chiara Braccini, Maria Perla Colombini PII:
S0143-7208(18)31529-8
DOI:
10.1016/j.dyepig.2018.08.046
Reference:
DYPI 6960
To appear in:
Dyes and Pigments
Received Date: 10 July 2018 Revised Date:
23 August 2018
Accepted Date: 24 August 2018
Please cite this article as: Degano I, Sabatini F, Braccini C, Colombini MP, Triarylmethine dyes: Characterization of isomers using integrated mass spectrometry, Dyes and Pigments (2018), doi: 10.1016/j.dyepig.2018.08.046. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
+ESI Product Ion CID @50.0 V
C22H24N3+ NH 2
223.121
CH3
208.099
RI PT
300.150
H3 C
120.079
181.088
283.122
314.164 330.199
H 2N
NH2
M AN U
SC
270.113
CH 3
CH 3 HN
209.107
285.138
243.103
180.080
TE D
314.168
272.130
299.146
330.199
AC C
208.100
CH3 CH 3 HN
287.154
272.131
223.124
315.172 299.149
157.089
330.199
CH 3 H2 N
120
140
160
CH 3
258.125
181.088
120.080
NH
HN
EP
167.075
180
200
220
240
260
280
Counts vs. Mass-to-Charge [m/z]
300
320
340
360
N CH 3
ACCEPTED MANUSCRIPT
Triarylmethine dyes: characterization of isomers using integrated mass spectrometry
Ilaria Deganoa a
University of Pisa, Department of Chemistry and Industrial Chemistry
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Via Moruzzi, 13, I-56126 Pisa (Italy)
Francesca Sabatinia
University of Pisa, Department of Chemistry and Industrial Chemistry
Via Moruzzi, 13, I-56126 Pisa (Italy)
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[email protected] Chiara Braccinia a
M AN U
[email protected]
a
RI PT
Authors
University of Pisa, Department of Chemistry and Industrial Chemistry
AC C
[email protected]
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Via Moruzzi, 13, I-56126 Pisa (Italy)
Maria Perla Colombinia,b a
University of Pisa, Department of Chemistry and Industrial Chemistry
Via Moruzzi, 13, I-56126 Pisa (Italy) b
ICVBC Institute for the Conservation and Valorization of Cultural Heritage, National Research Council of
Italy, Via Madonna del Piano, 10, I-50019 Sesto Fiorentino, Italy [email protected]
1
ACCEPTED MANUSCRIPT Abstract Triarylmethine dyes, such as methyl and crystal violets, diamond green and magentas, produced since the late nineteenth century, consist of complex mixtures of homologous compounds, often differing only for the presence or position of the same substituent on the aromatic rings. In this paper, Liquid
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Chromatography-High Resolution Mass Spectrometry (HPLC-HRMS) was used for the characterization of the profile of three dyes: methyl blue, methyl violet and fuchsine. The comprehension of dye synthesis and production strategies was often achieved by analyzing the single components by diode array detector.
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Nevertheless, the nature of the individual components needs to be confirmed by tandem mass spectrometric techniques through the solving of the structural issues.
M AN U
The analysis of standards and reference materials by tandem high resolution mass spectrometry allowed us to identify peculiar fragmentation pathways for the components of magentas and methyl violet dyes. The results highlighted the importance to undertake the measurements both in negative and positive ionization mode. The elucidation of specific fragmentation patterns allows the discrimination between
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different classes of compounds, while the identification of specific fragment ions allows one to distinguish isomers belonging to the same series.
EP
Keywords
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Triarylmethine dyes; CID fragmentation pattern; HPLC-ESI-Q-ToF
2
ACCEPTED MANUSCRIPT 1. Introduction One of the earliest classes of synthetic dyes ever produced were the triarylmethines (often historically referred to as triarylmethanes). Among them, the industrial process for producing Fuchsine was the first to be developed in 1859 by Verguin. Since then, triarylmethines have become the most widely used synthetic
RI PT
dyes thanks to their versatility and their bright colours ranging from red/purple to blue/green hues [1, 2]. The chromophore system of this class of dyes is constituted by three conjugated aromatic rings bonded to a central atom of carbon [3], while the exact hue depends on the number and nature of auxochrome groups.
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The current synthetic strategy consists in a condensation reaction between Michler’s ketone and a suitable aromatic amine [1]. Among the great number of synthetic dyes belonging to this class, some of them were
M AN U
widespread and their characterisation will be discussed in the present paper.
Among them, Fuchsine is composed by a mixture of four main compounds, differentiating by the number of methyl groups on the aromatic rings (Figure 1 a). Pararosaniline (also known as Basic Fuchsine or Magenta 0, C.I. 42500) is not methylated and it is currently used for staining polyacrylonitrile fibres and for detecting
TE D
aldehydes in biological samples[1, 4]; Fuchsin (also known as Rosaniline or Magenta I, C.I. 42510) contains one methyl substituent and it is used for dyeing silk, wool and paper [1]; Magenta II has two methyl substituents and it is the only one that cannot be synthesised as a pure compound; in the end New
ring [5].
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Fuchsine (Magenta III, C.I. 42520) is characterized by three methyl groups as substituents on the aromatic
AC C
Methyl Violet (C.I. 42535) consists in a mixture of tetra-, penta- and hexa-N-methyl pararosaniline (Figure 1 b) while Crystal Violet (C.I. 42555) contains N-hexamethyl pararosaniline only. Both Crystal and Methyl Violet have been historically used in dyeing textiles, and more recently have been broadly used in printing and ballpoint pen and felt tip inks [6, 7]; thus, they are studied in forensic science for the authentication and dating of documents [8, 9]. They can also be applied for dying polyacrylonitrile fibres [1]. Finally, Crystal Violet, due to its antifungal and cytotoxic properties [10], is employed to classify bacteria via Gram test [3].
3
ACCEPTED MANUSCRIPT Methyl blue (C.I. 42780), also known as Acid Blue 93, is characterized by three benzenesulfonyl substituents on amine groups (Figure 1c), which provide good solubility in water to the molecule [1]. Due to its fluorescence, it is used as probe in biological system studies [11, 12]. H 3C
NH2
a)
CH 3 N
Cl
b)
R'
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Cl
R'''
R'''
H3 C
NH 2
N
N
SC
H 2N
R'
R''
Tetra-N-methyl pararosaniline (Tetra MP): R’=R’’=R’’’=H Penta-N-methyl pararosaniline (Penta MP): R’=H; R’’=R’’’=CH3 Hexa-N-methyl pararosaniline (Hexa MP): R’=R’’=R’’’=CH3
M AN U
Pararosaniline (Mag 0): R’=R’’=R’’’=H Fuchsin (Mag I): R’=R’’=H; R’’’=CH3 Magenta II (Mag II): R’=R’’=CH3; R’’’=H New fuchsin (Mag III): R’=R’’=R’’’=CH3
R''
SO 3
c)
Na O 3S
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HN
N H
EP
N H
SO 3 Na
AC C
Figure 1: Chemical structures of the components of a) Fuchsine dye; b) Crystal and Methyl Violet dyes, and c) Methyl Blue.
As the issue of pollution of the environment by synthetic organic dyes has become progressively relevant, many studies have targeted the detection and identification of tryarylmethanes in wastewaters [13], and their removal using adsorption on activated surfaces [14-16] or photochemical degradation [17, 18]. Several studies adopted infrared [19, 20] and Raman [21] spectroscopies for triarylmethines detection. Nonetheless, the need to separate the different components in dye formulation or to detect their degradation products suggested the application of chromatographic techniques. Pyrolysis coupled with Gas Chromatography Mass Spectrometry (Py-GC/MS) was widely applied for the creation of databases fundamental for the identification of the main compounds constituting triarylmethine dyes [22-24]. 4
ACCEPTED MANUSCRIPT Nevertheless, Liquid Chromatography coupled with Diode Array or Mass Detector (LC-DAD, LC-MS) is is the method of choice due to the strong absorption of these dyes in the visible range, their low volatility and poor thermal stability [13]. Regarding the several promising approaches based on mass spectrometry, they are mostly focused on the optimization of ultrasensitive and ultrafast methods for the detection of a wide
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range of triarylmethines in aquaculture products or ballpoint inks mainly using High Performance Liquid Chromatography (HPLC) or Ultra High Performance Liquid Chromatography (UHPLC) coupled with quadrupole [25] or triple quadrupole (QqQ) analyzers [6, 11, 26]. Nevertheless, only few studies have been
SC
carried out using high resolution mass spectrometry [27] and using a Time of Flight ion Mass Spectrometry coupled with Secondary Ion Mass Spectrometry (TOF-SIMS) set-up [28,33]. Notably, in all the mentioned
M AN U
papers the identification of the minor compounds and the interpretation of fragmentation patterns are beyond the aim of the study.
In the present paper, HPLC-High Resolution Mass Spectrometry (HPLC-HRMS) was applied to investigate the composition of commercial Methyl Blue, Fuchsine, and Methyl Violet dyes both in terms of principal
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and in minor compounds. Both positive and negative modes and different voltages in the collision cell were tested to optimize mass spectrometric conditions for each dye and to obtain complementary information on fragmentation pathways. The several homologous compounds constituting the complex composition of
EP
these dyes have been separated and discriminated on the base of specific ions and fragmentation patterns. While Methyl Blue was chosen as representative of a sulfonated high molecular weight molecule, Fuchsine
AC C
and Methyl Violet were selected for being complex mixtures of compounds with similar structure but different position of substituents on the aromatic rings or amino groups. For Fuchsine, the results obtained at two different collision energies will be presented.
2. Experimental 2.1. Materials and reagents 5
ACCEPTED MANUSCRIPT Methyl Blue (95% purity) was purchased from Sigma-Aldrich (USA) and it was dissolved in DMSO (5 ppm). The wool yarns dyed with Fuchsine and Methyl Violet were kindly provided by Dr. Witold Nowik of the Département Recherche, Centre de Recherche et de Restauration des Musées de France-C2RMF within the IPERION project (www.iperionch.eu).
