Supporting Information for Environmental Science and Technology

Predictable LC-QTOF/MS Fragmentation of Ozone-Reactive N-Nitrosodimethylamine Precursors

Coupled with In Silico Fragmentation and IM–QTOF Facilitates Their Identification in Sewage

Klon D.C. Hinneh1*, Koji Kosaka2, Shinya Echigo3, Sadahiko Itoh1

1
Department of Environmental Engineering, Graduate School of Engineering, Kyoto University, Katsura,
Nishikyo-ku, Kyoto 615-8540, Japan
2
Department of Environmental Health, National Institute of Public Health, Wako, Saitama 351-0197,
Japan
3
Department of Global Environmentally-friendly Industries for Sustainable Development, Graduate
School of Global Environmental Studies, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-
8501, Japan

*Corresponding author

E-mail: hinneh.dc.58s@st.kyoto-u.ac.jp

This SI comprises of 24 Pages, 16 Figures, and 4 Tables.

S1
Contents

S1 Safety Information .................................................................................................................................... 3

S2 Reagents and Standard Solutions ............................................................................................................. 3
S3 LC-QTOF/MS Fragmentation Conditions for Model O3-Reactive NDMA Precursors ........................... 4
S4 MS/MS Spectra of Model O3-Reactive NDMA Precursors Bearing (CH3)2N–N–R Structures, where
R= –C=O or –C=S ......................................................................................................................................... 5
S5 MS/MS Spectra of 2-Furaldehydedimethylhydrazone (2-FDMH) with (CH3)2N–N=C– Structure ........ 6
S6 MS/MS Spectra of Compounds bearing N,N-dimethylsulfamide (DMS) [(CH3)2N(SO2)N]................... 6
S7 NDMAFP Test of 1,1-Dimethyl-4-phenyl-3-thiosemicarbazide (DMPTSC) by Ozonation ................... 7
S8 NDMA and NDMA Precursors at STPs with Combined Sewer Systems................................................ 8
S9 Occurrence of NDMA and NDMA Precursors in Sewage ....................................................................... 9
S10 Extraction and Recovery of NDMA Precursors in SW-2 Sample........................................................ 11
S11 HPLC Fractionation of SW-2 Samples ................................................................................................ 11
S12 Chloramination and Ozonation of Raw Sewage .................................................................................. 12
S13 NDMA Extraction, Measurement, and Recovery ................................................................................ 12
S14 Total Ion Current Chromatogram (TIC) of 15.5–16.5 min SW-2 Fraction after Infusion to LC-
QTOF/MS .................................................................................................................................................... 13
S15 Extracted Ion Chromatograms (EICs) of Peaks (m/z) Flagged by Diagnostic Fragments ................... 14
S16 Peaks (m/z) of Potential NDMA Precursors Flagged by m/z 61.0766 and 60.0688 Da in the 15.5–16.5
min SW-2 Fraction ...................................................................................................................................... 15
S17 MS/MS Spectra of Peaks (m/z) Flagged by Both Fragment m/z 61.0766 and 60.0688 Da.................. 16
S18 In silico-Fragmentation Tools and IM-QTOF/MS Acquisition ........................................................... 19
S19 Possible Structures of m/z 546.3846..................................................................................................... 20
S20 Total Ion Current Chromatogram (TIC) of the 15.5–16.5 min SW-2 Fraction by IM-QTOF/MS ...... 21
S21 Comparison of MS/MS Spectra of m/z 741.4313................................................................................. 22
S22 3-D Plots of the m/z 371.2195 Monomer by IM-QTOF ....................................................................... 23
S23 Reference .............................................................................................................................................. 24

S2
S1 Safety Information

N-Nitrosodimethylamine (NDMA) is a probable carcinogenic and mutagenic agent. NDMA is a

combustible liquid and is an irritant to the eye. When handling NDMA, one must wear appropriate
protective clothing, goggles, and gloves. In addition, one must read and strictly follow all safety protocols.
NDMA should be kept and stored in a tightly-closed container in a well-ventilated and cool place.

