Article
pubs.acs.org/ac
Untargeted Identification of Organo-Bromine Compounds
in Lake Sediments by Ultrahigh-Resolution Mass Spectrometry
with the Data-Independent Precursor Isolation and Characteristic
Fragment Method
Hui Peng,*,† Chunli Chen,† David M. V. Saunders,† Jianxian Sun,† Song Tang,‡ Garry Codling,†
Markus Hecker,†,‡ Steve Wiseman,† Paul D. Jones,†,‡ An Li,⊗ Karl J. Rockne,$ and John P. Giesy*,†,§,∥,⊥,#,○
†
Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, Saskatchewan Canada, S7N 5B3
School of Environment and Sustainability, 117 Science Place, Saskatoon, Saskatchewan Canada, S7N 5C8
§
Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan Canada S7N 5B3
∥
Zoology Department, Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824United States
⊥
School of Biological Sciences, University of Hong Kong, Hong Kong Special Administrative Region, Peoples Republic of China
#
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023,
People’s Republic of China
○
Biology Department, Hong Kong Baptist University, Hong Kong, Special Administrative Region, China
⊗
School of Public Health, University of Illinois at Chicago, Chicago, Illinois 60612, United States
$
Department of Civil and Materials Engineering (MC 246), University of Illinois at Chicago, 842 West Taylor Street, Chicago,
Illinois 60607-7023, United States
‡
S Supporting Information
*
ABSTRACT: While previous studies have found that unknown natural and synthetic organo-bromine compounds (NSOBCs)
contributed more than 99% of the total organic bromine (Br) in the environment, there was no efficient method for untargeted
screening to identify NSOBCs in environmental matrixes. A novel untargeted method for identifying NSOBCs, based
on ultrahigh-resolution mass spectrometry (UHRMS) with the Q Exactive instrument was developed. This method included
a data-independent precursor isolation and characteristic fragment (DIPIC-Frag) procedure to identify NSOBCs. A total of
180 successive 5-m/z-wide windows were used to isolate precursor ions. This resulted in a sufficient dynamic range and
specificity to identify peaks of Br fragment ions for analysis. A total of 2520 peaks of NSOBC compounds containing Br were
observed in sediments from Lake Michigan, United States. A new chemometric strategy which combined chromatographic
profiles, isotopic peaks, precursor isolation window information, and intensities was used to identify precursor ions and chemical
formulas for detecting NSOBCs. Precursor ions for 2163 of the 2520 NSOBCs peaks (86%) were identified, and chemical
formulas for 2071 NSOBCs peaks (82%) were determined. After exclusion of isotopic peaks, 1593 unique NSOBCs were
identified and chemical formulas derived for each. Most of the compounds identified had not been reported previously and had
continued
Received: April 16, 2015
Accepted: September 17, 2015
Published: September 17, 2015
© 2015 American Chemical Society
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intensities which were 100- to 1000-fold greater than the congeners of polybrominated diphenyl ethers (PBDEs). In extracts of
sediments, these compounds exhibited variations in intensities (<103 to ∼108), m/z values (170.9438−997.5217), retention times
on a C18 column (1.0−29.3 min), and the number of Br atoms (1−8). Generally, compounds with greater m/z values had longer
retention times and greater numbers of Br atoms. Three compounds were used in a proof-of-concept experiment to demonstrate
that structures of some of the screened NSOBCs could be further predicted by combining searching of database libraries and highresolution MS2 spectra.
N
and (3) due to the presence of large numbers of potential
interferences in environmental samples, specificity of methods
would need to be enhanced, which would increase the complexity
of data analysis (specificity).
To address these challenges, the goal of this study was to
develop a data-independent precursor isolation and characteristic
fragment (DIPIC-Frag) method to screen NSOBCs in environmental samples. This method was incorporated in the operation
of liquid chromatography (LC) coupled to an ultrahigh-resolution
mass spectrometer (UHRMS), the Q Exactive quadrupole,
Orbitrap MS, with atmospheric pressure chemical ionization
(APCI). To analyze multiplexed data sets produced by UHRMS,
a novel data mining strategy was developed to identify precursor
ions and predict chemical formulas or chemical structures of
NSOBCs. The method exhibited comprehensive coverage of
chemical structure diversity, large dynamic range, and specificity
and was successfully used to detect and identify 2 520 peaks
associated with NSOBCs in sediments from Lake Michigan,
the sole Laurentian Great Lake lying completely within the
United States.
atural and synthetic, organo-bromine compounds (NSOBCs)
are of concern due to their environmental persistence,
bioaccumulation, and potential for toxicity. Well-known NSOBCs,
such as polybrominated diphenyl ethers (PBDEs) and their
hydroxylated (OH-BDEs) and methoxylated (MeO-BDEs)
analogues have been reported to be ubiquitous in environmental
matrixes,1−3 wildlife,4,5 and humans.6−8 Results of both epidemiological investigations and controlled experiments suggest that
exposure to synthetic brominated compounds can cause various
adverse effects to humans and wildlife.7,9,10 Some naturally occurring, brominated compounds exhibit even greater toxic potencies
than synthetic compounds. For instance, OH-BDEs, which have
been reported to bind to the thyroid hormone receptor (THR),
exhibit greater potencies for neurotoxicity than their analogous
PBDEs.11,12 Identification and quantification of these NSOBCs in
the environment is therefore important for assessment of potential
effects on health of humans and/or wildlife.