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The solvents used for the HPLC analysis and sample pretreatment were: water (LC-MS grade, Sigma Aldrich, USA), acetonitrile (ACN, LC-MS grade, Sigma Aldrich, USA), formic acid (FA, J.T. Baker, USA), methanol (MeOH; Sigma-Aldrich, USA), acetone (Sigma-Aldrich, USA), oxalic acid dehydrate (99.8 % purity, Carlo Erba,
SC
Italy) and dimethyl sulfoxide (DMSO, J.T. Baker, Holland).
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2.2. Sample treatment
Fuchsine and Methyl Violet were extracted from wool yarns. In these cases, 0.7 mg of wool were treated with 600 μL oxalic acid 0.5 M/MeOH/acetone/H2O (1:30:40:40 v/v/v/v) and heated in ultrasonic bath for 30 min at 60 °C [34]. The dried extract was solubilized in 300 μL DMSO; the residual yarn was further treated
syringe filters and injected.
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with 200 μL DMSO in ultrasonic bath for 10 min at 60 °C. The two extracts are admixed, filtered with PTFE
2.3. High Performance Liquid Chromatography/Mass Spectrometry
EP
A HPLC 1200 Infinity, coupled with a Quadrupole-Time of Flight tandem mass spectrometer 6530 Infinity Q-
AC C
ToF detector by a Jet Stream ESI interface (Agilent Technologies, USA), was used. Drying and sheath gas were N2, purity > 98%.
ESI conditions were: drying gas temperature 350 °C, flow 10 L/min; capillary voltage 4.5 KV; nebulizer gas pressure 35 psi; sheath gas temperature 375 °C, flow 11 L/min. High resolution MS and MS/MS acquisition range was set from 100 to 1000 m/z in negative and positive mode. The acquisition rate was 1.04 spectra/sec for both MS and MS/MS; for the MS/MS experiments, voltages in the range 30-100 V were tested in the collision cell to obtain information on CID fragmentation pathways of selected analytes (collision gas N2, purity 99.999%). The FWHM (Full Width Half Maximum) of quadrupole mass bandpass
6
ACCEPTED MANUSCRIPT used during MS/MS precursor isolation was 4 m/z. The Agilent tuning mix HP0321 was used to calibrate the mass axis daily in positive and negative ionization modes in the 100-1700 m/z range. The HPLC conditions were: Poroshell 120 EC-C18 column (3.0 mm x 75 mm, 2.7 μm particle size) with a Zorbax Eclipse plus C-18 guard column (4.6 mm x 12.5 mm, 5 μm particle size), a flow rate of 0.4 mL/min,
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injection volume 4 µL. Separation was achieved at 30 °C using a gradient of FA 0.1% v/v in H2O (eluent A) and FA 0.1% v/v in CH3CN (eluent B). The elution gradient was programmed as follows: 85% A for 2.6 minutes, followed by a linear gradient to 50% B in 13 min, then to 70 %B in 5.2 min and to 100% B in 0.5
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min, maintained for 1 min. The re-equilibration time for each analysis was 10 min.
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2.4. Data elaboration
Both the HPLC and the mass spectrometer were controlled by MassHunter Workstation Software (B.04.00), which was also used for data acquisition, and for data analysis. The Extracted Ion Chromatograms were obtained by applying the “Find by Formula” algorithm embedded in the Workstation Qualitative Analysis
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Software; for both positive and negative ionization, the formula matching was set at 2 ppm tolerance with a limit extraction range of 1.5 min and an area filter of 500 counts. Formula having an isotopic pattern score
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lower than 25% were discarded. All data were acquired in replicates.
AC C
3. Results and discussion
The results obtained by HPLC-ESI-Q-ToF in full scan and tandem MS for the triarylmethine dyes Methyl Blue, Fuchsine and Methyl Violet will be discussed in the present section.
3.1. Methyl blue Methyl Blue is sold as a disodium salt, characterized by one protonated amino group and three negative sulfonated groups. Therefore, considering the acid/basic nature of this dye, both positive and negative ionization modes were tested, and the best results were achieved at 50 V for positive and 30 V for negative 7
ACCEPTED MANUSCRIPT ionization. Using acidified eluents for chromatographic separation, Methyl Blue will be mostly in the neutral form with protonated sulfonated groups; thus, we define M as the molecular weight of Methyl Blue in its neutral form. While in positive mode only the [M+H]+ and the corresponding ion without one sulfonic group was revealed (chromatogram in Figure 2 a and spectrum in Figure 2 b), even working at relatively high CID
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voltages (50 V), in negative mode a widely diagnostic profile of [M-2H]2- was collected at 30 V. The MS/MS spectrum of this ion (Figure 2 c) allowed us to assign consecutive losses of sulfonic, sulfate and further sulfonic groups. The higher intensity of the ion [M-2H-SO3]2- with respect to the ion at [M-2H]2- was
SC
predictable based on the reduced stability of quasimolecular ion due to the presence of sulfonated groups [29]. Several mass spectrometric studies were carried out on the characterization of sulfonated dyes by LC–
M AN U
MS [29-31]. Electrospray Ionization is considered the most suitable for this class of ionic and strongly polar compounds [29]. Nevertheless, to the best of our knowledge, this is the first time that Methyl Blue was
AC C
EP
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studied by mass spectrometric techniques.
8
ACCEPTED MANUSCRIPT a)
+ESI EIC(378.561, 379.062, 756.114, 757.117) Scan Frag=175.0V 10.60
C37H30N 3O9S3+ -ESI EIC(376.546, 377.048, 754.099, 755.102) Scan Frag=175.0V
10
x10 4
12
13
14
15 16 17 18 Acquisition Time [min]
19
20
21
22
23
24
25
26
+ESI Product Ion (10.19-10.83 min, 3 Scans) Frag=175.0V [email protected] (756.109[z=1] -> **) [M+H]+
SC
b)
11
RI PT
Counts
C37H28N3O9S3-
756.109
0.5
412.096
254.091
c)
x103
100
150
200
250
300
350
400
507.063
450 500 550 600 Mass-to-Charge [m/z]
[M+H-SO3]+ 676.150
650
700
750
800
TE D
0
M AN U
Counts
1
-ESI Product Ion (11.16-11.31 min, 3 Scans) Frag=175.0V [email protected] (376.547[z=1] -> **)
2.5
850
900
950
1000
[M-2H-SO3]2336.569
SO 3
2
O 3S
1
B
0.5
N H
AC C
Counts
1.5
EP
N
[M-2H-2SO3]2296.589
SO3 H
N H
A
[B]˙ -
[M-2H]2[M-2H-SO3-SO2]2-
[A]˙-
155.990 170.996
0
100
120
140
160
376.543
304.584
180
200
220 240 260 Mass-to-Charge [m/z]
280
300
320
340
360
380
Figure 2: a) HPLC-ESI-Q-ToF Extract Ion Chromatograms (EIC) of C37H29N3O9S3 acquired in positive and negative ionization mode from the DMSO solution of Methyl Blue (the EICs are reported in the same scale). Tandem mass spectra (Collision-Induced Dissociation CID at 50.0 V for positive and 30.0 V for negative ionization) of Methyl Blue standard solution: b) precursor ion m/z=756.109 (positive ionization); c) precursor ion m/z=376.547 (negative ionization). Isolation width = 4 m/z.
9
ACCEPTED MANUSCRIPT 3.2. Fuchsine The HPLC-ESI-Q-TOF analysis of Fuchsine extracted from wool yarns confirmed the presence of the four expected compounds (Figure 3): Pararosaniline (Mag 0), Fuchsin (Mag I), Magenta II (Mag II) and New Fuchsine (Mag III). Table 2 reports the chemical structures of all the compounds identified and
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characterized in Fuchsine. Considering the presence of the protonated amino group, the positive mode was the ionization method of choice. In Figure 4, the MS/MS spectra of the four compounds present in Fuchsine, acquired at two values
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of voltages in the collision cell (30.0 V and 50.0 V) are compared.
+ESI EIC(172.106; 173.114; 344.212; 345.220) Scan Frag=175.0V x10 6
1.5
Counts
13.81
1
C 22 H24 N3 +
C 19 H18N3 +
16.04
12.59
0 10
11
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0.5
M AN U
C 21 H22 N3 + C20 H20 N3 + 14.92
12
13
14
15 16 17 18 Acquisition Time [min]
19
20
+
21
22
23
24
25
+
26
+
Figure 3: HPLC-ESI-Q-ToF Extract Ion Chromatograms (EIC) of C19H18N3 (Mag 0), C20H20N3 , (Mag I), C21H22N3 (Mag II) +
AC C
EP
and C22H24N3 (Mag III) acquired in positive ionization mode from the extracted of Fuchsine dyed wool yarn.
10
ACCEPTED MANUSCRIPT +ESI Product Ion (12.59 min) Frag=175.0V [email protected] (288.149[z=1] -> **) 288.149 195.091
+ESI Product Ion (12.49 min) Frag=175.0V [email protected] (288.149[z=1] -> **) 195.091
Mag 0 C19H18N3+
151.054 178.064 167.073 115.052
271.121
+ESI Product Ion (13.81 min) Frag=175.0V [email protected] (302.165[z=1] ->**)
195.091 209.107
209.107 106.066
286.135 271.122
151.053
SC
300.150 286.134
120.082 167.075 193.085 223.122 181.086 106.066
M AN U
209.107 223.122
+ESI Product Ion (16.00 min) Frag=175.0V [email protected] (330.197[z=1] -> **)
223.122
223.122 208.098
120.082
300.153
208.100
286.136 300.149 269.108 316.187
+ESI Product Ion (16.04 min) Frag=175.0V [email protected] (330.197[z=1] -> **)
330.197
Mag III C 22H24N3+
302.165
254.091
209.107
316.180
Mag II C 21H22N3+
181.086
+ESI Product Ion (14.92 min) Frag=175.0V [email protected] (316.181[z=1] -> **)
+ESI Product Ion (14.90 min) Frag=175.0V [email protected] (316.180[z=1] -> **)
106.066
286.133 271.122
167.073
195.091 168.080
272.120 286.136
+ESI Product Ion (13.81 min) Frag=175.0V [email protected] (302.166[z=1] ->**)
302.165
Mag I C 20H20N3+
106.066
254.096
RI PT
168.080
283.122
314.164 330.197
120 140 160 180 200 220 240 260 280 300 320 340 Counts vs. Mass-to-Charge (m/z)
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120 140 160 180 200 220 240 260 280 300 320 340 Counts vs. Mass-to-Charge (m/z)
181.086
300.149
Figure 4: Tandem mass spectra of the four components of Fuchsine dye: Mag 0, Mag I, Mag II and Mag III acquired in positive mode, CID at 30.0 V (left) and CID at 50.0 V (right), isolation width =4 m/z.