S2 Reagents and Standard Solutions

All chemicals were of analytical grade. NDMA (1000 µg/mL in MeOH; 1 mL) and NDMA-d6 (1000 µg/mL
in methylene chloride, DCM; 1 mL) were purchased from AccuStandard, Inc. (Connecticut, USA). We
diluted both NDMA and NDMA-d6 to 10 mg/L and 1000 mg/L in MeOH, respectively, and separately
transferred them to Hi-sealed amber storage bottles (purchased from Kanto Kakagu; Tokyo, Japan). Both
solutions were stored at 4 ºC until use. We purchased acetonitrile (≥99.8%; for LC/MS), methanol (≥99.7%;
for LC/MS), and formic acid (≥98%) from Wako Pure Chemical Industries Ltd (Osaka, Japan). We also
purchased dichloromethane (5000; for Pesticide Residue and PCB Analysis) and hexane (5000; for
Pesticide Residue and PCB Analysis) from Wako Pure Chemical Industries Ltd. 1,1,1’,1’-tetramethyl-4,4’-
(methylene-di-p-phenylene)disemicarbazide (TMDS) (>95.0%), unsymmetrical dimethylhydrazine
(UDMH) (>98.0%), 4,4’-hexamethylenebis(1,1-dimethylsemicarbazide) (HDMS) (>98.0%), dacarbazine
(DTIC) (>98.0%), 1,1,5,5-tetramethylcarbohydrazide (TMCH) (>99.5%), 1,1-dimethylsemicarbazide
(DMSC) (98.5%), 2-furaldehydedimethylhydrazone (2-FDMH) (>97%), N,N-dimethylisopropylamine
(DMiPA) (>99.0%), daminozide (DMASA) (>98.0%), N,N-dimethylbenzylamine (DMBzA) (>98.0%),
and metformin hydrochloride (Met) (>98.0%) were purchased from Tokyo Chemical Industry (Tokyo,
Japan). We purchased ranitidine hydrochloride (97.0%) from Wako Pure Chemical Industries Ltd, 1,1-
dimethyl-4-phenyl-3-thiosemicarbazide (DMPTSC) (NA) from Sigma-Aldrich (Missouri, USA), N,N-
dimethyl-N’-tolylsufonyldiamide (DMST) (100 µg/mL in MeOH) and tolylfluanide (ToFD) (100 µg/mL in
MeOH) from AccuStandard® Inc. (Connecticut, USA), and 4-{[(dimethylamino)sulfonyl]amino}benzoic
acid (COOH-DMST) (98.0%) from Fluorochem Ltd (Derbyshire, UK).

S3
S3 LC-QTOF/MS Fragmentation Conditions for Model O3-Reactive NDMA Precursors

Table S1. LC-QTOF/MS conditions for fragmentation of model O3-reactive NDMA precursors

Model 1260 Infinity Series (Agilent Technologies, Singapore)

Column ACQUITY UPLC BEH C-18; 1.7 μm 100x2.1 mm (Waters)

Column Temp. 30 ºC

Injection volume 20 μL
LC
Mobile phase A: Milli-Q water (MQW)(0.1% formic acid)
B: Acetonitrile (ACN)

Flow rate 0.2 mL/min

Gradient 1 B:5% (0 min) 60% (3 min) 100% (8 min) 100% (9

min) 5% (10 min)

Gradient 2 B:5% (0 min) 60% (3 min) 95% (13 min) 95% (15
min) 5% (16 min)

Model 6560 Ion Mobility QTOF LC/MS (Agilent Technologies,

Singapore)

Ionization mode ESI (+)

Capillary voltage 3500 V

Fragmentor voltage 190 V

Collision energy (CE) 10, 20, or 30 eV

Nebulizer gas pressure 40 psi

QTOF-MS
Gas temperature 300 ºC

Sheath gas temperature 375 ºC

m/z range 40 – 1000

Scan mode Auto MS/MS or All ions MS/MS: (CE: 10, 20, 30 eV)

Sheath gas and dry gas 12; 12 L/min

flows

Reference mass Purine: m/z 121.0509; HP 921: m/z 922.0098

S4
S4 MS/MS Spectra of Model O3-Reactive NDMA Precursors Bearing (CH3)2N–N–R Structures,
where R= –C=O or –C=S