In natural waters, sediment can be a large depository of
persistent environmental pollutants. For chemicals that are
persistent and relatively hydrophobic, enrichment from water
into sediment often enables their detection and quantitation at
trace levels.3,13,14 Various NSOBCs such as PBDEs and some
other brominated flame retardants have been detected in sediments with concentrations commonly in the ∼ng/g, dry mass
(dm) range.13−16 However, concentrations of total organic
bromine (TOB) in samples of marine organisms and sediment
have been found to be in the ∼μg/g range,17 which suggests that
currently known and concerned NSOBCs contribute <0.1% to
TOB. Thus, identities of most TOB in sediments were unknown.17
Targeted ion monitoring, using single or triple quadrupole
mass spectrometry (MS) coupled to liquid (LC) or gas (GC)
chromatography is currently the main strategy to identify and
quantify organic compounds for which standards are available.18−20 To provide maximum selectivity and sensitivity, in
targeted ion monitoring, only characteristic ions or ion transitions
of targeted analytes are monitored. In contrast, ion-trap and timeof-flight (TOF) MS techniques are superior for screening of
unknown compounds.21 For example, an untargeted-method
using a scripting approach combined with GC × GC-TOF
MS has been developed and successfully used to identify new
chloro/bromo-carbazole compounds.22 However, in addition to
synthetic brominated compounds and their byproducts, more
than 2 200 natural brominated compounds produced by marine
organisms have been identified.23 Because of the number of
potential NSOBCs and the current difficulty in identifying novel
compounds, a more robust, untargeted method to identify
NSOBCs in the environment is needed. However, screening
NSOBCs in environmental samples faces several major challenges
including (1) NSOBCs exhibit a wide range of physical-chemical
properties which may lead to poor volatility for GC/MS or
low ionization efficiency for LC−MS, thus making it difficult
to develop a single, robust mass spectrometric method for all
potential compounds (coverage); (2) concentrations of individual NSOBCs can span several orders of magnitude (dynamic
range), thus necessitating a method with a large dynamic range;
■
MATERIALS AND METHODS
Chemicals and Reagents. Authentic standards of 10 native
PBDEs, three OH-BDEs, three MeO-BDEs, three diastereoisomes
of hexabromocyclododecane (HBCDs), tetrabromobisphenol A
(TBBPA), bis (2-ethylhexyl)-2,3,4,5-tetrabromophtalate (TBPH),
and 2-ethylhexyl-tetrabromobenzene (TBB) were purchased
from Wellington Laboratories Inc. (Guelph, ON, Canada).
5-Bromoindole and 4-bromophenol were purchased from SigmaAldrich Chemical Co. (St. Louis, MO). 1,3,6,8-Tetrabromocarbazole
was purchased from Toronto Research Chemicals Inc. (Toronto,
ON, Canada). Hydroxylated TBB (OH-TBB) and OH-TBPH were
purified from BZ-54 technical product as previously described.24
Florisil (6 cm3, 1 g, 30 μm) solid-phase extraction (SPE) cartridges
were purchased from Waters (Milford, MA). Methyl tert-butyl
ether (MTBE), dichloromethane (DCM), hexane, methanol, and
acetone were all of omni-Solv grade and were purchased from
EMD Chemicals (Gibbstown, NJ).
Collection of Sediments. Surface sediment samples were
collected from two locations in Lake Michigan in September 2010
(sampling map is shown in Figure S1, Supporting Information)
using a PONAR grab sample, as described previously.25 Samples
were separated into aliquots and stored in amber glass jars with
aluminum foil liner caps. Samples were transported on ice
and stored in the dark at −20 °C. Each sample was lyophilized,
manually homogenized, and passed through a 1 mm sieve.
Sample Pretreatment. Approximately 10 g of sediment was
extracted for identification of NSOBC. Methods for extraction
have been described previously.17 Briefly, samples were extracted
by use of an accelerated solvent extractor (Dionex ASE-200,
Sunnyvale, CA). Two solvents were used in the extraction: (1)
n-hexane/DCM (1:1) at 100 °C and 1500 psi, and (2) n-hexane/
MTBE (1:1) at 60 °C and 1000 psi. Two extraction cycles
(10 min each) were performed for each solvent per sample
(approximately 50 mL for each solvent). Following extraction,
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Instrument detection limits (IDLs) for the model chemicals
were defined as 5 times within 20% relative standard derivation
for the standards. Method detection limits (MDLs) for the
model chemicals were calculated based on six replicate analyses
of sample extracts at a concentration of approximately five times
the corresponding IDLs, and then MDLs were calculated as
previously described.26 Recoveries were determined by spiking
25 model NSOBCs into samples of sediment at 50 ng/g dw (n = 3).
The recoveries for these compounds ranged from 73 ± 7%
(BDE-183) to 98 ± 12% (OH-TBPH).
Formula Elucidation. Elemental compositions of detected
NSOBCs were calculated using a program written for R software
in which the mass tolerance was set to 5 ppm for compounds
greater than 200 m/z. Chemical formulas were set to contain
up to 100 C, 200 H, 5 N, 30 O, 5 I, and 2 S per molecule. The
number of Br or Cl atoms was constrained based on information
from isotopic peaks. All assigned formulas were required to meet
basic chemical criteria as described previously.27
Distribution of Intensities of Isotopic Peaks. Since the
pattern of isotopic peaks is important to narrow the list of
potential formulas for a given exact mass, numbers of bromine
and chlorine atoms in detected NSOBCs were calculated based
on patterns of isotopic peaks.28 Details of the method for making
the calculations are provided in the Supporting Information.
fractions were combined. Volumes of extracts were reduced to
∼1 mL by rotary evaporation and loaded onto Florisil cartridges
which had been previously conditioned by 6 mL of DCM.
NSOBCs were eluted from Florisil cartridges by use of 5 mL of
DCM and then 5 mL of methanol. Final extracts were blown to
dryness under a gentle stream of nitrogen and reconstituted in
400 μL of acetone. Acetone was selected as the reconstitution
solvent, considering the hydrophobicity of identified NSOBCs.
Because a limited number of NSOBCs were detected in
methanol fractions (data not shown), only the DCM fraction
from the cartridges were collected for screening of NSOBCs. The
use of Florisil cartridges, which have also been used in previous
sample pretreatments for halogenated compounds analysis,24
allowed for the removal of most of the yellow interferences in
extracts of sediment. Such a simple one-step sample cleanup
method was useful for untargeted screening of NSOBCs in
sediments to avoid potential loss of compounds, but more
efficient sample pretreatment methods such as gel permeation
chromatography (GPC) would be warranted if the DIPIC-Frag
method was applied to more complicated matrixes such as biotic
samples. To avoid potential background contamination during
sample pretreatment, all equipment rinses were carried out with
acetone and hexane, and procedural blank experiments were performed along with each batch of samples. A total of 113 NSOBC
peaks were detected in the blank, partly due to instrument carryover, but the peak abundances of these NSOBCs were at least
10-fold less than those of sediments samples. The background
contamination from the blanks was subtracted from sediment
samples for downstream data analysis.
LC-Q Exactive Data Acquisition. Aliquots of extracts were
analyzed using a Q Exactive UHRMS equipped with a Dionex
UltiMate 3000 UHPLC system (Thermo Fisher Scientific).
Separation of NSOBCs was compared among different types
of HPLC columns, and the Hypersil GOLD C18 column (3 μm;
2.1 mm × 50 mm; Thermo Fisher Scientific) was selected for
the present method considering the good separation ability
and sensitivity achieved with its use. Injection volume was 5 μL.
Ultrapure water (A) and methanol (B) were used as mobile
phases. Initially 20% B was increased to 80% in 3 min, then
increased to 100% at 8 min and held static for 19.5 min, followed
by a decrease to initial conditions of 20% B and held for 2 min
to allow for equilibration. Rate of flow was 0.20 mL/min. The
column and sample compartment temperatures were maintained
at 30 and 10 °C, respectively.
Data were acquired in data-independent acquisition (DIA) mode.
Parameters for DIA were one full MS1 scan (150−2 000 m/z)
recorded at resolution R = 70 000 (at m/z 200) with a maximum
of 3 × 106 ions collected within 100 ms, followed by six DIA
MS/MS scan recorded at a resolution R = 35 000 (at m/z 200)
with maximum of 1 × 105 ions collected within 60 ms. DIA data
were collected by use of 5-m/z-wide isolation windows per
MS/MS scan, although different combinations of isolation
windows could be used in future work. Each DIA MS/MS scan
was chosen for analysis from a list of all 5 m/z isolation windows.
In these experiments, 180, 5-m/z-wide windows between 100 and
1 000 m/z were grouped into nine separate methods, each of
which contained 20 windows. Small overlaps with neighboring
windows were used to reduce the likelihood of placing window
edges on critical target peaks. Mass spectrometric settings for
APCI (−) mode were as follows: discharge current, 10 μA;
capillary temperature, 225 °C; sheath gas, 20 L/h; auxiliary gas,
5 L/h; probe heater temperature, 350 °C.
■
RESULTS AND DISCUSSION
Principles and Workflow of the DIPIC-Frag Method. To
address challenges for identifying NSOBCs, an untargeted
DIPIC-Frag method was developed by combining several
techniques to address the challenges including coverage, dynamic
range, and specificity (workflow was shown in Figure 1). To test
the performance of the DIPIC-Frag method, 25 model NSOBCs
including 19 synthetic chemicals and 6 natural products were used
(Supporting Information Table S1). These compounds had
diverse chemical structures ranging from hydrophilic phenolic
compounds (e.g., TBBPA) to highly hydrophobic compounds
(e.g., BDE-209). To increase coverage of NSOBCs, APCI (−)
was used. Following optimization, all 25 model NSOBCs showed
sufficient sensitivity (IDLs ranged from 5 ng/L to 20 μg/L),
including the hydrophobic BDE-209. The MDLs were in the
range of <1 to 10 000 pg/g, dm and were less than concentrations
of the most well-known NSOBCs (typically ng/g) and also total
organic bromine (typically μg/g) in sediments. Thus, the DIPICFrag method would likely be sufficiently sensitive to identify
unknown NSOBCs in environmental samples. Compared to
the traditional GC/MS method, which is not compatible with
compounds of lesser volatility, and LC−ESI-MS method, which is
not compatible with less polar compounds, the use of APCI (−)
increased coverage of unknown NSOBCs of diverse chemical
structures. In addition, compared with the electron impact (EI)
ionization source used in GC/MS in which molecules are often
cleaved to fragments, the LC compatible APCI source is relatively
“softer” and preserves parent molecular ions, which allowed for
the calculation of molecular mass and chemical formulas.
To address the issue of specificity of NSOBCs and distinguish
from other interferences, characteristic fragment ions were
used to identify peaks associated with NSOBCs. Although the
25 model NSOBCs had diverse chemical structures, they all
produced a Br fragment ion (m/z = 78.9171) in negative mode at
relatively high HCD collision energies (>30 eV) (typical product
ion spectra of OH-TBB was shown in Figure S2). Preferential
cleavage of Br and its greater electronegativity has been well
documented in traditional GC/MS methods and enables greater
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Figure 1. Typical workflow of the DIPIC-Frag method to identify brominated compounds: (a) 180 successive 5-m/z-width precursor isolation windows
were used in the DIPIC-Frag method. (b) Bromine fragment peaks were detected in each precursor isolation window, with an average of 15 peaks
detected for each window. (c) Mass spectra in separated DIA windows at the same retention time as the bromine fragment peaks for precursor ions
alignment. Collisional energy was set to a wide range (10, 30, 60 eV) to produce both bromine fragments (left red dashed cycle of the spectra) and
precursor ions (right red dashed cycle of the spectra). (d) The chromatographic elution profiles of bromine fragments (top) and precursor candidates
(bottom) were used to identify precursor ions for each bromine fragment peak. Typically, 1−3 precursor ions were identified from 20 to 30 candidate
ions in the precursor ion region. (e) Isotopic peaks of the proposed precursor ions were further determined to confirm the identity of the precursor ions.
Intensity for each isotopic peak is shown in the top right of the figure. (f) The chemical formula was calculated based on multiple lines of evidence
including number of bromine atoms, intensity information and exact m/z values. (g) Chemical structures of some compounds were identified by
combining database searches and high-resolution MS2 spectra.
sediments had m/z > 1 000, the maximal mass range was set to
1 000 m/z.
Performance of the DIPIC-Frag method was evaluated by its
use to identify NSOBCs in sediments from Lake Michigan.
When extracted with a 7 ppm mass width, multiple characteristic
peaks for Br (m/z = 78.9171) were detected in multiple precursor isolation windows (Figure 1b). Confirmation was obtained
by monitoring a second isotopic peak of Br for the m/z = 80.9150.
Windows for isolation of precursors in the DIPIC-Frag method
were necessary to deconvolute peaks for NSOBCs, since multiple
NSOBCs exhibited similar retention times and thus their Br
fragment peaks could not be efficiently deconvoluted by using
single precursor isolation mode, as has been used previously in
untargeted methods. An intensity cutoff of 1000 was used in the
DIPIC-Frag method, and Br peaks exceeding this threshold
were identified as NSOBCs for subsequent data analysis.
Detected NSOBCs were distributed across multiple precursor
isolation windows and retention times. NSOBCs were detected
in all precursor isolation windows greater than 165 m/z with
retention times of 1.0−29.3 min. The elution of several NSOBCs
at ∼1.0 min indicated they could not be efficiently retained
by a C18 column. Future optimization of HPLC condition is
warranted to enhance coverage of these polar NSOBCs.
Finally, an average of 15 NSOBC peaks were detected in each of
the 180 5-m/z-width precursor isolation windows, with a total
2 520 peaks detected. The DIPIC-Frag method detected more
NSOBCs than did previous untargeted methods, which typically
identified fewer than 100.22
sensitivity with electron capture negative ionization (ECNI)
compared to EI ionization,29,30 which has also been observed
for MeO-BDEs and OH-BDEs using LC−MS/MS.31 Thus, the
[Br] ion can be used as a characteristic product ion to specifically
screen compounds containing Br with the use of APCI (−).
Based on this strategy, NSOBCs peaks could be easily distinguished from other interferences and the method provided
great convenience in subsequent data analysis.
To increase the dynamic range of the method, multiple
successive MS2 windows during the data-independent acquisition (DIA) were used in the DIPIC-Frag method (Figure 1a and
Figure S3). One full scan with a mass resolution of 70 000
followed by six cycles of DIA scans with mass resolutions of
35 000 were performed in the DIPIC-Frag method. A detailed
mass scanning scheme is shown in Figure S3. Performance of
DIPIC-Frag was closely related to the width of precursor
isolation windows. In principle, to reduce coeluted interferences,
narrower isolation windows (e.g., 50 2-m/z-width windows to
cover the mass range, 500−600 m/z) are preferable, compared to
wider isolation windows (e.g., 2 50-m/z windows to cover the
mass range, 500−600 m/z). However, because of the limited
scanning rates of the Q Exactive instrument, use of narrower
isolation windows would limit coverage of masses and decrease
throughput. Following optimization, the width of isolation
windows was set at 5-m/z (Figure 1a and Figure S3) and nine
individual methods with 100 m/z range with 20 windows for each
method and 180 windows for all nine methods with 900-m/z
mass ranges (100−1000 m/z) were used. Because primary
screening experiments showed that few NSOBCs detected in
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Figure 2. Identification of precursor ions by combining full scan spectra and precursor ion region information from separated DIA windows. (a) The
precursor ion at 248.8549 m/z was detected in the precursor ion isolation DIA window but could not be detected in full scan mode due to the high
abundance of interferences (total ion intensity was 6.08 × 109 in the bottom spectra) and limited dynamic range of full scan spectra; (b) ion at
247.8711 m/z was detected in full scan spectra but could not be detected in the precursor ion isolation DIA window; (c) two NSOBC compounds with a
monoisotopic peak at 435.7738 m/z and 433.7586 m/z produced overlapping bromine fragment peaks at a retention time (rt) = 12.40 min. (d)
Consistent with part c, mass spectra at rt = 12.40 min showed two isotopic peak clusters with mass derivations of ∼2 Da. Red arrows indicate
corresponding bromine fragment or precursor ions peaks.
Precursor Ion Alignment. Identification of precursor ions
for each of the 2 520 potential NSOBCs peaks was accomplished
by use of a novel data mining strategy, which was developed by
combining multiple lines of evidence (Figure S4). First, a stepped
collision energy procedure was used at lesser (10 eV) and greater
values (30 and 60 eV) during DIA scans. With this procedure,
precursor ions were fragmented with different energies but
injected to the C-trap for detection at the same time, which
allowed for collection of information on both precursor ions
and Br fragments simultaneously in the same spectrum. In the
portion of spectra (right side of the mass spectra from Figure 1c)
identified as the “precursor ion region”, relatively large signals
from precursor ions between the 5-m/z-width isolation windows
were observed. In addition to an expanded dynamic range,
the use of precursor isolation windows also reduced the time
to identify precursor ions because the width of the window
inherently limits the number of ions to be identified, which
typically contained 20−30 precursor ions candidates. Profiles of
retention times and shapes of peaks during chromatographic
elution of the 20−30 ions in the precursor ion region were
investigated, and the ions showed similar chromatographic
profiles with Br peaks were identified as potential precursor ions
of the corresponding NSOBC peaks. Use of chromatographic
elution profiles for alignment of precursor ions has been applied
previously in metabolomics and proteomics studies.32,33 In the
example shown in Figure 1 (bromoindole), the precursor ion
with m/z of 193.9599 was specifically identified as having
the same chromatographic elution profile as the corresponding
Br fragment peaks (Figure 1d). Information from the precursor
ion region in DIA windows and information from full scan
spectra was integrated to obtain the most accurate precursor ion
information (Figure S4). The m/z values identified in full scan
mode were used because of the greater mass resolution of the
method (R = 70 000 at 200 m/z) compared to separated windows
(R = 35 000 at 200 m/z). Most precursor ions were detected
by both full scan and DIA scan. However, approximately 15%
of lesser-abundance precursor ions were observed only in the
precursor ion region in separated DIA windows (Figure 2a). This
result was expected due to interferences in extracts that exceeded
the dynamic range of the full scan mode. Approximately 10% of
precursor ions were only observed in full scan mode (Figure 2b),
due likely to the greater maximal injection of ions, 3 × 106, in
full scan, which was 30-fold greater than for separated windows
(1 × 105). Following the first two steps, precursor ions were
specifically detected for most NSOBCs. Third, because they had
relatively large abundances and have been previously used to
characterize brominated compounds, isotopic peaks of NSOBCs
were used to further confirm results.22 For bromoindole, two
isotopic peaks (m/z = 193.9599 and 195.9579) were specifically
detected with similar chromatographic elution profiles, which
further confirmed that the ion at m/z = 193.9599 was the
precursor ion to the corresponding Br peaks (Figure 1e). Finally,
theoretically, intensities of precursor ions should be greater than
or similar to that of product ions divided by product ion number
(text in Supporting Information). Thus, intensities of precursor
ions in full scan were also calculated. This calculation can be
useful to identify overlaps of NSOBCs peaks which could not
be completely deconvoluted by DIA windows in a few cases.
For instance, a potential precursor ion with m/z 435.7738 and at
retention time 12.40 min was observed in the window centered at
435 ± 2.5 m/z and had a similar chromatographic elution profile
to corresponding Br fragment peaks (Figure 2c). However,
intensity of the proposed ion was less than that of the Br fragment
divided by Br number and monoisotopic peaks number (details
of the calculation are provided in Supporting Information).
Following inspection of the mass spectra at 12.40 min, another
ion with a m/z of 433.7586 (isotopic ion with m/z of 435.7565)
was observed (Figure 2d), which overlapped with the ion at
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Figure 3. Distribution of identified NSOBC compounds. (a) Distribution of intensities of 2 520 NSOBCs and their comparison to previously known
brominated compounds. Multiple analogues were detected for brominated carbazole, and the bromine number (x + y) ranged from 1 to 7, where x and y
indicate the number of bromines. (b) Distribution of intensities of the 2 520 NSOBC compounds with different ranges of m/z values. (c) Distribution of
the 2 520 NSOBC compounds by retention time and m/z values. The sizes of the dots are proportional to intensities. The colors of dots represent
numbers of bromines. Red represents numbers of bromines, and gray represents those precursor ions whose formula could not be identified. All
NSOBCs were determined in a surficial sediment sample (sed-32) from Lake Michigan.
m/z 435.7738 with a retention time of 12.40 min. This peak was
expected to be the dominant compound producing a Br fragment
at 12.40 min. By combining multiple lines of evidence, precursor
ions for 2 163 peaks corresponding to NSOBCs were identified,
which represented 86% of the 2 520 peaks originally identified as
being due to brominated compounds. Most of the unidentified
NSOBCs had relatively small peak intensities (<1 × 104) and
great molecular mass (>600 m/z). Therefore, their precursor ions
were expected to be obscured by coeluting greater-abundance
interferences. These unidentified NSOBC peaks could potentially
be identified by narrowing the range of the full scan or increasing
the amount of ions injected. Intensities of observed compounds
exhibited a large dynamic range from <103 to ∼108 (Figure 3a).
Such a dynamic range of NSOBCs has posed challenges to
previously used untargeted screening methods but was achieved
by the novel DIPIC-Frag method.
Because of the lack of commercial standards for most observed
compounds, it was not possible to accurately determine their
concentrations. However, peak intensities of NSOBCs were
100−1000-fold greater than several congeners of PBDEs and
OH-PBDEs. Another interesting result from the method is that
multiple isomers were observed for some NSOBCs. Similar
m/z values but with different retention times (Figure 2c) were
observed for some NSOBCs. Considering the resolution of the
Q Exactive, these NSOBCs should be multiple isomers with Br
atoms in various positions or even different chemical backbones,
and further studies are warranted to clarify their exact chemical
structures.34
Determination of Chemical Formulas. To further determine formulas of NSOBCs, a multiple-step strategy was used.
The first step was to use isotopic peaks to calculate numbers
of Br/Cl and to distinguish isotopic peaks from the primary
monoisotopic peak. For example, on the basis of distributions
of isotopes (Figure S5), relative intensities of the two isotopic
peaks in Figure 1e were ∼1:1, which indicated that there was
1 Br atom in the molecular formula. Isotopic peaks of Br
fragments from DIA precursor isolation windows also provided
important information on isotopic peaks observed for precursor
ions. For the Br fragment peak (retention time was 10.52 min)
from the precursor window at 350 ± 2.5 m/z, only the monoisotopic ion at m/z = 78.9171 was detected, which indicated that
the primary monoisotopic ion of the NSOBC precursor ion
should be detected in the second half of the precursor ion region
(350−352.5 m/z) (Figure S6). On the basis of this information,
the precursor ion at m/z 351.8977 was identified. From the next
precursor isolation window of 355 ± 2.5 m/z, the Br fragment
peak was still observed but showed similar intensities for the two
Br fragment ions (m/z = 78.9171 and 80.9151), which indicated
the presence of two or three isotope peaks in these windows
(ions at m/z = 353.8959 and m/z = 355.8939). Taken together,
this information allowed for identification of the monoisotopic
ion of the detected brominated compound, which contained
two Br atoms (C13H8NOBr2), at m/z 351.8977. Similarly, on
the basis of the detection of both the Br isotopic peak of m/z
78.9171 and 80.9151 with similar intensity, the primary
monoisotopic peak of the precursor ion of the NSOBC in
Figure 1e was narrowed to the first half of the isolation window
(192.5−195 m/z), further confirming that the ion with m/z
193.9599 was the primary monoisotopic peak of the NSOBC
compound (bromoindole). Information on numbers of Cl and
Br atoms of the compound was used in a program written by the
authors to calculate the elemental composition of the compounds.
Generally, after constraining the numbers of Br and Cl atoms and
mass tolerance (5 ppm), there were few compounds (<4) included
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Figure 4. Comparison of profiles of NSOBCs between two sediment samples from Lake Michigan: sediment-32 (a) and sediment-44 (b)
(chromatogram was extracted from 405 ± 2.5 m/z DIA window). (c) Intensity ratios of NSOBCs in sediment-44 to those in sediment-32. (d) Intensity
ratios of NSOBCs compounds between two sediment samples and their relationship between retention time.
on lists with m/z less than 600 m/z and fewer yet (≤2) on lists
with m/z less than 400 m/z. However, the list which included
compounds with larger m/z values (>600) was larger. Thus,
compounds with the least mass errors were used to predict
chemical formulas of NSOBCs. For example, after constraining
the number of Br atoms to 1 and for an m/z of 193.9599,
C8H5NBr was identified as the single formula with mass error of
−3.0 ppm (Figure 1f). Finally, by use of this data mining strategy,
chemical formulas were successfully identified for 2 071 of the
2 520 NSOBCs (82%), corresponding to 1593 of unique NSOBC
compounds after excluding isotopic peaks. Detected NSOBCs
had great variations in m/z values (170.9438−997.5217),
retention times (1.0−29.3 min), and numbers of Br atoms
(1−8); yet, these values were clearly associated, since compounds
with larger m/z generally had greater retention times and
numbers of Br atoms (Figure 3c). The smallest NSOBC was
identified as bromophenol (C6H5OBr) with m/z of 170.9438 and
retention time at 6.44 min. As shown in Figure S7, the MS1
spectra, retention time, and MS2 spectra for the DIA window
of the identified bromophenol peak were consistent with results
from the commercially available standard, 4-bromophenol.
Among the nine methods applied to various ranges of masses,
the number of NSOBCs and their intensities increased from the
mass range 100−200 m/z to 400−500 m/z and declined from the
mass range 500−600 m/z to 900−1000 m/z. This trend was
particularly apparent for precursor windows greater than 700 m/z
(Figure 3b). Most compounds detected had m/z values between
300 and 700 and retention times between 8 and 15 min. Masses
and retention times were similar to those of BDE-47 (molecular
mass = 564.7, retention time = 8.7 min), which is one of the most
bioaccumulative synthetic brominated compounds, indicating
the potential bioaccumulation of these unknown NSOBCs in
organisms.
Although identities of dominant NSOBCs were similar in
sediments from the two locations in Lake Michigan, profiles of
detected NSOBCs showed great differences. For example, take
the NSOBCs peaks extracted from the 405 ± 2.5 m/z DIA
window, compound peak 2, peaks 3/4, peak 5, and peak 6
were prominent in both sediment samples (Figure 4a,b), but
the relative contributions of the compounds varied. The relative
contribution of peak 5 was greater in sediment-32 (Figure 4a),
while the relative contributions of peaks 3/4 and peaks 8/9 were
greater in sediment-44 (Figure 4b). Ratios of intensities of all the
1593 detected NSOBCs compounds between the two sediment
samples showed great variation which ranged from 10−5 to 103
(Figure 4c), especially for compounds with retention times
between 10 and 15 min (Figure 4d). Previous studies have
reported possible natural and anthropogenic emission sources
of NSOBCs.35,36 The variation of the NSOBC profiles among
sediment locations indicated different emission source patterns
(e.g., different microorganism communities), and future studies
are warranted to clarify the source emission of NSOBCs.
Determination of Chemical Structure by Combining
MS/MS Spectra and Chemical Database Information.
Most of the compounds detected had not been reported
previously. While the present study focused on screening for
the presence of compounds, chemical structures of some of the
novel NSOBCs could be further predicted by combining
database search information and high-resolution MS2 spectra.
For example, the chemical formula of the molecular ion [M − H]−
of one of the NSOBCs was determined to be C8H5NBr
(Figure 1f). The most likely structure for the chemical with the
formula C8H6NBr was determined to be bromoindole by use
of the Chemspider database (Figure 1g). By use of a commercial
5-bromoindole standard, the NSOBC peak was successfully
validated as bromoindole (Figure S8). The class of chemicals
to which this NSOBC belonged was recently reported to be
produced by a marine microorganism.35,36 The chemical structure
of another detected NSOBC peak with m/z of 399.7975 and
retention time at 10.7 min was also identified. The chemical
formula, which was determined to be C12H5NBr3, was expected to
represent the molecular ion of the compound. Thus, the chemical
formula, C12H6NBr3, and the Chemspider database were used to
identify the compound as tribromo-9H-carbazole. On the basis
of a similar strategy, we have identified more than 50 isomers/
analogues of halogenated carbazoles with different chlorine,
bromine, and iodine atom numbers. To validate the results, one of
the identified halogenated carbazole peaks (tetrabromocarabazole,
with similar structure to PCDF) was compared with the commercial standard, 1,3,6,8-tetrabromocarbazole. As shown in
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Figure 5. Determination of structures of a novel compound by combining MS/MS spectra and information from a publicly available compound
database. Shown are (a) mass spectra in 350 ± 2.5 m/z and 355 ± 2.5 m/z window at rt = 10 min (precursor ion was 351.8977 m/z, potential product ion
was 336.8743 m/z); (b) chromatographic elution profiles and isotopic peaks to confirm product ions; (c) proposed chemical structures and
fragmentation routes. The precursor ion might represent the displacement of [M − Br + O] with addition of oxygen in the APCI source.
Figure S9, the MS1, retention time, and MS2 spectra from the
DIA window were consistent between the standard and sediment
sample. Since Zhu et al. reported the occurrence of brominated
carbazoles in sediment cores from Lake Michigan in 2005,37 a
recent study has also identified iodine analogues of this class of
compounds in sediment samples from Lake Michigan.22,38
In most cases, a direct search of publicly available databases
using predicted formulas did not yield results. Therefore, in these
cases, a different strategy was adopted to achieve greater hit rates
for chemical structures. Taken the compound from Figure S6,
for example, from the isomer clusters of compounds in the
350 ± 2.5 m/z window, with retention times of 9−11 min,
the monoisotopic peak of the compound was determined to be
351.8977 m/z (Figure 5). There were at least four compounds
detected with the same m/z values and similar retention times,
which indicated the potential existence of isomers for these
compounds. Formulas of compounds were predicted to be
C13H8NOBr2 with a mass error of 1.1 ppm. Compounds with
this formula have not been previously reported to occur in the
environment. Several ions with similar m/z values to the
precursor ions with m/z = 336.8743, 338.8721, and 340.8700
were detected in the same DIA mass spectra (Figure 5a). These
might be product ions of the compounds. To further confirm
these results, the chromatographic elution profiles of the three
ions (Figure 5b), were investigated. All three ions had the same
chromatographic profiles as Br fragment peaks and the precursor
ion at m/z = 351.8977. These results indicated that the three
peaks are likely isotopic peaks of a fragment from the compound.
The chemical formula of the fragment was predicted to be
C12H5NOBr2, which would represent the loss of a methyl group
(−CH3) from the precursor compound in the HCD collision
cell. To further identify chemical structures, the Chemspider
database was used to further identify potential structures for
C13H9NOBr2. Reasonable structures that contained a −CH3
group were not obtained by direct search of Chemspider
database. Thus, the chemical formula was changed to exclude
the Br atom (query of C13H11NO rather than C13H9NOBr2), and
methyl- and hydroxylated polybrominated carbazoles were
identified (Figure 5c). On the basis of the chemical structure,
the 336.8743 m/z fragment with loss of a methyl group from the
precursor, it was identified as bromo-carbazole. One limitation of
this type of identification is the potential displacement reaction that has been reported to occur in the APCI (−) source.39
This reaction might result in formation of the [M − Br + O]− ion.
Therefore, there are two potential chemical structures for
chemicals which incorporate oxygen, as shown in Figure 5c. In
principle, the two possible chemical structures could be further
distinguished by use of ESI ionization or methylation by use of a
1,4-diazabicyclo[2.2.2]octane (DABCO) catalyst or dansylation
derivatization, but this is beyond the scope of the present paper.
These three examples have demonstrated that the combination
of database searches and high-resolution MS2 spectra can be used
to identify chemical structures of previously unknown NSOBCs.
However, the identification of chemical structures of compounds
is time-consuming and challenging, and it is recommended
to first limit the number of NSOBCs based on intensity or
effects observed in bioassays and then identify structures of target
NSOBCs.
Overall, the present study proposed a novel, untargeted
DIPIC-Frag method to screen NSOBCs in the environment and
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presented numerous advantages in coverage, specificity, and
dynamic range. A systematic data mining strategy was developed
to identify the precursor ions, chemical formulas, and chemical
structures of detected NSOBCs. On the basis of the results of
this study, the largest known mass spectrometry library of
NSOBCs (2520 NSOBC peaks, Supplementary Data Table) was
established which could be adopted in low-resolution mass
spectrometry. Future studies are warranted to investigate emission
sources, environmental behaviors, and potential ecological risks
of novel NSOBCs identified in this study. Additionally, their
contribution to total organic Br in the environment should be
investigated.
■
ASSOCIATED CONTENT
S Supporting Information
*
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.analchem.5b01435.
Calculation of isotopic peaks intensities distribution,
information on 25 model NSBCs, sampling map, product
ion spectra of OH-TBB, scanning scheme of the DIPIC-Frag
method, workflow to identify precursor ions of NSBCs
compounds, distribution of isotopic peaks of NSBCs, isotopic
peaks of bromine fragment to help to identify bromine
number and precursor ions, and validations of bromophenol,
bromoindole, and tetrabromocarbazole (PDF)
Precursor ion DIA window, retention time, peak intensity,
exact mass, predicted formula, and calculated mass error of
all the 2520 NSOBC peaks identified by the DIPIC-Frag
method (XLSX)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail: huisci@gmail.com.
*Phone: 306-966-2096; 306-966-4680 (secretary). Fax: 306966-4796. E-mail: jgiesy@aol.com
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This research was part of the Great Lakes Sediment Surveillance
Program funded by a Cooperative Agreement from the U.S. EPA
Great Lakes Restoration Initiative with Assistance Grant GL00E00538 (U.S. EPA Program Officer Todd Nettesheim), a
Discovery Grant from the Natural Science and Engineering
Research Council of Canada (Project No. 326415-07) and a
grant from the Western Economic Diversification Canada
(Project Nos. 6578, 6807, and 000012711). The authors wish
to acknowledge the support of an instrumentation grant from the
Canada Foundation for Infrastructure. Prof. Giesy was supported
by the Canada Research Chair program, and the 2012 “High
Level Foreign Experts” (Grant GDT20143200016) program,
funded by the State Administration of Foreign Experts Affairs,
the P.R. China to Nanjing University, and the Einstein Professor
Program of the Chinese Academy of Sciences.
■
REFERENCES
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1
Untargeted Identification of Organo-bromine Compounds in Lake Sediments by Ultra-
2
High Resolution Mass Spectrometry with Data-Independent Precursor Isolation and
3
Characteristic Fragment (DIPIC-Frag) Method
4
5
Hui PENG, Chunli CHEN, David M.V. Saunders, Jianxian SUN, Song TANG, Garry Codling,
6
Markus Hecker, Steve Wiseman, Paul D. Jones, An Li, Karl J. Rockne, John. P. Giesy
7
8
9
10
Tables
Figures
1
9
11
12
This supporting information provides text, figures and tables addressing (1) Calculation
13
of isotopic peaks intensities distribution; (2) Information on 25 model NSBCs; (3) Sampling
14
map; (4) Product ion spectra of OH-TBB; (5) Scanning scheme of the DIPIC-Frag method;
15
(6) workflow to identify precursor ions of NSBCs compounds; (7) distribution of isotopic
16
peaks of NSBCs; (8) isotopic peaks of bromine fragment would help to identify bromine
17
number and precursor ions; (9) Validation of bromophenol; (10) Validation of bromoindole;
18
(11) validation of tetrabromocarbazole.
1
Isotopic Peaks Intensities Distribution. Intensities of isotopic peaks for precursor ions and Br
fragments were compared semi-quantitatively to further confirm the identities of precursor ions.
Since the relative abundances of chlorine and bromine isotopes were much greater than those of
other elements, only isotopic peaks of bromine and chlorine were considered for semiquantitative calculation of isotopic peak. For a given NSOBC with formula CxHyOzNiClnBrm, (x,
y, z, and i are usually not available), isotopic peaks of the compound were assumed to have a
known distribution which follow Pascal’s triangle (Equation 1).
( a + b ) m (c + d ) n
(1)
Where: a = 0.51 and b = 0.49 are the relative abundances of Br isotopes 79Br (m/z=78.9183) and
81
Br (m/z=80.9163), respectively. m indicates the number of bromine contained in the compound.
c = 0.76 and d = 0.24 are the relative abundances of Cl isotopes
35
Cl (m/z=34.9689) and
37
Cl
(m/z=36.9659), respectively. n indicates the number of chlorine contained in the compound.
Based on the binomial distribution of the isotopic peaks from Equation 1, the relative
abundance of each isotopic peak (with k 79Br and j 35Cl) to total abundance could be calculated
as Equation 2.
Abundancei =
m ! k m−k n! j n− j
ab × cd
k!
j!
(2)
Where: Abundancei indicates the relative abundance of the ith isotopic peak of the compound, k
is the number of 79Br and j is the number of 35Cl in the isotopic peak.
Since the isotopic peaks of bromine fragments were also useful information for precursor
ion alignment, the relative abundance of the peak for monoisotopic
79
Br at m/z=78.9183 was
calculated for each isotopic peak by multiplying the relative abundance of the monoisotopic peak
(Equation 2).
2
Bri =Abundancei × (k + 1) × 2 / m
(3)
Where Bri is the relative abundance of 79Br from the isotopic peak of the compound, (k+1)×2/m
is the relative proportion of
79
Br in the isotopic peak to total
79
Br. Based on Equation 3, the
relative abundance of 79Br from each isotopic peak could be calculated.
Intensities of precursor ion and bromine fragments ions. Since the relative abundances of
precursor ion candidates and bromine fragments are also important information for precursor ion
alignment, the threshold of the ratio between abundances of precursor ions (indicated by peak
intensities in the present study) and the 79Br fragment at m/z=78.9183 was predicted. Intensity of
the precursor ions should be greater than that of product ions divided by fragment number
(Equation 4).
Intensity precursor >Intensity product / m
(4)
Where: Intensityprecursor is the intensity of precursor ions, Intensityproduct is the intensity of product
ions, m is the number of product ion fragments contained in the formulae (the number of Br atom
in the NSOBC).
Because the bromine fragments were monitored from a 5-m/z precursor isolation window,
which might contain up to 3 isotopic precursor ions for the same NSOBC, the summed
intensities of isotopic precursor ions and bromine fragment were predicted according to Equation
5.
∑ Intensity
precursor
>
∑
Intensityi / m = Intensity bromine / m
(5)
k=1,2,3
Where: ∑Intensityprecursor is total intensity of isotopic precursor ions from the same NSOBC in
the precursor isolation window. Intensityi is intensity of Br fragment ions from the ith isotopic
3
precursor ion, which was expected to be less than the intensity of the corresponding precursor
ion, and Intensitybromine is the summed intensity of total Br fragments detected in the
corresponding precursor isolation window. Because the precursor isolation window was 5-m/z,
which allowed at most 3 isotopic peaks for brominated compounds, the intensity of the maximal
precursor ion in the window should be greater than one third of the summed intensity of all
isotopic peaks of precursor ions (Equation 6).
Intensitymax >Intensitybromine / (3 × m)
(6)
Where: Intensitymax is the intensity of the most abundant isotopic precursor ion peak in the
isolation window.
4
Table S1. Name, molecular weight, KOW value, and instrument sensitivity of 25 model
brominated chemicals.
Compounds
HBCD-α
HBCD-β
HBCD-γ
TBBPA
TBB
OH-TBB
OH-TBPH
TBPH
6-OH-BDE-47
4’-OH-BDE-49
2-OH-BDE-123
6-MeO-BDE-47
4’-MeO-BDE-49
4’-MeO-BDE-99
BDE-47
BDE-49
BDE-66
BDE-85
BDE-99
BDE-100
BDE-153
BDE-154
BDE-183
BDE-209
a
Formula
C12H18Br6
C12H18Br6
C12H18Br6
C15H12Br4O2
C15H18Br4O2
C15H19Br3O3
C24H35Br3O5
C24H34Br4O4
C12H6O2Br4
C12H6O2Br4
C12H5O2Br5
C13H8O2Br4
C13H8O2Br4
C13H7O2Br5
C12H6Br4O
C12H6Br4O
C12H6Br4O
C12H5Br5O
C12H5Br5O
C12H5Br5O
C12H4Br6O
C12H4Br6O
C12H3Br7O
C12Br10O
MW
635.6509
635.6509
635.6509
539.7571
545.8040
483.8884
640.0035
701.9191
497.7101
497.7101
575.6206
511.7258
511.7258
589.6363
481.7152
481.7152
481.7152
559.6257
559.6257
559.6257
637.5362
637.5362
715.4467
949.1783
KOW values were from references1-5
5
Log KOWa
5.07
5.12
5.47
4.5
8.8
9.56
11.95
6.4
6.4
7.2
7.3
7.3
8.2
6.8
6.8
6.8
7.7
7.7
7.7
8.6
8.6
9.4
12.1
IDLs (µg/L)
0.2
0.3
0.1
0.05
2.0
0.005
0.01
5.0
0.05
0.05
0.05
0.3
0.3
0.3
3.0
4.0
2.0
0.8
0.9
2.0
2.0
3.0
5.0
20
Figure S1. Sampling locations of two sediment samples (sed-32 and sed-44) from Lake
Michigan.
6
Figure S2. Product ion of a bromine fragment from brominated compounds (hydroxylated TBB
in this sample) under relatively high collision energy (>30eV).
7
Figure S3. Scheme for data independent precursor isolation and characteristic fragment (DIPICFrag) method. Nine different methods (Method1 - Method 9) were performed for a single
sample, each method covered a mass range of 100 Da. For each method, the full scan was used
for each 7 cycles, and then 6 following successive data independent isolation (DIA) windows (5
m/z) was scanned. 20 DIA window was used for each method to cover the 100 Da mass range.
Stepped collision energy at 10, 30 and 60 eV was used for the DIA scanning to simultaneously
record information on bromine fragment and precursor ions in the same mass spectra.
8
Figure S4. Workflow to identify precursor ions of NSOBCs. The first step is to get the
chromatographic profiles of candidate ions from precursor ion regions in separated precursor
isolation DIA windows (there are typically 20-30 ions). If we detected an ion with the same
chromatographic profile with the bromine fragment, we further used full scan spectra to get more
accurate m/z values. If we could not detect precursor ion in the precursor ion region, we
searched precursor ion from full scan spectra. For the potential precursor ions detected in full
scan or precursor ion region, we calculated the intensity of the ions to further make sure that
intensities of precursor ions were greater than that of the fragment divided by number of atom.
Then, we also checked the isotopic peaks of the precursor ions. If the potential precursor ions’
intensities were low or no isotopic peaks were detected, we moved to the next candidate ions in
the precursor ion region for the next round of data analysis. Finally, the list of likely precursor
ions was produced.
9
Figure S5. Distribution of isotopic peaks of brominated compounds with different compositions
of bromine/chlorine. Y-axis indicated the relative intensities of the isotopic peaks to the maximal
intensity of the peak.
11
Figure S6. Isotopic peaks of bromine fragment in separated DIA windows could help to identify
bromine numbers and molecular ions of brominated compounds. As shown in the bottom figure,
only the bromine ion at m/z=78.9171 was detected in the window at 350±2.5 m/z, which meant
that the primary monoisotopic ion of the compounds should be between 350-352 m/z. If the
molecular ion was lower than 350 m/z, we should have observed the isotopic peaks of bromine at
m/z=80.9151 because the mass span of brominated compounds is ~2 m/z. As shown in the top
figure, two isotopic peaks were observed at m/z=353.8959 and m/z=355.8939 respectively. In
this figure, isotopic peak of bromine at m/z=80.9151 were detected at similar intensity to the ion
at m/z=78.9171. By combining the isotopic peaks of the bromine fragment in different precursor
isolation windows and the distribution of the isotopic peaks of the precursor ions, the compound
was identified as C13H8NOBr2 with mass error of 1.1 ppm and a monoisotopic molecular ion at
m/z=351.8977.
12
Figure S7. Validation of an identified bromophenol by use of the commercially available
standard 4-bromophenol. (a) Extracted ion chromatogram at m/z=170.9445 (10 ppm mass width)
for a standard of 4-bromophenol (100 µg/L). (b) MS2 spectra for 4-bromophenol from a 170±2.5
DIA window. (c) Extracted ion chromatogram at m/z=170.9445 (10 ppm mass width) from
sediment extract. (d) MS2 spectra for a bromophenol peak in sediment extract from a 170±2.5
DIA window.
13
Figure S8. Validation of an identified bromoindole by use of commercially available standard of
5-bromoindole. (a) Extracted ion chromatogram at m/z=193.9605 (10 ppm mass width) for a
standard of 5-bromoindole (100 µg/L). (b) MS2 spectra for 5-bromoindole from a 195±2.5 DIA
window. (c) Extracted ion chromatogram at m/z=193.9605 (10 ppm mass width) from sediment
extract. (d) MS2 spectra for a 5-bromoindole peak in sediment extract from a 195±2.5 DIA
window.
14
Figure S9. Validation of an identified brominated carbazole by use of the commercially available
standard of 1,3,6,8-tetrabromocarbazole. (a) Extracted ion chromatogram at m/z=477.7077 (10
ppm mass width) for standard of 1,3,6,8-tetrabromocarbazole (100 µg/L). (b) MS2 spectra for
1,3,6,8-tetrabromocarbazole from a 480±2.5 DIA window. (c) Extracted ion chromatogram at
m/z=477.7077 (10 ppm mass width) from sediment extract. NSOBC at 13.94 min was an isomer
of 1,3,6,8-tetrabromocarbazole with different bromine positions on the aromatic ring. (d) MS2
spectra for a tetrabromocarbazole peak in sediment extract from 480±2.5 DIA window.
15
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16