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The interpretation of the tandem mass spectra of these homologous compounds highlights the differences
AC C
that occur in the loss of small radicals but some main common fragments are characteristic of the series. While there is not an evident correlation in the first losses of the magentas (-NH2˙,CH4, CH3˙) and their structure, interpretations have been proposed to justify the presence of the most intense peaks in the mass spectra of the different compounds. As expected, the precursor ion, which is abundant in the tandem mass spectra acquired at 30 V, is very low for CID at 50 V. In the latter case, a more extensive fragmentation occurs, resulting in several diagnostic peaks, which are missing in the spectra acquired at 30 V. In detail, the tandem mass spectra acquired at 50 V (Figure 4) show the following unique base peaks at: m/z =195.091 for Mg 0, m/z =209.107 for Mg I and Mg II and m/z =223.122 for Mg III. The peaks correspond to methylenedianiline for Mg 0, or a substituted methylenedianiline with one or two methyl groups, 11
ACCEPTED MANUSCRIPT respectively. In line with these considerations, the ions observed at m/z =167.080, ascribable to biphenylmethylium, and at m/z =181.086, ascribable to phenyl(m-tolyl)methylium, are characteristic of Mg 0-I-II and Mg I-II-III, respectively. The fragmentation proposed to distinguish the different homologues is shown in Figure 5 and the corresponding m/z are summarized in Table 1.
RI PT
R' NH 2
H 2N
SC
R' = R'' = H R'' R' = CH 3, R'' = H R' = R'' = CH3
m/z = 106.065
M AN U
R''' = CH3
m/z = 195.091 m/z = 209.107 m/z = 223.122
R''' NH 2
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Figure 5: Fragmentation pathway of Fuchsine.
Table 1: Fragments identified in MS/MS spectra of the four components of Fuchsine: Mag 0, Mag I, Mag II and Mag III (See Figure 5 for the chemical structure hypothesized for the m/z value reported).
X
Mag II
X
Mag III
X
X
AC C
Mag I
m/z =120.082
m/z =167.080
EP
m/z =106.065 Mag 0
m/z =181.086
X
m/z =195.091
m/z =209.107
m/z =223.122
X
X
X
X
X X
X
X X
X X
3.3. Methyl Violet
The complexity of the formulation of methyl violet is highlighted by the several peaks of homologues compounds and isomers detected in the wool yarn extracts by HPLC-ESI-Q-ToF (Figure 6). Extracting the five chemical formula relating to hexa-N-methyl pararosanilines (Hexa MP), penta- (Penta MP), tetra- (Tetra MP), tri- (Tri MP) and bi- (Bi MP), two isomers were found for Tetra MP and bi MP, three isomers for Tri MP and one peak for Hexa MP and Penta MP, respectively. The tandem mass spectra relative to the detected compounds are provided in Figure 7. Consistently with our considerations on fuchsine, the tandem mass 12
ACCEPTED MANUSCRIPT spectra collected using CID at 50 V provided more information than those obtained at 30 V and were thus selected for further discussion. In a previous study focused on Crystal Violet degradation, Hexa MP, Penta MP, Tetra MP, Tri MP and bi MP have been detected in not irradiated Crystal Violet solutions by HPLC-PDA and LC-MS (ion trap), but neither the isomers were detected, nor the mass spectra were presented or
RI PT
discussed [32]. In another study, the novel and ultra-rapid Surface Acoustic Wave Nebulisation (SAWN) has revealed the presence of the compounds mentioned above in textiles dyed with Crystal Violet, providing the relative mass-mass spectra only for of Hexa MP [33]. SAWN-MS is an ambient ionization technique, and
SC
consequently it does not involve prior separation not allowing us to discriminate any isomers. Moreover, the production of ions and ionization mechanism are different than in conventional ESI. The fragments
M AN U
obtained in the mass-mass spectra of Hexa MP (m/z 372. 244) in the paper of Astefanei et Al. [33] are consistent with the ones reported in this study: m/z 356.2 is the most intense product ion, followed by m/z 340.1, and m/z 235.1. The differences consist in 2 additional peaks (m/z 208.1 and 312.1) reported in the SAWN-MS/MS spectrum that were not observed in this study.
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Through the comparison of the corresponding tandem mass spectra reported in Figure 7 with those in Figure 4, Mg II and Mg III were identified as isomers of Bi MP and Tri MP, respectively, while the other peaks ascribed to the chemical formula of Tri MP and Tetra MP are positional isomers of methyl substituted
AC C
EP
pararosanilines.
13
ACCEPTED MANUSCRIPT x106
+ESI EIC(186.121; 187.129; 372.243; 373.251) Scan Frag=175.0V
C 25 H30N3+ C24H28N3 +
4
23.09
21.78
C 22H24N3+ C 23 H26N3+
2
20.37
C21H22N3 + 1
16.43 16.87
18.85 18.41 19.71
0 11
12
13
14
15
16
17
18
Acquisition Time [min]
19
20
21
22
23
SC
10
RI PT
Counts
3
24
25
26
+
+
+
+
+
M AN U
Figure 6: HPLC-ESI-Q-ToF Extract Ion Chromatograms (EIC) of the components of Methyl violet dye: C21H22N3 , C22H24N3 , C23H26N3 , C24H28N3 and C25H30N3 acquired in positive ionization mode from the extracted of Methyl violet
AC C
EP
TE D
dyed wool yarn
14
ACCEPTED MANUSCRIPT +ESI Product Ion (15.25 min) Frag=175.0V [email protected] (316.181[z=1] -> **) 209.106
Mag II C21H22N3+
+ESI Product Ion (19,71 min) Frag=175.0V CID@50,0 (344,214[z=1] -> **) 328.188
Tetra MP I C23H26N3+
286.136 300.148 193.084 223.121 167.073 269.108 181.086 106.062 316.181 245.107 120.078
207.099 180.086
Bi MP 209.107 C21H22N3+ 181.087 243.104
Tetra MP II C23H26N3+
230.095 120.081
+ESI Product Ion (16.52 min) Frag=175.0V [email protected] (330.199[z=1] -> **) 223.121
Mag III C22H24N3+
181.088
283.122 270.113
314.164 330.199
+ESI Product Ion (18.41 min) Frag=175.0V [email protected] (330.199[z=1] -> **) 314.168
Tri MP I C22H24N3+
221.107
237.138
326.165
358.229
+ESI Product Ion (23.25 min) Frag=175.0V [email protected] (372.244[z=1] -> **) 356.211
TE D
Hexa MP C25H30N3+
209.107 285.138 180.080 194.089 221.109243.103 272.130 299.146 167.075
330.199
AC C
194.095
EP
+ESI Product Ion (18.85 min) Frag=175.0V [email protected] (330.199[z=1] -> **) 287.154 208.100 Tri MP II 258.125 C22H24N3+ 181.088 272.131 315.172 223.124 299.149 120.080 157.089
344.214
+ESI Product Ion (21.75 min) Frag=175.0V [email protected] (358.229[z=1] -> **) 342.198
M AN U
120.079
313.160 221.108 258.128 287.154
Penta MP C24H28N3+
208.099 300.150
194.096
SC
106.067 157.089 131.071
312.157 344.214
256.118 284.139
+ESI Product Ion (20.40 min) Frag=175.0V [email protected] (344.214 [z=1] -> **) 328.183
301.157 285.131 316.181
223.129
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+ESI Product Ion (16.85 min) Frag=175.0V [email protected] (316.181[z=1] -> **) 273.138 167.073 194.085 258.118
238.157
340.179 235.122
372.244
328.190
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 Counts vs. Mass-to-Charge [m/z]
330.199
120 140 160 180 200 220 240 260 280 300 320 340 360 Counts vs. Mass-to-Charge [m/z]
Figure 7: Tandem mass spectra of the several components of Methyl violet dyes identified in Figure 6 in positive mode,
CID at 50.0 V, isolation width = 4 m/z.
A first evaluation of the mass spectra of Hexa MP, Penta MP and Tetra MP I and Tetra MP II evidences that for all the four compounds the base peak corresponds to the loss of CH4. This neutral loss only occurs for those compounds in which at least one nitrogen is substituted with two methyl groups. 15
ACCEPTED MANUSCRIPT In order to match the molecular structure of Tetra MP I and Tetra MP II with their mass spectra, m/z= 120.081 and m/z= 238.146 were chosen as peculiar ion for tetra MP I and tetra MP II, respectively, as shown in the fragmentation scheme reported in Figure 8 a and b. The compounds with chemical formula C23H26N3+ can be distinguished considering that Mag III presents a
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different fragmentation pattern respect to other tri-methyl pararosanilines. Moreover, Tri MP II spectrum presents a characteristic loss of -C2H5N, associated with the formation of m/z= 287.154 (Figure 8 c). The tandem mass spectrum of Bi MP shows some fragments characterized by same m/z value than those of
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Mag II but with higher relative intensity. This could be due to the lower bond energy required to break a CN bond (methyl group on the amino nitrogen) than to break a C-C one (methyl group on the aromatic ring).
proposed in Figure 8 d, to identify Bi MP.
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Two typical m/z values (273.138 and 258.118) have thus been chosen on the base of the fragmentation
In Table 2 the chemical structures of all the compounds identified and characterized in Fuchsine and Methyl
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Violet are reported.
Figure 8: Fragmentation pathway of a) Tetra MP I; b) Tetra MP II, c) Tri MP II and d) Bi MP.
16
ACCEPTED MANUSCRIPT Table 2: Identified compounds in the HPLC-ESI-Q-ToF chromatograms of Fuchsine and Methyl Violet analyzed in positive mode (in bold the most intense ions are reported). 1
Name
2
MS
Assigned structure
Raw Formula
MS
Precursor ion
NH2
+
Mag 0
C19H18N3
H2N
288.149
NH2 CH 3
(CID= 50.0 V)
272.136; 254.096; 195.091; 178.016; 167.073; 151.054
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NH 2
Product ions and fragmentation
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Molecules
286.135; 271.122; 254.091;
+ C20H20N3
Mag I
302.165
209.107; 195.091; 181.086;
H2N
NH 2 NH2
M AN U
167.080; 151.053;106.065
300.149; 286.136; 269.108;
CH 3
+
C21H22N3
Mag II H2N
NH 2 CH 3
+
C21H22N3
Bi MP
223.122; 209.107; 193.085; 181.086; 167.075; 120.082; 106.065
CH 3
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HN
316.180
301.157; 285.131; 273.138; 316.180
258.118; 243.104; 230.095; 209.107; 194.085; 181.087; 167.073; 106.062
NH
H 2N
EP
CH3
NH2
AC C
CH 3
314.164; 300.149; 283.122; + C22H24N3
Mag III
330.197
H 3C
223.121; 208.098; 181.086; 120.082
H2N
NH 2 CH 3
CH3
HN
314.168; 299.146; 285.138; +
C22H24N3
Tri MP I
330.197
272.130; 243.103; 221.109; 209.107; 194.084; 180.080
HN CH3
NH CH 3
17
ACCEPTED MANUSCRIPT CH 3 HN
315.172; 299.149; 287.154; + C22H24N3
Tri MP II
330.197
272.131; 258.125; 223.124; 208.100; 181.088; 120.080
CH 3 N
H2N
CH3 CH3
H3C N
Tetra MP I
344.214
H3C NH
N H
H3 C
CH 3
238.157; 223.129; 180.086
SC
N
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328.188; 312.157; 256.118; + C23H26N3
328.183; 313.160; 287.154;
+ C23H26N3
Tetra MP II
344.214
258.128; 221.108; 194.096; 120.081
N
NH2
CH3 H 3C
CH3 N
+
C24H28N3
Penta MP H3C N
NH
CH3
CH3 H3 C
CH 3
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N
+
Hexa MP
C25H30N3
H3 C
358.229
356.211
342.198; 326.165; 237.138; 221.107
356.211; 340.179; 328.190; 235.122
CH3
N
N
CH3
AC C
EP
CH3
4. Conclusions
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H3 C
The proposed analytical approach, based on the use of positive and negative HPLC-ESI-Q-ToF, allowed us to fully characterize the chemical composition of all the references dyes under study. The reduced injection volume (4 µL) allows us to perform subsequent analyses in positive and negative mode on the same sample’s extract. The main features of Methyl Blue, Fuchsine and Methyl Violet can be summarized as follows. Methyl blue, due to its structure, is better ionized in negative mode at 30.0 V as double-charged pseudomolecular ion and losses ascribed to sulfonic and sulfate have been detected in the tandem mass spectrum. 18
ACCEPTED MANUSCRIPT As regards the several homologues contained in Fuchsine and Methyl Violet, it is interesting noticing as the use of a 50.0 V voltage in the collision cell allowed us to get diagnostic profiles, useful to distinguish the different isomers. While for Fuchsine some marker ions have been identified to characterize the spectra of the different Magentas, resolving the complex mixture of magentas and pararosalines isomers in Methyl
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Violet required a more thoughtful interpretation of the fragmentation patterns. We demonstrated that liquid chromatography coupled with high resolution mass spectrometry is the best
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approach to separate and characterize such complex mixtures of early synthetic dye formulations.
Acknowledgments
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This work was performed in the framework of the European research infrastructure for conservation of Cultural Heritage IPERION (www.iperionch.eu), Task 7.2 Diagnostic of material changes in organic materials in Cultural Heritage (7.2c-Degradation of synthetic dyes and organic pigments). Authors would like to thank Dr. Witold Nowik (Département Recherche, Centre de Recherche et de Restauration des Musées de France-
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C2RMF) for the preparation of the wool yarns.
The work was partially founded by the project PRA_2016_13 “Analytical chemistry applications for deepening the knowledge of materials and techniques in modern and contemporary art”, supported by the
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University of Pisa.
19
ACCEPTED MANUSCRIPT References Gessner, T., Mayer, U.: Triarylmethane and Diarylmethane Dyes. (2000)
2.
Abrahart, E.N.: Dyes and their intermediates. (1977)
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aromatic amines, organic dyes, and related exposures. Elsevier; 2008.
Germinario G., Garrappa S., D’Ambrosio V., van der Werf I.D., Sabbatini L.: Chemical composition of
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felt-tip pen inks. Analytical and Bioanalytical Chemistry. 410, 1079-1094 (2018) Moretti P., Germinario G., Doherty B., van der Werf I., Sabbatini L., Mirabile, A., Sgamellotti A., Miliani C.: Disclosing the composition of historical commercial felt-tip pens used in art by integrated vibrational spectroscopy and pyrolysis-gas chromatography/mass spectrometry. Journal of Cultural
8.
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Heritage. (2018) in press https://doi.org/10.1016/j.culher.2018.03.018. Akhmerova, D., Krylova, A., Stavrianidi, A., Shpigun, O., Rodin, I.: Forensic Identification of Dyes in Ballpoint Pen Inks Using LC–ESI–MS. Chromatographia. 80, 1701-1709 (2017) Weyermann, C., Kirsch, D., Costa Vera, C., Spengler, B.: Evaluation of the photodegradation of
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forensic sciences. 54, 339-345 (2009) Au, W., Pathak, S., Collie, C.J., Hsu, T.C.: Cytogenetic toxicity of gentian violet and crystal violet on mammalian cells in vitro. Mutation Research/Genetic Toxicology. 58, 269-276 (1978) 11.
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Hou, X., Tong, X., Dong, W., Dong, C., Shuang, S.: Synchronous fluorescence determination of human serum albumin with methyl blue as a fluorescence probe. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 66, 552-556 (2007) 20
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Hurtaud-Pessel, D., Couëdor, P., Verdon, E.: Liquid chromatography–tandem mass spectrometry method for the determination of dye residues in aquaculture products: Development and validation. Journal of Chromatography A. 1218, 1632-1645 (2011)
14.
Gupta, V.K., Mittal, A., Gajbe, V., Mittal, J.: Adsorption of basic fuchsin using waste materials—
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bottom ash and deoiled soya—as adsorbents. Journal of Colloid and Interface Science. 319, 30-39 (2008) 15.
de O. Martins, A., Canalli, V.M., Azevedo, C.M.N., Pires, M.: Degradation of pararosaniline (C.I. Basic
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Red 9 monohydrochloride) dye by ozonation and sonolysis. Dyes and Pigments. 68, 227-234 (2006) Kosanić, M.M., Tričković, J.S.: Degradation of pararosaniline dye photoassisted by visible light.
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Journal of Photochemistry and Photobiology A: Chemistry. 149, 247-251 (2002) Iqbal, M.J., Ashiq, M.N.: Adsorption of dyes from aqueous solutions on activated charcoal. Journal of Hazardous Materials. 139, 57-66 (2007) 18.
Wu, T., Cai, X., Tan, S., Li, H., Liu, J., Yang, W.: Adsorption characteristics of acrylonitrile, p-
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toluenesulfonic acid, 1-naphthalenesulfonic acid and methyl blue on graphene in aqueous solutions. Chemical Engineering Journal. 173, 144-149 (2011) 19.
Kumar, C.G., Mongolla, P., Basha, A., Joseph, J., Sarma, V., Kamal, A.: Decolorization and
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biotransformation of triphenylmethane dye, methyl violet, by Aspergillus sp. isolated from Ladakh, India. J. Microbiol. Biotechnol. 21, 267-273 (2011) Ayed, L., Chaieb, K., Cheref, A., Bakhrouf, A.: Biodegradation and decolorization of
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triphenylmethane dyes by Staphylococcus epidermidis. Desalination. 260, 137-146 (2010) 21.
Guineau, B.: Non-destructive analysis of organic pigments and dyes using Raman microprobe, microfluorometer or absorption microspectrophotometer. Studies in Conservation. 34, 38-44 (1989)
22.
Ghelardi, E., Degano, I., Colombini, M.P., Mazurek, J., Schilling, M., Learner, T.: Py-GC/MS applied to the analysis of synthetic organic pigments: characterization and identification in paint samples. Analytical and Bioanalytical Chemistry. 407, 1415-1431 (2015) 21
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Russell, J., Singer, B.W., Perry, J.J., Bacon, A.: The identification of synthetic organic pigments in modern paints and modern paintings using pyrolysis-gas chromatography–mass spectrometry. Analytical and Bioanalytical Chemistry. 400, 1473 (2011)
24.
Germinario, G., Rigante, E.C.L., van der Werf, I.D., Sabbatini, L.: Pyrolysis gas chromatography–mass
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spectrometry of triarylmethane dyes. Journal of Analytical and Applied Pyrolysis. 127, 229-239 (2017) 25.
Doerge, D.R., Churchwell, M.I., Gehring, T.A., Pu, Y.M., Plakas, S.M.: Analysis of malachite green and
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metabolites in fish using liquid chromatography atmospheric pressure chemical ionization mass spectrometry. Rapid Communications in Mass Spectrometry. 12, 1625-1634 (1998) López-Gutiérrez, N., Romero-González, R., Plaza-Bolaños, P., Martínez-Vidal, J.L., Garrido-Frenich,
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26.
A.: Simultaneous and Fast Determination of Malachite Green, Leucomalachite Green, Crystal Violet, and Brilliant Green in Seafood by Ultrahigh Performance Liquid Chromatography–Tandem Mass Spectrometry. Food Analytical Methods. 6, 406-414 (2013)
Villar-Pulido, M., Gilbert-López, B., García-Reyes, J.F., Martos, N.R., Molina-Díaz, A.: Multiclass
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27.
detection and quantitation of antibiotics and veterinary drugs in shrimps by fast liquid chromatography time-of-flight mass spectrometry. Talanta. 85, 1419-1427 (2011) Coumbaros, J., Kirkbride, K.P., Klass, G., Skinner, W.: Application of time of flight secondary ion
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28.
mass spectrometry to the in situ analysis of ballpoint pen inks on paper. Forensic Science
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International. 193, 42-46 (2009)
Holčapek, M., Volná, K., Vaněrková, D.: Effects of functional groups on the fragmentation of dyes in electrospray and atmospheric pressure chemical ionization mass spectra. Dyes and Pigments. 75, 156-165 (2007)
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Gosetti, F., Gianotti, V., Angioi, S., Polati, S., Marengo, E., Gennaro, M.: Oxidative degradation of food dye E133 Brilliant Blue FCF: liquid chromatography–electrospray mass spectrometry identification of the degradation pathway. Journal of Chromatography A. 1054, 379-387 (2004)
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Holčapek, M., Jandera, P., Přikryl, J.: Analysis of sulfonated dyes and intermediates by electrospray mass spectrometry. Dyes and Pigments. 43, 127-137 (1999)
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Confortin, D., Neevel, H., Brustolon, M., Franco, L., Kettelarij, A.J., Williams, R.M., et al., editors. Crystal violet: study of the photo-fading of an early synthetic dye in aqueous solution and on paper
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with HPLC-PDA, LC-MS and FORS. Journal of Physics: Conference Series; 2010: IOP Publishing. Astefanei, A., van Bommel, M., Corthals, G.L.: Surface Acoustic Wave Nebulisation Mass Spectrometry for the Fast and Highly Sensitive Characterisation of Synthetic Dyes in Textile
Manhita, A., Ferreira, T., Candeias, A., Barrocas Dias, C.: Extracting natural dyes from wool – an
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evaluation of extraction methods. Analytical and Bioanalytical Chemistry. 400, 1501–1514 (2011).
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34.
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Samples. Journal of The American Society for Mass Spectrometry. 28, 10, 2108–2116 (2017).
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ACCEPTED MANUSCRIPT Highlights
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we fully characterized the composition of three triarylmethine dyes by LC-ESI-Q-ToF we studied the positive and negative tandem mass spectra of methyl blue we rationalized the MS/MS fragmentation of crystal violet and fuchsine components
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• • •
S0143-7208(18)31529-8
DOI:
10.1016/j.dyepig.2018.08.046
Reference:
DYPI 6960
To appear in:
Dyes and Pigments
Received Date: 10 July 2018 Revised Date:
23 August 2018
Accepted Date: 24 August 2018
Please cite this article as: Degano I, Sabatini F, Braccini C, Colombini MP, Triarylmethine dyes: Characterization of isomers using integrated mass spectrometry, Dyes and Pigments (2018), doi: 10.1016/j.dyepig.2018.08.046. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
+ESI Product Ion CID @50.0 V
C22H24N3+ NH 2
223.121
CH3
208.099
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300.150
H3 C
120.079
181.088
283.122
314.164 330.199
H 2N
NH2
M AN U
SC
270.113
CH 3
CH 3 HN
209.107
285.138
243.103
180.080
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314.168
272.130
299.146
330.199
AC C
208.100
CH3 CH 3 HN
287.154
272.131
223.124
315.172 299.149
157.089
330.199
CH 3 H2 N
120
140
160
CH 3
258.125
181.088
120.080
NH
HN
EP
167.075
180
200
220
240
260
280
Counts vs. Mass-to-Charge [m/z]
300
320
340
360
N CH 3
ACCEPTED MANUSCRIPT
Triarylmethine dyes: characterization of isomers using integrated mass spectrometry
Ilaria Deganoa a
University of Pisa, Department of Chemistry and Industrial Chemistry
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Via Moruzzi, 13, I-56126 Pisa (Italy)
Francesca Sabatinia
University of Pisa, Department of Chemistry and Industrial Chemistry
Via Moruzzi, 13, I-56126 Pisa (Italy)
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[email protected] Chiara Braccinia a
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[email protected]
a
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Authors
University of Pisa, Department of Chemistry and Industrial Chemistry
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[email protected]
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Via Moruzzi, 13, I-56126 Pisa (Italy)
Maria Perla Colombinia,b a
University of Pisa, Department of Chemistry and Industrial Chemistry
Via Moruzzi, 13, I-56126 Pisa (Italy) b
ICVBC Institute for the Conservation and Valorization of Cultural Heritage, National Research Council of
Italy, Via Madonna del Piano, 10, I-50019 Sesto Fiorentino, Italy [email protected]
1
ACCEPTED MANUSCRIPT Abstract Triarylmethine dyes, such as methyl and crystal violets, diamond green and magentas, produced since the late nineteenth century, consist of complex mixtures of homologous compounds, often differing only for the presence or position of the same substituent on the aromatic rings. In this paper, Liquid
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Chromatography-High Resolution Mass Spectrometry (HPLC-HRMS) was used for the characterization of the profile of three dyes: methyl blue, methyl violet and fuchsine. The comprehension of dye synthesis and production strategies was often achieved by analyzing the single components by diode array detector.
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Nevertheless, the nature of the individual components needs to be confirmed by tandem mass spectrometric techniques through the solving of the structural issues.
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The analysis of standards and reference materials by tandem high resolution mass spectrometry allowed us to identify peculiar fragmentation pathways for the components of magentas and methyl violet dyes. The results highlighted the importance to undertake the measurements both in negative and positive ionization mode. The elucidation of specific fragmentation patterns allows the discrimination between
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different classes of compounds, while the identification of specific fragment ions allows one to distinguish isomers belonging to the same series.
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Keywords
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Triarylmethine dyes; CID fragmentation pattern; HPLC-ESI-Q-ToF
2
ACCEPTED MANUSCRIPT 1. Introduction One of the earliest classes of synthetic dyes ever produced were the triarylmethines (often historically referred to as triarylmethanes). Among them, the industrial process for producing Fuchsine was the first to be developed in 1859 by Verguin. Since then, triarylmethines have become the most widely used synthetic
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dyes thanks to their versatility and their bright colours ranging from red/purple to blue/green hues [1, 2]. The chromophore system of this class of dyes is constituted by three conjugated aromatic rings bonded to a central atom of carbon [3], while the exact hue depends on the number and nature of auxochrome groups.
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The current synthetic strategy consists in a condensation reaction between Michler’s ketone and a suitable aromatic amine [1]. Among the great number of synthetic dyes belonging to this class, some of them were
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widespread and their characterisation will be discussed in the present paper.
Among them, Fuchsine is composed by a mixture of four main compounds, differentiating by the number of methyl groups on the aromatic rings (Figure 1 a). Pararosaniline (also known as Basic Fuchsine or Magenta 0, C.I. 42500) is not methylated and it is currently used for staining polyacrylonitrile fibres and for detecting
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aldehydes in biological samples[1, 4]; Fuchsin (also known as Rosaniline or Magenta I, C.I. 42510) contains one methyl substituent and it is used for dyeing silk, wool and paper [1]; Magenta II has two methyl substituents and it is the only one that cannot be synthesised as a pure compound; in the end New
ring [5].
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Fuchsine (Magenta III, C.I. 42520) is characterized by three methyl groups as substituents on the aromatic
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Methyl Violet (C.I. 42535) consists in a mixture of tetra-, penta- and hexa-N-methyl pararosaniline (Figure 1 b) while Crystal Violet (C.I. 42555) contains N-hexamethyl pararosaniline only. Both Crystal and Methyl Violet have been historically used in dyeing textiles, and more recently have been broadly used in printing and ballpoint pen and felt tip inks [6, 7]; thus, they are studied in forensic science for the authentication and dating of documents [8, 9]. They can also be applied for dying polyacrylonitrile fibres [1]. Finally, Crystal Violet, due to its antifungal and cytotoxic properties [10], is employed to classify bacteria via Gram test [3].
3
ACCEPTED MANUSCRIPT Methyl blue (C.I. 42780), also known as Acid Blue 93, is characterized by three benzenesulfonyl substituents on amine groups (Figure 1c), which provide good solubility in water to the molecule [1]. Due to its fluorescence, it is used as probe in biological system studies [11, 12]. H 3C
NH2
a)
CH 3 N
Cl
b)
R'
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Cl
R'''
R'''
H3 C
NH 2
N
N
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H 2N
R'
R''
Tetra-N-methyl pararosaniline (Tetra MP): R’=R’’=R’’’=H Penta-N-methyl pararosaniline (Penta MP): R’=H; R’’=R’’’=CH3 Hexa-N-methyl pararosaniline (Hexa MP): R’=R’’=R’’’=CH3
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Pararosaniline (Mag 0): R’=R’’=R’’’=H Fuchsin (Mag I): R’=R’’=H; R’’’=CH3 Magenta II (Mag II): R’=R’’=CH3; R’’’=H New fuchsin (Mag III): R’=R’’=R’’’=CH3
R''
SO 3
c)
Na O 3S
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HN
N H
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N H
SO 3 Na
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Figure 1: Chemical structures of the components of a) Fuchsine dye; b) Crystal and Methyl Violet dyes, and c) Methyl Blue.
As the issue of pollution of the environment by synthetic organic dyes has become progressively relevant, many studies have targeted the detection and identification of tryarylmethanes in wastewaters [13], and their removal using adsorption on activated surfaces [14-16] or photochemical degradation [17, 18]. Several studies adopted infrared [19, 20] and Raman [21] spectroscopies for triarylmethines detection. Nonetheless, the need to separate the different components in dye formulation or to detect their degradation products suggested the application of chromatographic techniques. Pyrolysis coupled with Gas Chromatography Mass Spectrometry (Py-GC/MS) was widely applied for the creation of databases fundamental for the identification of the main compounds constituting triarylmethine dyes [22-24]. 4
ACCEPTED MANUSCRIPT Nevertheless, Liquid Chromatography coupled with Diode Array or Mass Detector (LC-DAD, LC-MS) is is the method of choice due to the strong absorption of these dyes in the visible range, their low volatility and poor thermal stability [13]. Regarding the several promising approaches based on mass spectrometry, they are mostly focused on the optimization of ultrasensitive and ultrafast methods for the detection of a wide
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range of triarylmethines in aquaculture products or ballpoint inks mainly using High Performance Liquid Chromatography (HPLC) or Ultra High Performance Liquid Chromatography (UHPLC) coupled with quadrupole [25] or triple quadrupole (QqQ) analyzers [6, 11, 26]. Nevertheless, only few studies have been
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carried out using high resolution mass spectrometry [27] and using a Time of Flight ion Mass Spectrometry coupled with Secondary Ion Mass Spectrometry (TOF-SIMS) set-up [28,33]. Notably, in all the mentioned
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papers the identification of the minor compounds and the interpretation of fragmentation patterns are beyond the aim of the study.
In the present paper, HPLC-High Resolution Mass Spectrometry (HPLC-HRMS) was applied to investigate the composition of commercial Methyl Blue, Fuchsine, and Methyl Violet dyes both in terms of principal
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and in minor compounds. Both positive and negative modes and different voltages in the collision cell were tested to optimize mass spectrometric conditions for each dye and to obtain complementary information on fragmentation pathways. The several homologous compounds constituting the complex composition of
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these dyes have been separated and discriminated on the base of specific ions and fragmentation patterns. While Methyl Blue was chosen as representative of a sulfonated high molecular weight molecule, Fuchsine
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and Methyl Violet were selected for being complex mixtures of compounds with similar structure but different position of substituents on the aromatic rings or amino groups. For Fuchsine, the results obtained at two different collision energies will be presented.
2. Experimental 2.1. Materials and reagents 5
ACCEPTED MANUSCRIPT Methyl Blue (95% purity) was purchased from Sigma-Aldrich (USA) and it was dissolved in DMSO (5 ppm). The wool yarns dyed with Fuchsine and Methyl Violet were kindly provided by Dr. Witold Nowik of the Département Recherche, Centre de Recherche et de Restauration des Musées de France-C2RMF within the IPERION project (www.iperionch.eu).
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The solvents used for the HPLC analysis and sample pretreatment were: water (LC-MS grade, Sigma Aldrich, USA), acetonitrile (ACN, LC-MS grade, Sigma Aldrich, USA), formic acid (FA, J.T. Baker, USA), methanol (MeOH; Sigma-Aldrich, USA), acetone (Sigma-Aldrich, USA), oxalic acid dehydrate (99.8 % purity, Carlo Erba,
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Italy) and dimethyl sulfoxide (DMSO, J.T. Baker, Holland).
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2.2. Sample treatment
Fuchsine and Methyl Violet were extracted from wool yarns. In these cases, 0.7 mg of wool were treated with 600 μL oxalic acid 0.5 M/MeOH/acetone/H2O (1:30:40:40 v/v/v/v) and heated in ultrasonic bath for 30 min at 60 °C [34]. The dried extract was solubilized in 300 μL DMSO; the residual yarn was further treated
syringe filters and injected.
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with 200 μL DMSO in ultrasonic bath for 10 min at 60 °C. The two extracts are admixed, filtered with PTFE
2.3. High Performance Liquid Chromatography/Mass Spectrometry
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A HPLC 1200 Infinity, coupled with a Quadrupole-Time of Flight tandem mass spectrometer 6530 Infinity Q-
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ToF detector by a Jet Stream ESI interface (Agilent Technologies, USA), was used. Drying and sheath gas were N2, purity > 98%.
ESI conditions were: drying gas temperature 350 °C, flow 10 L/min; capillary voltage 4.5 KV; nebulizer gas pressure 35 psi; sheath gas temperature 375 °C, flow 11 L/min. High resolution MS and MS/MS acquisition range was set from 100 to 1000 m/z in negative and positive mode. The acquisition rate was 1.04 spectra/sec for both MS and MS/MS; for the MS/MS experiments, voltages in the range 30-100 V were tested in the collision cell to obtain information on CID fragmentation pathways of selected analytes (collision gas N2, purity 99.999%). The FWHM (Full Width Half Maximum) of quadrupole mass bandpass
6
ACCEPTED MANUSCRIPT used during MS/MS precursor isolation was 4 m/z. The Agilent tuning mix HP0321 was used to calibrate the mass axis daily in positive and negative ionization modes in the 100-1700 m/z range. The HPLC conditions were: Poroshell 120 EC-C18 column (3.0 mm x 75 mm, 2.7 μm particle size) with a Zorbax Eclipse plus C-18 guard column (4.6 mm x 12.5 mm, 5 μm particle size), a flow rate of 0.4 mL/min,
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injection volume 4 µL. Separation was achieved at 30 °C using a gradient of FA 0.1% v/v in H2O (eluent A) and FA 0.1% v/v in CH3CN (eluent B). The elution gradient was programmed as follows: 85% A for 2.6 minutes, followed by a linear gradient to 50% B in 13 min, then to 70 %B in 5.2 min and to 100% B in 0.5
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min, maintained for 1 min. The re-equilibration time for each analysis was 10 min.
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2.4. Data elaboration
Both the HPLC and the mass spectrometer were controlled by MassHunter Workstation Software (B.04.00), which was also used for data acquisition, and for data analysis. The Extracted Ion Chromatograms were obtained by applying the “Find by Formula” algorithm embedded in the Workstation Qualitative Analysis
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Software; for both positive and negative ionization, the formula matching was set at 2 ppm tolerance with a limit extraction range of 1.5 min and an area filter of 500 counts. Formula having an isotopic pattern score
EP
lower than 25% were discarded. All data were acquired in replicates.
AC C
3. Results and discussion
The results obtained by HPLC-ESI-Q-ToF in full scan and tandem MS for the triarylmethine dyes Methyl Blue, Fuchsine and Methyl Violet will be discussed in the present section.
3.1. Methyl blue Methyl Blue is sold as a disodium salt, characterized by one protonated amino group and three negative sulfonated groups. Therefore, considering the acid/basic nature of this dye, both positive and negative ionization modes were tested, and the best results were achieved at 50 V for positive and 30 V for negative 7
ACCEPTED MANUSCRIPT ionization. Using acidified eluents for chromatographic separation, Methyl Blue will be mostly in the neutral form with protonated sulfonated groups; thus, we define M as the molecular weight of Methyl Blue in its neutral form. While in positive mode only the [M+H]+ and the corresponding ion without one sulfonic group was revealed (chromatogram in Figure 2 a and spectrum in Figure 2 b), even working at relatively high CID
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voltages (50 V), in negative mode a widely diagnostic profile of [M-2H]2- was collected at 30 V. The MS/MS spectrum of this ion (Figure 2 c) allowed us to assign consecutive losses of sulfonic, sulfate and further sulfonic groups. The higher intensity of the ion [M-2H-SO3]2- with respect to the ion at [M-2H]2- was
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predictable based on the reduced stability of quasimolecular ion due to the presence of sulfonated groups [29]. Several mass spectrometric studies were carried out on the characterization of sulfonated dyes by LC–
M AN U
MS [29-31]. Electrospray Ionization is considered the most suitable for this class of ionic and strongly polar compounds [29]. Nevertheless, to the best of our knowledge, this is the first time that Methyl Blue was
AC C
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studied by mass spectrometric techniques.
8
ACCEPTED MANUSCRIPT a)
+ESI EIC(378.561, 379.062, 756.114, 757.117) Scan Frag=175.0V 10.60
C37H30N 3O9S3+ -ESI EIC(376.546, 377.048, 754.099, 755.102) Scan Frag=175.0V
10
x10 4
12
13
14
15 16 17 18 Acquisition Time [min]
19
20
21
22
23
24
25
26
+ESI Product Ion (10.19-10.83 min, 3 Scans) Frag=175.0V [email protected] (756.109[z=1] -> **) [M+H]+
SC
b)
11
RI PT
Counts
C37H28N3O9S3-
756.109
0.5
412.096
254.091
c)
x103
100
150
200
250
300
350
400
507.063
450 500 550 600 Mass-to-Charge [m/z]
[M+H-SO3]+ 676.150
650
700
750
800
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0
M AN U
Counts
1
-ESI Product Ion (11.16-11.31 min, 3 Scans) Frag=175.0V [email protected] (376.547[z=1] -> **)
2.5
850
900
950
1000
[M-2H-SO3]2336.569
SO 3
2
O 3S
1
B
0.5
N H
AC C
Counts
1.5
EP
N
[M-2H-2SO3]2296.589
SO3 H
N H
A
[B]˙ -
[M-2H]2[M-2H-SO3-SO2]2-
[A]˙-
155.990 170.996
0
100
120
140
160
376.543
304.584
180
200
220 240 260 Mass-to-Charge [m/z]
280
300
320
340
360
380
Figure 2: a) HPLC-ESI-Q-ToF Extract Ion Chromatograms (EIC) of C37H29N3O9S3 acquired in positive and negative ionization mode from the DMSO solution of Methyl Blue (the EICs are reported in the same scale). Tandem mass spectra (Collision-Induced Dissociation CID at 50.0 V for positive and 30.0 V for negative ionization) of Methyl Blue standard solution: b) precursor ion m/z=756.109 (positive ionization); c) precursor ion m/z=376.547 (negative ionization). Isolation width = 4 m/z.
9
ACCEPTED MANUSCRIPT 3.2. Fuchsine The HPLC-ESI-Q-TOF analysis of Fuchsine extracted from wool yarns confirmed the presence of the four expected compounds (Figure 3): Pararosaniline (Mag 0), Fuchsin (Mag I), Magenta II (Mag II) and New Fuchsine (Mag III). Table 2 reports the chemical structures of all the compounds identified and
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characterized in Fuchsine. Considering the presence of the protonated amino group, the positive mode was the ionization method of choice. In Figure 4, the MS/MS spectra of the four compounds present in Fuchsine, acquired at two values
SC
of voltages in the collision cell (30.0 V and 50.0 V) are compared.
+ESI EIC(172.106; 173.114; 344.212; 345.220) Scan Frag=175.0V x10 6
1.5
Counts
13.81
1
C 22 H24 N3 +
C 19 H18N3 +
16.04
12.59
0 10
11
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0.5
M AN U
C 21 H22 N3 + C20 H20 N3 + 14.92
12
13
14
15 16 17 18 Acquisition Time [min]
19
20
+
21
22
23
24
25
+
26
+
Figure 3: HPLC-ESI-Q-ToF Extract Ion Chromatograms (EIC) of C19H18N3 (Mag 0), C20H20N3 , (Mag I), C21H22N3 (Mag II) +
AC C
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and C22H24N3 (Mag III) acquired in positive ionization mode from the extracted of Fuchsine dyed wool yarn.
10
ACCEPTED MANUSCRIPT +ESI Product Ion (12.59 min) Frag=175.0V [email protected] (288.149[z=1] -> **) 288.149 195.091
+ESI Product Ion (12.49 min) Frag=175.0V [email protected] (288.149[z=1] -> **) 195.091
Mag 0 C19H18N3+
151.054 178.064 167.073 115.052
271.121
+ESI Product Ion (13.81 min) Frag=175.0V [email protected] (302.165[z=1] ->**)
195.091 209.107
209.107 106.066
286.135 271.122
151.053
SC
300.150 286.134
120.082 167.075 193.085 223.122 181.086 106.066
M AN U
209.107 223.122
+ESI Product Ion (16.00 min) Frag=175.0V [email protected] (330.197[z=1] -> **)
223.122
223.122 208.098
120.082
300.153
208.100
286.136 300.149 269.108 316.187
+ESI Product Ion (16.04 min) Frag=175.0V [email protected] (330.197[z=1] -> **)
330.197
Mag III C 22H24N3+
302.165
254.091
209.107
316.180
Mag II C 21H22N3+
181.086
+ESI Product Ion (14.92 min) Frag=175.0V [email protected] (316.181[z=1] -> **)
+ESI Product Ion (14.90 min) Frag=175.0V [email protected] (316.180[z=1] -> **)
106.066
286.133 271.122
167.073
195.091 168.080
272.120 286.136
+ESI Product Ion (13.81 min) Frag=175.0V [email protected] (302.166[z=1] ->**)
302.165
Mag I C 20H20N3+
106.066
254.096
RI PT
168.080
283.122
314.164 330.197
120 140 160 180 200 220 240 260 280 300 320 340 Counts vs. Mass-to-Charge (m/z)
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120 140 160 180 200 220 240 260 280 300 320 340 Counts vs. Mass-to-Charge (m/z)
181.086
300.149
Figure 4: Tandem mass spectra of the four components of Fuchsine dye: Mag 0, Mag I, Mag II and Mag III acquired in positive mode, CID at 30.0 V (left) and CID at 50.0 V (right), isolation width =4 m/z.
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The interpretation of the tandem mass spectra of these homologous compounds highlights the differences
AC C
that occur in the loss of small radicals but some main common fragments are characteristic of the series. While there is not an evident correlation in the first losses of the magentas (-NH2˙,CH4, CH3˙) and their structure, interpretations have been proposed to justify the presence of the most intense peaks in the mass spectra of the different compounds. As expected, the precursor ion, which is abundant in the tandem mass spectra acquired at 30 V, is very low for CID at 50 V. In the latter case, a more extensive fragmentation occurs, resulting in several diagnostic peaks, which are missing in the spectra acquired at 30 V. In detail, the tandem mass spectra acquired at 50 V (Figure 4) show the following unique base peaks at: m/z =195.091 for Mg 0, m/z =209.107 for Mg I and Mg II and m/z =223.122 for Mg III. The peaks correspond to methylenedianiline for Mg 0, or a substituted methylenedianiline with one or two methyl groups, 11
ACCEPTED MANUSCRIPT respectively. In line with these considerations, the ions observed at m/z =167.080, ascribable to biphenylmethylium, and at m/z =181.086, ascribable to phenyl(m-tolyl)methylium, are characteristic of Mg 0-I-II and Mg I-II-III, respectively. The fragmentation proposed to distinguish the different homologues is shown in Figure 5 and the corresponding m/z are summarized in Table 1.
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R' NH 2
H 2N
SC
R' = R'' = H R'' R' = CH 3, R'' = H R' = R'' = CH3
m/z = 106.065
M AN U
R''' = CH3
m/z = 195.091 m/z = 209.107 m/z = 223.122
R''' NH 2
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Figure 5: Fragmentation pathway of Fuchsine.
Table 1: Fragments identified in MS/MS spectra of the four components of Fuchsine: Mag 0, Mag I, Mag II and Mag III (See Figure 5 for the chemical structure hypothesized for the m/z value reported).
X
Mag II
X
Mag III
X
X
AC C
Mag I
m/z =120.082
m/z =167.080
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m/z =106.065 Mag 0
m/z =181.086
X
m/z =195.091
m/z =209.107
m/z =223.122
X
X
X
X
X X
X
X X
X X
3.3. Methyl Violet
The complexity of the formulation of methyl violet is highlighted by the several peaks of homologues compounds and isomers detected in the wool yarn extracts by HPLC-ESI-Q-ToF (Figure 6). Extracting the five chemical formula relating to hexa-N-methyl pararosanilines (Hexa MP), penta- (Penta MP), tetra- (Tetra MP), tri- (Tri MP) and bi- (Bi MP), two isomers were found for Tetra MP and bi MP, three isomers for Tri MP and one peak for Hexa MP and Penta MP, respectively. The tandem mass spectra relative to the detected compounds are provided in Figure 7. Consistently with our considerations on fuchsine, the tandem mass 12
ACCEPTED MANUSCRIPT spectra collected using CID at 50 V provided more information than those obtained at 30 V and were thus selected for further discussion. In a previous study focused on Crystal Violet degradation, Hexa MP, Penta MP, Tetra MP, Tri MP and bi MP have been detected in not irradiated Crystal Violet solutions by HPLC-PDA and LC-MS (ion trap), but neither the isomers were detected, nor the mass spectra were presented or
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discussed [32]. In another study, the novel and ultra-rapid Surface Acoustic Wave Nebulisation (SAWN) has revealed the presence of the compounds mentioned above in textiles dyed with Crystal Violet, providing the relative mass-mass spectra only for of Hexa MP [33]. SAWN-MS is an ambient ionization technique, and
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consequently it does not involve prior separation not allowing us to discriminate any isomers. Moreover, the production of ions and ionization mechanism are different than in conventional ESI. The fragments
M AN U
obtained in the mass-mass spectra of Hexa MP (m/z 372. 244) in the paper of Astefanei et Al. [33] are consistent with the ones reported in this study: m/z 356.2 is the most intense product ion, followed by m/z 340.1, and m/z 235.1. The differences consist in 2 additional peaks (m/z 208.1 and 312.1) reported in the SAWN-MS/MS spectrum that were not observed in this study.
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Through the comparison of the corresponding tandem mass spectra reported in Figure 7 with those in Figure 4, Mg II and Mg III were identified as isomers of Bi MP and Tri MP, respectively, while the other peaks ascribed to the chemical formula of Tri MP and Tetra MP are positional isomers of methyl substituted
AC C
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pararosanilines.
13
ACCEPTED MANUSCRIPT x106
+ESI EIC(186.121; 187.129; 372.243; 373.251) Scan Frag=175.0V
C 25 H30N3+ C24H28N3 +
4
23.09
21.78
C 22H24N3+ C 23 H26N3+
2
20.37
C21H22N3 + 1
16.43 16.87
18.85 18.41 19.71
0 11
12
13
14
15
16
17
18
Acquisition Time [min]
19
20
21
22
23
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10
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Counts
3
24
25
26
+
+
+
+
+
M AN U
Figure 6: HPLC-ESI-Q-ToF Extract Ion Chromatograms (EIC) of the components of Methyl violet dye: C21H22N3 , C22H24N3 , C23H26N3 , C24H28N3 and C25H30N3 acquired in positive ionization mode from the extracted of Methyl violet
AC C
EP
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dyed wool yarn
14
ACCEPTED MANUSCRIPT +ESI Product Ion (15.25 min) Frag=175.0V [email protected] (316.181[z=1] -> **) 209.106
Mag II C21H22N3+
+ESI Product Ion (19,71 min) Frag=175.0V CID@50,0 (344,214[z=1] -> **) 328.188
Tetra MP I C23H26N3+
286.136 300.148 193.084 223.121 167.073 269.108 181.086 106.062 316.181 245.107 120.078
207.099 180.086
Bi MP 209.107 C21H22N3+ 181.087 243.104
Tetra MP II C23H26N3+
230.095 120.081
+ESI Product Ion (16.52 min) Frag=175.0V [email protected] (330.199[z=1] -> **) 223.121
Mag III C22H24N3+
181.088
283.122 270.113
314.164 330.199
+ESI Product Ion (18.41 min) Frag=175.0V [email protected] (330.199[z=1] -> **) 314.168
Tri MP I C22H24N3+
221.107
237.138
326.165
358.229
+ESI Product Ion (23.25 min) Frag=175.0V [email protected] (372.244[z=1] -> **) 356.211
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Hexa MP C25H30N3+
209.107 285.138 180.080 194.089 221.109243.103 272.130 299.146 167.075
330.199
AC C
194.095
EP
+ESI Product Ion (18.85 min) Frag=175.0V [email protected] (330.199[z=1] -> **) 287.154 208.100 Tri MP II 258.125 C22H24N3+ 181.088 272.131 315.172 223.124 299.149 120.080 157.089
344.214
+ESI Product Ion (21.75 min) Frag=175.0V [email protected] (358.229[z=1] -> **) 342.198
M AN U
120.079
313.160 221.108 258.128 287.154
Penta MP C24H28N3+
208.099 300.150
194.096
SC
106.067 157.089 131.071
312.157 344.214
256.118 284.139
+ESI Product Ion (20.40 min) Frag=175.0V [email protected] (344.214 [z=1] -> **) 328.183
301.157 285.131 316.181
223.129
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+ESI Product Ion (16.85 min) Frag=175.0V [email protected] (316.181[z=1] -> **) 273.138 167.073 194.085 258.118
238.157
340.179 235.122
372.244
328.190
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 Counts vs. Mass-to-Charge [m/z]
330.199
120 140 160 180 200 220 240 260 280 300 320 340 360 Counts vs. Mass-to-Charge [m/z]
Figure 7: Tandem mass spectra of the several components of Methyl violet dyes identified in Figure 6 in positive mode,
CID at 50.0 V, isolation width = 4 m/z.
A first evaluation of the mass spectra of Hexa MP, Penta MP and Tetra MP I and Tetra MP II evidences that for all the four compounds the base peak corresponds to the loss of CH4. This neutral loss only occurs for those compounds in which at least one nitrogen is substituted with two methyl groups. 15
ACCEPTED MANUSCRIPT In order to match the molecular structure of Tetra MP I and Tetra MP II with their mass spectra, m/z= 120.081 and m/z= 238.146 were chosen as peculiar ion for tetra MP I and tetra MP II, respectively, as shown in the fragmentation scheme reported in Figure 8 a and b. The compounds with chemical formula C23H26N3+ can be distinguished considering that Mag III presents a
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different fragmentation pattern respect to other tri-methyl pararosanilines. Moreover, Tri MP II spectrum presents a characteristic loss of -C2H5N, associated with the formation of m/z= 287.154 (Figure 8 c). The tandem mass spectrum of Bi MP shows some fragments characterized by same m/z value than those of
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Mag II but with higher relative intensity. This could be due to the lower bond energy required to break a CN bond (methyl group on the amino nitrogen) than to break a C-C one (methyl group on the aromatic ring).
proposed in Figure 8 d, to identify Bi MP.
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Two typical m/z values (273.138 and 258.118) have thus been chosen on the base of the fragmentation
In Table 2 the chemical structures of all the compounds identified and characterized in Fuchsine and Methyl
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Violet are reported.
Figure 8: Fragmentation pathway of a) Tetra MP I; b) Tetra MP II, c) Tri MP II and d) Bi MP.
16
ACCEPTED MANUSCRIPT Table 2: Identified compounds in the HPLC-ESI-Q-ToF chromatograms of Fuchsine and Methyl Violet analyzed in positive mode (in bold the most intense ions are reported). 1
Name
2
MS
Assigned structure
Raw Formula
MS
Precursor ion
NH2
+
Mag 0
C19H18N3
H2N
288.149
NH2 CH 3
(CID= 50.0 V)
272.136; 254.096; 195.091; 178.016; 167.073; 151.054
SC
NH 2
Product ions and fragmentation
RI PT
Molecules
286.135; 271.122; 254.091;
+ C20H20N3
Mag I
302.165
209.107; 195.091; 181.086;
H2N
NH 2 NH2
M AN U
167.080; 151.053;106.065
300.149; 286.136; 269.108;
CH 3
+
C21H22N3
Mag II H2N
NH 2 CH 3
+
C21H22N3
Bi MP
223.122; 209.107; 193.085; 181.086; 167.075; 120.082; 106.065
CH 3
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HN
316.180
301.157; 285.131; 273.138; 316.180
258.118; 243.104; 230.095; 209.107; 194.085; 181.087; 167.073; 106.062
NH
H 2N
EP
CH3
NH2
AC C
CH 3
314.164; 300.149; 283.122; + C22H24N3
Mag III
330.197
H 3C
223.121; 208.098; 181.086; 120.082
H2N
NH 2 CH 3
CH3
HN
314.168; 299.146; 285.138; +
C22H24N3
Tri MP I
330.197
272.130; 243.103; 221.109; 209.107; 194.084; 180.080
HN CH3
NH CH 3
17
ACCEPTED MANUSCRIPT CH 3 HN
315.172; 299.149; 287.154; + C22H24N3
Tri MP II
330.197
272.131; 258.125; 223.124; 208.100; 181.088; 120.080
CH 3 N
H2N
CH3 CH3
H3C N
Tetra MP I
344.214
H3C NH
N H
H3 C
CH 3
238.157; 223.129; 180.086
SC
N
RI PT
328.188; 312.157; 256.118; + C23H26N3
328.183; 313.160; 287.154;
+ C23H26N3
Tetra MP II
344.214
258.128; 221.108; 194.096; 120.081
N
NH2
CH3 H 3C
CH3 N
+
C24H28N3
Penta MP H3C N
NH
CH3
CH3 H3 C
CH 3
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N
+
Hexa MP
C25H30N3
H3 C
358.229
356.211
342.198; 326.165; 237.138; 221.107
356.211; 340.179; 328.190; 235.122
CH3
N
N
CH3
AC C
EP
CH3
4. Conclusions
M AN U
H3 C
The proposed analytical approach, based on the use of positive and negative HPLC-ESI-Q-ToF, allowed us to fully characterize the chemical composition of all the references dyes under study. The reduced injection volume (4 µL) allows us to perform subsequent analyses in positive and negative mode on the same sample’s extract. The main features of Methyl Blue, Fuchsine and Methyl Violet can be summarized as follows. Methyl blue, due to its structure, is better ionized in negative mode at 30.0 V as double-charged pseudomolecular ion and losses ascribed to sulfonic and sulfate have been detected in the tandem mass spectrum. 18
ACCEPTED MANUSCRIPT As regards the several homologues contained in Fuchsine and Methyl Violet, it is interesting noticing as the use of a 50.0 V voltage in the collision cell allowed us to get diagnostic profiles, useful to distinguish the different isomers. While for Fuchsine some marker ions have been identified to characterize the spectra of the different Magentas, resolving the complex mixture of magentas and pararosalines isomers in Methyl
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Violet required a more thoughtful interpretation of the fragmentation patterns. We demonstrated that liquid chromatography coupled with high resolution mass spectrometry is the best
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approach to separate and characterize such complex mixtures of early synthetic dye formulations.
Acknowledgments
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This work was performed in the framework of the European research infrastructure for conservation of Cultural Heritage IPERION (www.iperionch.eu), Task 7.2 Diagnostic of material changes in organic materials in Cultural Heritage (7.2c-Degradation of synthetic dyes and organic pigments). Authors would like to thank Dr. Witold Nowik (Département Recherche, Centre de Recherche et de Restauration des Musées de France-
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C2RMF) for the preparation of the wool yarns.
The work was partially founded by the project PRA_2016_13 “Analytical chemistry applications for deepening the knowledge of materials and techniques in modern and contemporary art”, supported by the
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University of Pisa.
19
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2.
Abrahart, E.N.: Dyes and their intermediates. (1977)
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Zollinger, H. John Wiley & Sons, (2003)
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Ewen, A.B.: An Improved Aldehyde Fuchsin Staining Technique for Neurosecretory Products in
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1.
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Baan R., Straif K., Grosse Y., Secretan B., El Ghissassi F., Bouvard V., et al. Carcinogenicity of some
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aromatic amines, organic dyes, and related exposures. Elsevier; 2008.
Germinario G., Garrappa S., D’Ambrosio V., van der Werf I.D., Sabbatini L.: Chemical composition of
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felt-tip pen inks. Analytical and Bioanalytical Chemistry. 410, 1079-1094 (2018) Moretti P., Germinario G., Doherty B., van der Werf I., Sabbatini L., Mirabile, A., Sgamellotti A., Miliani C.: Disclosing the composition of historical commercial felt-tip pens used in art by integrated vibrational spectroscopy and pyrolysis-gas chromatography/mass spectrometry. Journal of Cultural
8.
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Heritage. (2018) in press https://doi.org/10.1016/j.culher.2018.03.018. Akhmerova, D., Krylova, A., Stavrianidi, A., Shpigun, O., Rodin, I.: Forensic Identification of Dyes in Ballpoint Pen Inks Using LC–ESI–MS. Chromatographia. 80, 1701-1709 (2017) Weyermann, C., Kirsch, D., Costa Vera, C., Spengler, B.: Evaluation of the photodegradation of
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9.
crystal violet upon light exposure by mass spectrometric and spectroscopic methods. Journal of
10.
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forensic sciences. 54, 339-345 (2009) Au, W., Pathak, S., Collie, C.J., Hsu, T.C.: Cytogenetic toxicity of gentian violet and crystal violet on mammalian cells in vitro. Mutation Research/Genetic Toxicology. 58, 269-276 (1978) 11.
Song, S., Hou, X., Shuang, S., Dong, C.: Study on the interaction between Methyl Blue and hsa in the presence of β-CD/HP-β-CD by molecular spectroscopy. World Scientific, (2012)
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Hou, X., Tong, X., Dong, W., Dong, C., Shuang, S.: Synchronous fluorescence determination of human serum albumin with methyl blue as a fluorescence probe. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 66, 552-556 (2007) 20
ACCEPTED MANUSCRIPT 13.
Hurtaud-Pessel, D., Couëdor, P., Verdon, E.: Liquid chromatography–tandem mass spectrometry method for the determination of dye residues in aquaculture products: Development and validation. Journal of Chromatography A. 1218, 1632-1645 (2011)
14.
Gupta, V.K., Mittal, A., Gajbe, V., Mittal, J.: Adsorption of basic fuchsin using waste materials—
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bottom ash and deoiled soya—as adsorbents. Journal of Colloid and Interface Science. 319, 30-39 (2008) 15.
de O. Martins, A., Canalli, V.M., Azevedo, C.M.N., Pires, M.: Degradation of pararosaniline (C.I. Basic
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we fully characterized the composition of three triarylmethine dyes by LC-ESI-Q-ToF we studied the positive and negative tandem mass spectra of methyl blue we rationalized the MS/MS fragmentation of crystal violet and fuchsine components
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