Figure S1. MS/MS spectra of five model NDMA precursors bearing (CH3)2N–N: DMSC, HDMS,
DMASA, TMCH, and DMPTSC. Fragmentation patterns are consistent with that of TMDS. TMCH and
DMASA formed [M+Na]+ and [M+Na–H]+ adducts, respectively. All other adducts are [M+H]+. It is
noteworthy that DMSC (first from top) formed many adducts: [M+H]+, [M+K]+, [M+CaAc]+, and 13C-
[2M+Cu–H]+. However, only [M+H]+ and 13C-[2M+Cu–H]+ showed m/z 61.0766. It was surprising to
see that 13C-[2M+Cu–H]+ (m/z 269.0743) was more intense than [M+H]+ (m/z 104.0824). K, Ca, Cu,
and Ac presumably originated from the formic acid used as mobile phase additives. Ac is acetate.

S5
S5 MS/MS Spectra of 2-Furaldehydedimethylhydrazone (2-FDMH) with (CH3)2N–N=C– Structure

Figure S2. MS/MS spectrum of 2-FDMH ([M+H]+) shows that hydrazone bearing (CH3)2N–N generates
a loss of (CH3)2N–N=CH2 (72.0688 Da).

S6 MS/MS Spectra of Compounds bearing N,N-dimethylsulfamide (DMS) [(CH3)2N(SO2)N]

Figure S3. MS/MS spectra of ToFD, DMST, and COOH-DMST show a consistent fragment loss of
(CH3)2NSO2 (108.0119 Da). All adducts are [M+H]+.

S6
S7 NDMAFP Test of 1,1-Dimethyl-4-phenyl-3-thiosemicarbazide (DMPTSC) by Ozonation

Ozonation: The procedure for ozonation of DMPTSC was similar to those used by Kosaka et al.1,2 We used
1 L glass bottles in a semi-batch system with an ozone generator (AZH-3S, Hamamatsu Vegetable, Japan)
and prepared ozone from pure oxygen gas. Ozonation conditions were as follows: DMPTSC, 0.01 mM (in
500 mL MQW); ozone concentration in gas phase, 10 mg/L; ozone gas flow rate, 1 L/min; reaction time,
20 min; and temperature, 20 ºC; pH, 7 (5 mM phosphate buffer). We stooped the reaction by adding a small
amount of 6 g/L of sodium thiosulfate (Na 2S2O3). NDMA measurement: The analytical method for the
measurement of NDMA and NDMA-d6 is the same as those by Hinneh et al.3 Briefly, we measured NDMA
and NDMA-d6 by LC-MS/MS analysis. An ACQUITY UPLC BEH C18 analytical column (Waters
Corporation, USA) was used for LC separation on a Shimadzu prominence 20 A UPLC system (Shimadzu,
Kyoto, Japan) interfaced to a triple quadrupole mass spectrometer (4000 QTRAP, SCIEX, USA). The
mobile phases A and B were MQW (0.1% formic acid) and acetonitrile, respectively.

S7
S8 NDMA and NDMA Precursors at STPs with Combined Sewer Systems

Chloramination: Primary- and secondary-effluents were chloraminated to assess the occurrence of NDMA
and NDMA precursors in combined sewer overflows (CSO). The chloramination procedure was similar to
that employed in Hinneh et al.3 with few modifications. For brevity, chloramination conditions are
summarized: sample volume, 120 mL; chloramine (i.e., NH2Cl) dose, 6.2 mg/L as Cl2 to maintain a residual
chloramine concentration of 3.0±0.5 mg/L as Cl2; reaction time, 24 h; temperature, 24 ºC pH, 7 (5 mM
phosphate buffer). After 24 h, we stopped the reaction by adding a small amount of 6 g/L of sodium
thiosulfate (Na2S2O3).

Figure S4. NDMA formation potentials (FPs) of sewage samples at STP-A and STP-B. We collected
two samples: CSO and normal. CSO is a combined sewer overflow sample, and normal is a sample
collected under no rainfall. We measured NDMAFP as a surrogate of NDMA precursor (s). NDMAFP
is the difference between NDMA before and after chloramination. We chloraminated all samples at the
laboratory except the final effluent (FE), collected after ozonation at STP-B. PE is primary effluent, and
SE is secondary effluent; ND is not detected (i.e., <limit of quantification (LOQ) (2 ng/L)).

S8
S9 Occurrence of NDMA and NDMA Precursors in Sewage

Figure S5. NDMA concentration of sewage in feeder pipes of STP-B. Except for the SW-1 sample and
the raw SW-2 sample (without oxidant), the error bar represents relative error after measurements of two
samples collected on different sampling dates (see Table S2).

Table S2. Water quality parameters and NDMA concentration before and after chloramination or ozonation
of water samples.

DOC NDMA Before NDMA After

Sample Sampling Date (mg/L) pH (ng/L) (ng/L)

SW-1 6.30.2019 4.45 7.50 11.6 21.8ª (304)

SW-2 6.30.2019 19.4 7.48 316 7,200ª (8,050)

7.08.2019 78.1 6.83 316 4050ª (75,000)

SW-3 6.30.2019 8.59 6.83 9.15 11.8ª (78.0)

7.08.2019 NM NM 10.9 120ª (21.6)

ªNDMA concentration after chloramination; NDMA concentrations in parentheses are NDMA formed
after ozonation; NM is not measured.

S9
Figure S6. NDMA concentration of SW-2 sample after chloramination or ozonation – (A) 5-min fractions
from 0.5–40.5 min and (B) 1-min fractions from 10.5–20.5 min. Chloramination conditions: NH2Cl
concentration, 7.2 mg/L; reaction time, 24 h; pH, 7 (5 mM phosphate buffer); temperature, 20 ºC;
Ozonation conditions: O3 in gas phase, 20 mg/L; O3 gas flow rate, 1 L/min; reaction time, 20 min; pH, 7
(5 mM phosphate buffer); temperature, 20 ºC.

S10
S10 Extraction and Recovery of NDMA Precursors in SW-2 Sample

We pretreated the NDMA precursors by evaporation or solid-phase extraction (SPE). Evaporation: We

evaporated 500 mL of sewage samples with a rotary evaporator to 5 mL (100x concentration) at 50 ºC with
a vacuum. SPE: We adopted the procedure from Hanigan et al.4 with a few modifications. We conducted
duplicate serial extraction using Sep-Pak Plus mixed-mode cation exchange (MCX) cartridges (500 mg x
2; Waters). The sample volume was 500 mL. The pH of the sample was adjusted to 3 by sequential addition
of ≤1M H2SO4. The SPE flow rate was 5 mL/min. We preconditioned the cartridges by passing through
them: 5 mL of dichloromethane, 5 mL of methanol, and 5 mL of ultrapure water, respectively. The MCX
cartridges were then dried under nitrogen gas and eluted with 10 mL of ammonia and methanol (5:95; v/v)
solution at 2−3 mL/min. Finally, we concentrated the eluate to 0.5 mL (1000x concentration) under a gentle
stream at 50 ºC. Because we performed duplicate extraction (0.5 mL each), we mixed the sample to a 1
mL composite sample. Further pH adjustment was not necessary because we purged the ammonia from the
eluate with nitrogen gas. The pH of the eluate was ∼7, confirmed by a pH test paper.

S11 HPLC Fractionation of SW-2 Samples

The fractionation experiments on SW-2 samples were similar to those described by Kosaka et al.1,2 We
firstly collected eight 5-min pooled fractions (0.5–5.5, 5.5–10.5, 10.5–15.5, 15.5–20.5, 20.5–25.5, 25.5–
30.5, 30.5–35.5, and 35.5–40.5 min) in the order of elution. The volume of each fraction was 5 mL, so each
pooled fraction was 25 mL. We diluted the pooled fractions to 1 L in MQW and designated them as diluted
pooled fractions. We adjusted the pH of the diluted pooled fractions to 7 (5 mM phosphate buffer) because
the original pH of the SW-2 sample was 7.3. Methanol volume before dilution varied considerably (up to
25 mL) and was adjusted appropriately. Then, we subjected each diluted pooled fraction of 0.7 L to zonation
and 0.3 L to chloramination. Secondly, after identification of the 5-min pooled fractions (i.e., 10.5–15.5
and 15.5–20.5 min) with high NDMA concentrations by NDMA formation potential (FP) tests, the two 5-
min fractions were fractionated to ten 1-min fractions (10.5–11.5, 11.5–12.5,12.5–13.5, 13.5–14.5, 14.5–
15.5, 15.5–16.5, 16.5–17.5, 17.5–18.5, 18.5–19.5, 19.5–20.5 min). We diluted each 10 mL fraction to 1 L
in MQW and designated them as diluted fractions. We also adjusted the pH and methanol volume of the
diluted fractions to 7 (5 mM phosphate buffer) and 25 mL, respectively. We then subjected each diluted
fraction of 0.7 L to ozonation and 0.3 L to chloramination. An HPLC (Shimadzu Prominence 20 A UFLC)
coupled with a Zorbax SB-Aq column (4.6 mmx150 mm; Agilent Technologies, USA) was used for the
fractionation experiments. The column temperature was at 30 ºC. Mobile phases A and B were MQW and
methanol, respectively. The flow rate was 1.0 mL/min; injection volume was 50 μL; and the gradient
condition was B: 10% (0 min) 100% (20 min) 100% (40 min) 10% (45 min).

S11
 Figure S7. Extraction and recovery of NDMA precursors by evaporation or solid-phase extraction (SPE)
with mixed-mode cation exchanged cartridges (MCX) of SW-2 grab samples. The recovery (%) equals
(C/Co) multiplied by 100. The NDMA formation potentials (FP) of O3-reactive precursor (Co), measured
as a surrogate of NDMA precursors, was 25,350 ng/L, and the NDMAFP of the NH2Cl-reactive precursor
(Co) was 4,050 ng/L. The sample volume for evaporation or SPE was 500 mL.

S12 Chloramination and Ozonation of Raw Sewage

Chloramination: Sewage samples were chloraminated similar to primary and secondary effluents with
some modifications (see section S8). Chloramination conditions were as follows: sample volume, 200 or
300 mL; chloramine dose: 7.2 mg/L as Cl2 to maintain slight excess of residual chloramine; reaction time,
24 h; pH, 7 (5 mM phosphate buffer); temperature, 20 ºC. After the 24 h, we added a small amount of 6 g/L
of sodium thiosulfate (Na2S2O3) to stop the reaction. Ozonation: The procedures for ozonation were similar
to those used by Kosaka et al.1,2 Sewage samples were ozonated in 1 L glass bottles using a semi-batch
system with an ozone generator (AZH-3S, Hamamatsu Vegetable, Japan). We prepared ozone from pure
oxygen gas. Ozonation conditions were as follows sample volume, 600 or 700 mL; ozone concentration in
the gas phase, 10 or 20 mg/L; ozone gas flow rate, 1 L/min; reaction time, 10−20 min; and temperature,
20 ºC. We adjusted the pH to 7 (5 mM phosphate buffer) for fractions of concentrated sewage. After 10 or
20 min, we added a small amount of 6 g/L of sodium thiosulfate (Na2S2O3) to stop the reaction.

S13 NDMA Extraction, Measurement, and Recovery

We extracted NDMA and NDMA-d6 with procedures from Kosaka et al.2 with a few modifications. Sample
volumes were generally 500 mL for surface waters and STP effluents and 100 mL for all other samples.
For samples containing extremely high NDMA concentration, we reduced the sample volumes.

S12
Preconditioned AC-2 cartridges (400 mg x 2; Waters) trapped NDMA and NDMA-d6. We then used gentle
nitrogen gas (N2) to dry the cartridges. We used 10 mL of dichloromethane (DCM)/diethyl ether (50:50;
v/v) to elute the coupled cartridges, and the eluent was then passed through a preconditioned Sep-Pak Vac
Florisil cartridge (1g; Waters) for cleanup. The eluate was then concentrated to 0.2 mL under N2 gas and
then diluted to 0.5 mL with DCM. The analytical method for the measurement of NDMA and NDMA-d6 is
the same as those by Hinneh et al.3 The gradient condition was modified as follows: B: 5% (0 min) 5%
(5.0 min) 95% (5.5 min) 95% (8.5 min) 5% (9.0 min) 5% (14.0 min). The LOQ for primary
effluent, secondary effluent, and sewer samples was 2.0 ng/L. The recoveries of NDMA in MQW, tap water,
river water, and sewage were 126% [n=2; NDMA addition = 2 ng/L; relative standard deviation (RSD) =
10%], 115% (NDMA = 2 ng/L; RSD =16%), 121% (NDMA = 2 ng/L; RSD = 8%), 118% (NDMA = 10
ng/L; RSD = 9%), respectively.

S14 Total Ion Current Chromatogram (TIC) of 15.5–16.5 min SW-2 Fraction after Infusion to LC-
QTOF/MS

Figure S8. LC-QTOF/MS total ion current chromatogram (TIC) of the 15.5–16.5 min SW-2 fraction.

S13
S15 Extracted Ion Chromatograms (EICs) of Peaks (m/z) Flagged by Diagnostic Fragments

Figure S9. The combined EICs of peaks flagged by diagnostic fragment (A) m/z 61.0766, and (B) a
neutral loss of 60.0688 Da. Peaks colored green are dimers ([2M+H]+) of peaks in red ([M+H]+) with the
same retention time and similar MS/MS spectra (see Table S3).

S14
S16 Peaks (m/z) of Potential NDMA Precursors Flagged by m/z 61.0766 and 60.0688 Da in the 15.5–
16.5 min SW-2 Fraction

Table S3. The peaks (m/z) of potential NDMA precursors flagged from the 15.5-16.5 min SW-2 fraction
b
Retention Time Accurate Mass Diagnostic Flagging Mass Match Collision Energy
(min) Peaks Fragment Tolerance (eV)
(m/z) (Da loss or m/z) (ppm)

NA No Peak 60.0688 Da/m/z 61.0766 10;20 10;20;30

6.155 371.2208 60.0688 50; 100; 200 10

6.153 371.2202 60.0688 50; 100; 200 20

6.531 380.2979 60.0688 200 30

6.153 371.2208 m/z 61.0766 50;100 10

5.826 546.3846 61.0766 50;100 10

6.155 371.2202 61.0766 50;100 20

6.162 371.2185 61.0766 50;100 30

6.162 741.4363ª 61.0766 50;100 10

6.162 741.4318ª 61.0766 50;100 20

6.173 741.4275ª 61.0766 50;100 30

a
The m/z 741.4313 peaks are dimers [2M+H]+ of their respective m/z 371.2195 ([M+H]+) monomers. The m/z of
peaks are the accurate masses during fragmentation. It is noteworthy that we searched all peaks in the blank (MQW)
using 60.0688 Da and m/z 61.0766 as the diagnostic fragment, but we found no compound corresponding to either
fragment. The lowest allowable mass match tolerance = 10 ppm. bThe mass match tolerance is for the neutral loss
or fragment ion in the “Find by Auto MS/MS” tool. It removes any compound if the MS/MS spectrum does not
contain the specified neutral loss or fragment ion.

S15
S17 MS/MS Spectra of Peaks (m/z) Flagged by Both Fragment m/z 61.0766 and 60.0688 Da

Figure S10. MS/MS spectra of peaks in the 15.5–16.5 min fractionated SW-2 sample flagged by fragment
m/z 61.0766 and 60.0688 Da loss – (A) peaks flagged by fragment 60.0688 Da at 10, 20, and 30 eV,
respectively; (B) peaks flagged by m/z 61.0766 at 10 and 20 eV, respectively. The mass match tolerance
was 50 ppm (see the bottom of Table S3 for explanation).

S16
Figure S11. Peaks flagged by m/z 61.0766 at 10, 20, and 30 eV (top to bottom). The mass match tolerance
was 50 ppm (see the bottom of Table S3 for explanation).

S17
Figure S12. MS/MS spectra of m/z 546.3846 ([M+NH4]+) at 10, 15, and 20 eV. Fragment m/z 61.0766
was generated only at 10 eV. The m/z 546.3846 completely diminished at 30 eV.

S18
S18 In silico-Fragmentation Tools and IM-QTOF/MS Acquisition

In silico-Fragmentation Tools. We selected three in silico fragmenters (molecular structure correlator,

MSC, MetFrag2.2, and competitive fragmentation modeling-ID 3.0, CFM-ID 3.0).5–8 We used both MSC
and MetFrag2.2 as complementary fragmenters to help elucidate the structure of precursors.
Their complementary use is because, for example, the version of MSC allows only [M+H]+ adduct, whereas
MetFrag2.2 allows other adducts such as [M+NH4]+ and [M+Na]+.5,6 Both MSC and MetFrag2.2 use
accurate m/z values and chemical formulae, if available, to query online databases for structures. Then
through a logical, systematic bond-breaking approach, the fragmenters use the MS/MS data to score and
arrange the structures according to their likelihood of generating such MS/MS fragments. Furthermore, the
fragmenters permit the filtering of possible structures based on substructures (using SMILES arbitrary
target specification, SMARTS, for substructure queries),6 functional groups, MS/MS peaks, etc. They
further improve scoring based on the inclusion of patent and references criteria. CFM-ID 3.0 also operates
similarly to MSC and MetFrag2.2. However, in our study, we used CFM-ID 3.0 for spectral prediction to
manually compare the generated fragments.6,7 It is noteworthy that we did not intend to show which of these
tools is better than the other (s). But to demonstrate the usefulness of their combination in probing the
identification of the peaks. The MS tolerance = 5 ppm to match that of QTOF/MS. For MS/MS tolerance,
we set the value at 5 or 50 ppm. We exported the accurate MS and MS/MS data of all flagged peaks in the
15.5–16.5 min SW-2 fraction to a compound exchange file (*CEF) and imported them to the MSC for
processing. It is noteworthy that MSC allows the input of only [M+H]+. We manually inputted the
MS/MS data and chemical formulae on MetFrag2.2 and CFM-ID 3.0. IM-QTOF/MS. IM-QTOF was
operated in all ions MS/MS because auto MS/MS is not available in IM-QTOF. We recalibrated the axis of
each spectrum by IM-Data Processing Utility, which does the equivalent of the internal reference mass in
the QTOF-only mode.

S19
S19 Possible Structures of m/z 546.3846

Table S4. MetFrag2.2 results for m/z 546.3846 (M: C33H44N4O2), an [M+NH4]+ adduct, from PubChem
database.
PubChem CID (PCCID) 67658148 53651166 20088012 67785887 69822922
SCFrag+SCSubs (5 ppm)/Rank 2.0/1 1.9023/2 1.9001/3 1.9001/4 1.9001/4
(n=6)

Peaks explained (n=297) 39 37 37 37 37

SCFrag+SCSubs (50 – 1.4761/2 1.9369/1 1.1381/4 1.1381/4

ppm)/Rank (n=5)

Peaks explained (n=297) – 121 121 121 121

SCPCR/Rank (n=5) 1.02/4 1.4423/2 1.9001/1 1.001/3 1.001/3

Substructure inclusion [NX3][NX3] [NX3][NX3] [NX3][NX3] [NX3][NX3] [NX3][NX3]

[$([NX3]N(C)C)] [$([NX3]N(C)C)] [$([NX3]N(C)C)] [$([NX3]N(C)C)] [$([NX3]N(C)C)]

Structures filter by – (CH3)2N+–NH2 (CH3)2N+–NH2 (CH3)2N+–NH2 (CH3)2N+–NH2

explained MS/MS peaks

Remarks Lacks DMH Can coelute Top-most hit Stereoisomer Stereoisomer

The total retrieved candidates were 175. Bolded and italicized text represents the highest score and rank. It is
noteworthy that we used only MetFrag2.2 because it permits [M+NH4]+adduct, whereas MSC does not.

Figure S13. Possible structures of m/z 546.3846. PCCID is PubChem chemical identifier.

S20
S20 Total Ion Current Chromatogram (TIC) of the 15.5–16.5 min SW-2 Fraction by IM-QTOF/MS

Figure S14. Total Ion Current Chromatograms (TICs) of samples via IM-QTOF acquisition. Top – TIC of
15.5–16.5 min SW-2 fraction; middle – fortified 15.5–16.5 min SW-2 fraction (a known amount of TMDS
standard was spiked into the 15.5–16.5 min SW-2 fraction); bottom – TIC of TMDS standard spiked into
MQW.

S21
S21 Comparison of MS/MS Spectra of m/z 741.4313

Figure S15. MS/MS spectra of Dimers ([2M+H]+: m/z 741.4313) of (A) Isomer A in 15.5–16.5 min SW-
2 Fraction (B) TMDS in 15.5–16.5 min SW-2 Fraction (C) TDMS standard in MQW. We note that
Isomer A was identified as PCCID 141210417.

S22
S22 3-D Plots of the m/z 371.2195 Monomer by IM-QTOF

Figure S16. m/z 371.2195, an [M+H]+ adduct, showed one isomer with drift time 22.17 ms. Top: 15.5–
16.5 min SW-2 fraction; middle: fortified 15.5–16.5 min SW-2 fraction (a known amount of TMDS
standard was spiked into the 15.5–16.5 min SW-2 fraction); bottom: TMDS standard spiked into MQW.

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S23 Reference

(1) Kosaka, K.; Asami, M.; Konno, Y.; Oya, M.; Kunikane, S. Identification of Antiyellowing Agents
as Precursors of N-Nitrosodimethylamine Production on Ozonation from Sewage Treatment Plant
Influent. Environ. Sci. Technol. 2009, 43 (14), 5236–5241.

(2) Kosaka, K.; Asami, M.; Ohkubo, K.; Iwamoto, T.; Tanaka, Y.; Koshino, H.; Echigo, S.; Akiba, M.
Identification of a New N-Nitrosodimethylamine Precursor in Sewage Containing Industrial
Effluents. Environ. Sci. Technol. 2014, 48 (19), 11243–11250.

(3) Hinneh, K. D. C.; El Hanafi, A.; He, K.; Kosaka, K.; Echigo, S.; Asada, Y.; Itoh, S. Formation of
N-Nitrosodimethylamine by Chloramination of Anthropogenic Nitrogenous Compounds with
Dimethylamine Monitored by Japanese Water Authorities. J. Hazard. Mater. 2019, 367, 620–628.

(4) Hanigan, D.; Liao, X.; Zhang, J.; Herckes, P.; Westerhoff, P. Sorption and Desorption of Organic
Matter on Solid-Phase Extraction Media to Isolate and Identify N -Nitrosodimethylamine Precursors.
J. Sep. Sci. 2016, 39 (14), 2796–2805.

(5) Wolf, S.; Schmidt, S.; Müller-Hannemann, M.; Neumann, S. In Silico Fragmentation for Computer
Assisted Identification of Metabolite Mass Spectra. BMC Bioinformatics 2010, 11 (148), 1471–2105.

(6) Ruttkies, C.; Schymanski, E. L.; Wolf, S.; Hollender, J.; Neumann, S. MetFrag Relaunched:
Incorporating Strategies beyond in Silico Fragmentation. J. Cheminform. 2016, 8 (1), 1–16.

(7) Djoumbou-Feunang, Y.; Pon, A.; Karu, N.; Zheng, J.; Li, C.; Arndt, D.; Gautam, M.; Allen, F.;
Wishart, D. S. Cfm-Id 3.0: Significantly Improved Esi-Ms/Ms Prediction and Compound
Identification. Metabolites 2019, 9 (4), 1–23.

(8) Little, J. L.; Williams, A. J.; Pshenichnov, A.; Tkachenko, V. Identification of “Known Unknowns”
Utilizing Accurate Mass Data and Chemspider. J. Am. Soc. