Sensitive Method for the Identification of Potential Sensitizing Impurities in Reaction Mixtures by Fluorescent Nitrobenzoxadiazole-Labeled Glutathione

Article abstract of DOI:10.1021/acs.chemrestox.0c00327

Allergic contact dermatitis is a critical issue in the development of new chemicals. Minor impurities with strong skin-sensitizing properties can be generated as byproducts. However, it is very difficult to identify these skin sensitizers in product mixtures. In this study, fluorescent nitrobenzoxadiazole-labeled glutathione (NBD-GSH) was synthesized to identify small amounts of skin sensitizers in reaction mixtures. Twelve known skin sensitizers and three nonsensitizers were reacted with NBD-GSH. Adducts formed only with the skin sensitizers, which allowed for their detection by a fluorescence detector. Liquid chromatography-mass spectrometry (LC-MS) analyses showed that NBD-GSH reacted with the skin sensitizers via its thiol and amino groups. An adduct of NBD-GSH with the strong skin sensitizer 1-chloro-2,4-dinitrobenzene was detected with a limit of detection of 6 × 10-8 mol/L by high-performance liquid chromatography with fluorescence detection. When a reaction mixture from primary alcohol oxidation was incubated with NBD-GSH, a NBD-GSH adduct formed with skin-sensitizing aldehyde impurities and could be specifically detected by LC-MS with fluorescence detection. This method will be useful for detection and identification of small amounts of skin sensitizers in raw materials, intermediates, reaction mixtures, and end products in the chemical industry.

Full text of DOI:10.1021/acs.chemrestox.0c00327

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Article
Sensitive Method for the Identification of Potential Sensitizing
Impurities in Reaction Mixtures by Fluorescent
Nitrobenzoxadiazole-Labeled Glutathione
Takashi Tokunaga,* Gaku Yamamoto, Teruki Takahashi, Makiko Mukumoto, Masayuki Sato,
and Masahiko Okamoto
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ABSTRACT: Allergic contact dermatitis is a critical issue in the
development of new chemicals. Minor impurities with strong skin-
sensitizing properties can be generated as byproducts. However, it
is very difficult to identify these skin sensitizers in product
mixtures. In this study, fluorescent nitrobenzoxadiazole-labeled
glutathione (NBD-GSH) was synthesized to identify small
amounts of skin sensitizers in reaction mixtures. Twelve known
skin sensitizers and three nonsensitizers were reacted with NBD-GSH. Adducts formed only with the skin sensitizers, which allowed
for their detection by a fluorescence detector. Liquid chromatography-mass spectrometry (LC-MS) analyses showed that NBD-GSH
reacted with the skin sensitizers via its thiol and amino groups. An adduct of NBD-GSH with the strong skin sensitizer 1-chloro-2,4-
dinitrobenzene was detected with a limit of detection of 6 × 10−8 mol/L by high-performance liquid chromatography with
fluorescence detection. When a reaction mixture from primary alcohol oxidation was incubated with NBD-GSH, a NBD-GSH
adduct formed with skin-sensitizing aldehyde impurities and could be specifically detected by LC-MS with fluorescence detection.
This method will be useful for detection and identification of small amounts of skin sensitizers in raw materials, intermediates,
reaction mixtures, and end products in the chemical industry.
restricted.12 Many alternatives to animal testing have been
INTRODUCTION
■
developed on the basis of the key events from adverse outcome
In the chemical industry, the risk of allergic contact dermatitis
pathways.13 The direct peptide reactivity assay (DPRA)14−16
is a critical issue in the development of new chemicals. For the
and the amino acid derivative reactivity assay14,17 can be used
protection of workers and consumers, skin-sensitizing
potentials of chemicals in manufacturing processes, including
raw materials, intermediates, and products, must be assessed at
an early stage of product development. It is known that some
unexpected minor impurities with strong skin-sensitizing
properties are generated as byproducts and can contaminate
the nonsensitizing target product, which can then give the
entire sample strong skin-sensitizing properties.1 In such cases,
it is not easy to identify the component that is the main cause
of skin sensitization. In previous research with the commercial
dye Sudan III, each component was fractionated by column
chromatography and skin sensitization with each fraction was
evaluated to identify the main skin sensitizer.1 Other studies
have also used chromatographic separation before evaluation
to identify the skin-sensitizing components in a German
chamomile (Matricaria chamomilla) extract2 and a Commi-
phora myrrha (myrrh) extract used as a natural skin remedy.3
The guinea pig maximization test,4−6 Buehler test,4,7,8 and
the murine local lymph node assay9−11 have been validated by
the Organization for Economic Co-operation and Develop-
ment as animal tests for evaluating skin sensitization. In recent
years, because of increasing public awareness of animal welfare,
the use of test animals for scientific purposes has been
to assess the skin-sensitizing potentials of chemicals on the
basis of key event 1 of covalent binding of the chemical to skin
proteins. These tests evaluate the reactivity of chemicals
toward model synthetic peptides as a surrogate for proteins.
The KeratinoSens18,19 and LuSens18,20 tests have been
designed around key event 2, the activation of keratinocytes,
to evaluate activation of the Keap1-Nrf2-ARE pathway by
oxidative stress in keratinocytes. Another set of tests are based
on a third key event, the activation of dendritic cells, and
include the human cell line activation test (h-CLAT),21,22 U-
SENS,21,23 and IL-8 Luc assay.21,24 All types of testing,
including both animal tests and alternative methods, require
time-consuming steps for chromatographic fractionation,
Received: August 6, 2020
Published: December 1, 2020
https://dx.doi.org/10.1021/acs.chemrestox.0c00327
© 2020 American Chemical Society
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Figure 1. Synthetic scheme for NBD-GSH.
benzenesulfonic acid (TNBS) sodium salt dihydrate (purity ≥98.0%),
formaldehyde solution (35% solution, w/w), methyl salicylate (purity
≥98.0%), phenol (purity ≥99.0%), sodium dodecyl sulfate (≥95.0%),
sodium dihydrogen phosphate (purity ≥99.0%), and sodium
cyanoborohydride (NaBH3CN, purity ≥95%) were obtained from
FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Acetic
acid (purity ≥99.7%), 1,4-dioxane (purity ≥98.0%), distilled water,
trifluoroacetic acid (purity ≥98.0%), 0.1 mol/L sodium thiosulfate
solution, sodium tungstate dehydrate (purity >98.0%), hydroquinone
(purity ≥99.0%), disodium hydrogen phosphate, and heptahydrate
(≥99.0%) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan).
Acetonitrile (purity >99.5%), benzoquinone (purity >98.0%), and
trans-cinnamaldehyde (purity >97.0%) were obtained from Kanto
Chemical Co., Inc. (Tokyo, Japan). Triethylamine (purity >99.0%),
triisopropylsilane (purity >98.0%), N-(tert-butoxycarbonyl)-1,2-dia-
minoethane (purity >97.0%), 4 mol/L hydrogen chloride in 1,4-
dioxane, tetrabutylammonium bromide (purity >98.0%), 2-mercapto-
benzothiazole (purity >99.0%), trimellitic anhydride (>98.0%), α-
hexylcinnamaldehyde (purity >90.0%), and 2-phenyl-1-propanol
(purity >98.0%) were sourced from Tokyo Chemical Industry Co.,
Ltd. (Tokyo, Japan). Deuterium oxide (99.9% D) and 4-chloro-7-
nitrobenzofurazan (purity >98.0%) were purchased from Sigma-
Aldrich (St. Louis, MO). 1-Ethyl-3-(3-(dimethylamino)propyl)-
carbodiimide was purchased from the Peptide Institute, Inc. (Osaka,
Japan).
Instrumentation. Analysis of adducts with known skin sensitizers
and nonsensitizers was performed using high-performance liquid
chromatography (HPLC, LC-20A, Shimadzu Corporation, Kyoto,
Japan) with fluorescence detection (RF-20AXS, Shimadzu Corpo-
ration). The excitation and emission wavelengths were 470 and 540
nm, respectively. An XBridge C18 column (150 × 4.6 mm i.d., 3.5
μm; Waters Corporation, Milford, MA) was used with a gradient
elution with a mobile phase flow rate of 1.0 mL/min. The mobile
phase was a mixture of 0.1% acetic acid in water (mobile phase A) and
0.1% acetic acid in acetonitrile (mobile phase B). The initial mobile
phase composition was 95% of mobile phase A and 5% of mobile
phase B, and the percentage of mobile phase B was increased linearly
to 95% over 20 min. An Applied Biosystems QSTAR XL mass
spectrometer (Thermo Fisher Scientific, Waltham, MA) was used for
LC-MS of adducts of known skin sensitizers. The LC-MS experiments
were carried out under the following conditions: electrospray
ionization mode, positive; capillary voltage, 5.5 kV; ion source
temperature, 300 °C; and mass range, m/z 50−1200. For detecting
and identifying minor skin-sensitizing impurities in the reaction
mixture, a Waters ACQUITY UPLC with a FLR fluorescence detector
evaluation of the skin-sensitizing potential of each fraction, and
identification of the main skin-sensitizing substance.
Some methods that use peptides to trap skin sensitizers in a
mixture have been reported. In one study, the synthetic
peptide Ac-RFAACAA, the cysteine peptide in DPRA, was
incubated with a commercial fragrance (Azurone) and an
adduct with unexpected skin-sensitizing impurity was identified
by liquid chromatography-mass spectrometry (LC-MS).25
Glutathione (GSH, γ-glutamyl cysteinylglycine) is an endog-
enous tripeptide containing a thiol group. It plays a central role
in biological detoxication by trapping electrophilic xenobiotics
and their metabolites via its thiol group under catalysis by
glutathione-S-transferase.26 With use of this property of GSH,
a method for screening chemicals for their skin-sensitization
potentials was developed on the basis of the detection of GSH
adducts of the analytes by matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry and LC-MS.27 In
addition, dansyl glutathione has been widely used as a trapping
agent for identifying reactive metabolites in pharmaceutical
drug development.28 However, this method was designed for
trapping soft electrophilic skin sensitizers on thiol groups and
are not suitable for hard electrophiles such as aldehydes.
In this study, we designed a new trapping agent to identify a
wide variety of skin-sensitizing impurities. For this purpose, we
synthesized a fluorescent, nitrobenzoxadiazole (NBD)-labeled
GSH (NBD-GSH) with two reactive sites: a thiol group for
soft electrophiles and an amino group for hard electrophiles.
Because of its fluorescent group, both NBD-GSH itself and its
adducts can be selectively detected by a fluorescence detector.
This report describes the synthesis of NBD-GSH, reactivity of
NBD-GSH with known skin sensitizers, limit of detection of
NBD-GSH, and trapping and identification of skin-sensitizing
byproducts in a reaction mixture.
EXPERIMENTAL PROCEDURES
■
Materials. Glutathione (purity ≥99.0%), di-tert-butyl dicarbonate
(purity ≥97.0%), tetrahydrofuran (purity ≥99.5%), hydrogen
peroxide (35% aqueous solution, w/w), ethyl acetate (purity
≥99.5%), 1-chloro-2,4-dinitrobenzene (DNCB, purity ≥99.0%),
glutaraldehyde (50% aqueous solution w/w), p-phenylenediamine
(purity ≥97.0%), maleic anhydride (purity ≥99.0%), 2,4,6-trinitro-
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Figure 2. Structures of the tested compounds.
(Waters Corporation, Milford, MA) and ZORBAX Eclipse XDB-C18
column (50 × 3 mm, 1.8 μm; Agilent Technologies, Santa Clara, CA)
was directly hyphenated with a Waters ACQUITY TQD mass
spectrometer. The composition of the mobile phase of water/
acetonitrile was increased linearly from 95:5 (v/v) to 5:95 (v/v) in
4.33 min. The mobile phase flow rate was 0.86 mL/min. The LC flow
was split before the mass spectrometer with a split ratio of 4:1. A
Waters ACQUITY UPLC H-class and a Waters Synapt G2-Si mass
spectrometer equipped with a Waters ACQUITY UPLC BEH C18
column (100 × 2.1 mm, 1.7 μm) were used to obtain high-resolution
mass spectra for structure elucidation of the NBD-GSH adducts and
skin-sensitizing impurities in the reaction mixture. The chromato-
graphic conditions used for the high-resolution MS were the same as
those for LC-MS with fluorescence detection. High-resolution mass
spectra were recorded under the following conditions: electrospray
ionization mode, positive; capillary voltage, 0.25 kV; ion source
temperature, 100 °C; desolvation gas temperature, 450 °C;
desolvation gas flow rate, 1200 L/h; and mass range, m/z 50−1500.
Masslynx software (version 4.2, Waters Corporation) was used for
data analysis. A Bruker AVANCE II 600 MHz NMR spectrometer
the surface of the diamond ATR crystal and pressure was applied
using a metal rod to obtain the spectrum. The spectral range was
350−4000 cm−1. A F-2700 fluorescence spectrophotometer (Hitachi
High-Tech Science Corporation, Tokyo, Japan) was used. The
absorption spectrum of a NBD-GSH solution in water/acetonitrile
(1:1, v/v) with the concentration of 4 × 10−6 mol/L was obtained
from 220 to 730 nm. The excitation wavelength was set to 470 nm
and the emission wavelength was scanned from 220 to 730 nm in 0.5
nm increments.
Synthesis of Fluorescent NBD-GSH. NBD-GSH was synthe-
sized from GSH as shown in Figure 1. 1H NMR (600 MHz, D2O): δ
8.30 (br d, J = 9.0 Hz, 1H, benzofurazan H-5), 6.21 (d, J = 9.0 Hz,
1H, benzofurazan H-6), 4.33 (t, J = 6.6 Hz, 1H, Cys-α-methine), 3.90
(t, J = 6.6 Hz, 1H, Glu-α-methine), 3.78 (s, 2H, Gly-α-methylene),
3.61 (br s, 2H, −CH2CH2−), 3.51 (br t, J = 6.6 Hz, 2H,
−CH2CH2−), 2.74 (d, J = 6.0 Hz, 1H, Cys-β-methylene), 2.51−
2.40 (m, 2H, Glu-γ-methylene), 2.15−2.05 (m, 1H, Glu-β-
methylene). 13C NMR (100 MHz, D2O): δ 174.5, 172.4, 172.2,
171.7, 146.4, 144.2, 143.8, 139.0, 120.5, 99.8, 55.7, 52.8, 42.7, 42.5,
38.0, 30.8, 25.5, 25.2. HRMS (ESI+): calcd for C18H25N8O8S [M +
H]+, 513.1516; found, 513.1519. IR: 3276, 3072, 2946, 1641, 1618,
1590, 1525, 1413, 1354, 1242, 1187, 1132, 836, 721, 594, 517, 448
cm−1. The detailed procedure for the preparation of NBD-GSH is
given in the Supporting Information, section 1.1−1.5.
1
(Bruker BioSpin Corporation, Billerica, MA) was used for H NMR
measurements. The ATR FTIR spectra were measured using a Jasco
FT/IR-6300 spectrometer (Jasco Corporation, Tokyo, Japan)
equipped with a single reflection diamond ATR accessory (ATR
Pro One, Jasco Corporation). Aliquots of the samples were placed on
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a
Table 1. Comparison of Skin -Sensitizing Activities and NBD-GSH Adduct Formation for Different Compounds
skin-sensitizing activity
chemical
benzoquinone
1-chloro-2,4-dinitrobenzene
glutaraldehyde
hydroquinone
p-phenylenediamine
maleic anhydride
guinea pig maximization test
local lymph node assay
DPRA
NBD-GSH adduct
references
+
+
N/A
+
+
+
N/A
+
+
+
+ extreme
+ extreme
+ strong
+ strong
+ strong
+ strong
+ strong
+ strong
+ moderate
+ moderate
+ moderate
+ weak
+
+
+
+
+
+
+
+
+
+
+
−
−
N/A
−
D
D
D
D
D
D
D
D
D
D
D
D
ND
ND
ND
11,36
11,36
36
11,36
11,36
35,36
38
11,36
11,36
11,36
11,36
11,36
11,36
37
trinitrobenzenesulfonic acid
formaldehyde
2-mercaptobenzothiazole
trans-cinnamaldehyde
trimellitic anhydride
α-hexylcinnamaldehyde
methyl salicylate
+
+
−
N/A
−
phenol
sodium dodecyl sulfate
−
−
b
−
11,36
a
b
Symbols and abbreviations: +, sensitizing; −, nonsensitizing; N/A, not available; D, detected; ND, not detected. Characterized as a false positive
in LLNA.39
Figure 3. Chromatograms from liquid chromatography with fluorescence detection of incubated solutions of 1-chloro-2,4-dinitrobenzene (DNCB),
formaldehyde (FA), trinitrobenzenesulfonic acid (TNBS), and hydroquinone (HQ) with NBD-GSH.
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Incubation of NBD-GSH with Known Skin Sensitizers and
Nonsensitizers. NBD-GSH was dissolved in 0.2 mol/L phosphate
buffer (pH 7.0) at a concentration of 20 μmol/mL (0.6 mg/mL). The
test chemicals, 12 skin sensitizers, and 3 nonsensitizers were dissolved
in acetonitrile or acetonitrile/water (1:1, v/v) at 20 μmol/mL. NBD-
GSH and the chemical solutions were then mixed in a 1:1 molar ratio
(NBD-GSH/chemical). After incubation for 1 h at 37 °C, the reaction
mixtures were analyzed.
produced new peaks, whereas the 3 skin nonsensitizers did
not give any peaks. The prohaptens hydroquinone and p-
phenylenediamine did not form adducts with NBD-GSH
within the 1 h reaction time. Adduct peaks were detected when
the reaction time was extended to 24 h. It was presumed that
benzoquinone and quinonediimine formed in the reaction
solution by autoxidation.33,34
Limit of Detection of NBD-GSH Adducts with Skin
Sensitizers. For an estimation of the limit of detection, 1-chloro-
2,4-dinitrobenzene was dissolved in acetonitrile in the range from 4 ×
10−5 to 4 μmol/L. These solutions were reacted with the NBD-GSH
solution (4 μmol/L). Adduct peaks were detected using a
fluorescence detector at 540 nm (λem = 470 nm). The limit of
detection was calculated using the signal-to-noise ratio method.29 The
magnitude of the noise and the adduct peak height were measured
manually on the chromatogram and a signal-to-noise ratio of 3 was
used for the limit of detection.
Incubation of NBD-GSH with a Reaction Mixture. According
to the procedure described by Giacomini and co-workers,30 the
oxidation of 2-phenyl-1-propanol was carried out to obtain 2-
phenylpropionic acid, which is a substructure of ibuprofen. A mixture
of 2-phenyl-1-propanol (1.36 g, 10 mmol), 35% aqueous H2O2 (0.2
mL), Na2WO4·2H2O (33 mg, 0.1 mmol), tetrabutylammonium
bromide (32 mg, 0.1 mmol), and 10 mol % KHSO4 was stirred at
1000 rpm for 4 h at 90 °C. After the reaction was quenched with
sodium thiosulfate, 40 μL of the obtained organic layer was diluted
10-fold with acetonitrile. Then 50 μL of the diluted solution was
reacted for 10 min at 37 °C with 50 μL of a 4 mmol/L solution of
NBD-GSH in 100 mmol/L phosphate buffer (pH 7.0)/acetonitrile
(1:1). Finally, 10 μL of 400 mmol/L NaBH3CN aqueous solution was
added and the mixture was reacted for 50 min at 37 °C to trap any
skin-sensitizing byproducts. The reactant was analyzed by LC-MS
with fluorescence detection.
The results are summarized in Table 1. Results reported for
guinea pig maximization tests, local lymph node assays, and
DPRA results in previous research studies are provided for
comparison.
LC-MS Structure Confirmation of the Adducts of
Known Skin Sensitizers with NBD-GSH. The structures of
the NBD-GSH adducts with skin sensitizers were confirmed by
LC-MS. Chromatograms of the reaction mixtures of DNCB,
formaldehyde, and TNBS with NBD-GSH are shown in Figure
3.
DNCB reacted completely with NBD-GSH within 1 h and a
single adduct peak was observed. The adduct peak gave m/z
679 in LC-MS, and its isotope peak pattern did not indicate
the presence of a chlorine atom in the molecule. Therefore, the
chlorine atom of DNCB was substituted by NBD-GSH (Figure
4).
RESULTS AND DISCUSSION
■
Synthesis and Characterization of NBD-GSH. NBD-
GSH was synthesized from GSH in five steps with a total yield
of 12%. It is known that thiol groups and primary amino
groups in proteins react with electrophilic skin sensitizers.31
Therefore, in the DPRA method, the cysteine peptide with a
thiol group and the lysine peptide with a primary amino group
were used for trapping different types of skin sensitizers.14−16
NBD-GSH was designed to contain both a thiol group and a
primary amino group. NBD was bound to the carboxyl group
of GSH via an ethylene diamine linker to retain the free amino
group and reduce steric hindrance of the reactive thiol and
amino group caused by introduction of the NBD group. The
NMR spectrum indicated that NBD was introduced selectively
at the carboxyl group of the glycyl moiety (Figure S1,
Supporting Information). This selectivity might be caused by
steric hindrance of the butoxycarbonyl protective group toward
the glutamyl moiety. NBD-GSH was dissolved in the HPLC
mobile phase H2O/CH3CN (1:1, v/v). The maximum
excitation wavelength was 467 nm and the maximum emission
wavelength was 539 nm (Figure S2). These wavelengths were
used to detect all adducts. The emission wavelength of NBD32
is long enough to prevent overlap with the absorption
wavelengths of a wide variety of chemicals and to give high
selectivity in detection.
Figure 4. Mass spectrum of the NBD-GSH adduct with 1-chloro-2,4-
dinitrobenzene (DNCB). Adduct structure was proposed according to
the molecular weight.
Formaldehyde gave three peaks for adducts formed with
NBD-GSH: one peak (m/z 543) was for an adduct formed via
the thiol group and the other two peaks (m/z 525 and 537)
were for adducts formed via the amino group (Figure 5).
Peaks for three adducts were observed with TNBS: Adducts
A and B (m/z 724) with one TNBS and adduct C (m/z 935)
with two TNBS (Figure 6). These results confirmed that NBD-
GSH had two binding sites, at the thiol group and the primary
amino group, as designed. The LC-MS results for the other
chemicals are shown in Figures S3−S12.
Limit of Detection of the Adduct of NBD-GSH with
DNCB. A DNCB concentration of 6 × 10−8 mol/L in the
acetonitrile solution was required to give a NBD-GSH adduct
peak 3 times the height of the noise by HPLC with
fluorescence detection.
Trapping Known Skin Sensitizers with NBD-GSH.
NBD-GSH was incubated with 12 known skin sensitizers and 3
nonsensitizers (Figure 2) for 1 h to obtain fluorescent adducts.
After the reaction, the obtained reaction mixtures were
analyzed with HPLC with fluorescence detection to confirm
generation of the adduct peaks. All 12 skin sensitizers
Trapping of Skin-Sensitizing Impurities Using NBD-
GSH in a Reaction Mixture from Oxidization of 2-
Phenyl-1-propanol. For obtainment of a phenyl propionic
acid, 2-phenyl-1-propanol was oxidized by hydrogen peroxide
with a tungsten catalyst. The obtained reaction mixture was
reacted with NBD-GSH for 1 h to trap unknown skin-
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Figure 6. Mass spectra of the NBD-GSHs adducts with
trinitrobenzenesulfonic acid (TNBS). Adduct structures were
proposed according to the molecular weight.
adduct formation (m/z 629) occurred without using a
reducing agent, the amount formed was very small because
of the equilibrium between the amine and imine. Therefore,
for breaking of the chemical equilibrium between the amine
and imine and for production of the reduced adduct of NBD-
GSH irreversibly, a reducing agent41 was added following a
previous report.42 Even a small amount of an aldehyde
generated as a strong skin-sensitizing impurity can give the
final product strong skin-sensitizing properties; thus, the
addition of a reducing agent is of great importance to allow
for detection of the reduced adduct of NBD-GSH with high
sensitivity and for identification of the cause of skin
sensitization.
CONCLUSIONS
■
A method was developed for identifying small amounts of
impurities with skin-sensitizing potential in reaction mixtures.
Fluorescent NBD-GSH was synthesized from commercial
GSH in five steps with a total yield of 12%. NBD-GSH formed
adducts with 12 known skin sensitizers through its thiol group
and/or amino group. These adducts were detected selectively
using a fluorescence detector. The limit of detection of this
method was determined as 6 × 10−8 mol/L of DNCB in the
acetonitrile solution. For evaluation of the practicality of this
method, a reaction mixture from alcohol oxidation of 2-
phenylpropionic acid was assessed using the NBD-GSH
binding assay. Under fluorescence detection, the obtained
mixture produced a peak for a NBD-GSH adduct with skin-
sensitizing potential impurity. Analysis of the peak by LC-MS
analysis with fluorescence detection and HPLC-MS identified
2-phenyl-1-propanal as the skin-sensitizing impurity in the
reaction mixture. This method eliminates the requirement for
chromatographic separation and assessment of each fraction
using animal or nonanimal tests to identify the main cause of
Figure 5. Mass spectra of the NBD-GSH adducts with formaldehyde
(FA). Adduct structures were proposed according to the molecular
weight.
sensitizing byproducts and then analyzed by LC-MS with
fluorescence detection. One peak that was considered a
fluorescent NBD-GSH adduct with unknown skin-sensitizing
impurities was detected (Figure 7).
The mass spectrum of the adduct (m/z 631) and its
elemental composition, C27H34N8O8S, from a high-resolution
mass spectrum strongly suggested that the reduced adduct was
formed by reaction of the amino group of NBD-GSH with the
aldehyde byproduct 2-phenyl-1-propanal, which is known to be
a skin sensitizer.38−40 This aldehyde formed as an intermediate
between the corresponding alcohol and carboxylic acid and
remained in the reaction system as an impurity. Although
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Figure 7. Trapping of skin-sensitizing impurities by NBD-GSH from the reaction mixture for oxidization of 2-phenyl-1-propanol. (A)
Chromatogram from liquid chromatography with fluorescence detection of the incubated solution of the reaction mixture with NBD-GSH and
NaBH3CN. (B) Mass spectrum of the NBD-GSH adduct with the skin sensitizer 2-phenyl-1-propanal in the reaction mixture. The adduct structure
was proposed according to the molecular weight.
the skin-sensitizing property in the mixture. Consequently, it
will be useful for identifying small amounts of skin sensitizers
in raw materials, intermediates, reaction mixtures, and end
products in the chemical industry.
Masayuki Sato − Environmental Health Science Laboratory,
Sumitomo Chemical Co., Ltd., Osaka 554-8558, Japan
Masahiko Okamoto − Environmental Health Science
Laboratory, Sumitomo Chemical Co., Ltd., Osaka 554-8558,
Japan
ASSOCIATED CONTENT
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.chemrestox.0c00327
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.chemrestox.0c00327.
Funding
The authors received no financial support for this research.
Detailed procedure of NBD-GSH synthesis; introduc-
tion of NBD to GSH; fluorescence spectra of NBD-
GSH; chromatograms from liquid chromatography with
fluorescence detection; and LC mass spectra of the
NBD-GSH adducts (PDF)
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Mr. Kohei Atsumi for his assistance with infrared
spectrometry, Ms. Akiko Sumi and Ms. Kayo Fujita for help
with the HPLC analysis, and Dr. Mitsunobu Kawamura and
Mr. Daisuke Sasayama for their advice about the oxidation
reactions.
AUTHOR INFORMATION
■
Corresponding Author
Takashi Tokunaga − Environmental Health Science
Laboratory, Sumitomo Chemical Co., Ltd., Osaka 554-8558,
Japan; orcid.org/0000-0002-9876-0563;
Email: tokunagat2@sc.sumitomo-chem.co.jp
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Gaku Yamamoto − Environmental Health Science Laboratory,
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Teruki Takahashi − Environmental Health Science
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Makiko Mukumoto − Environmental Health Science
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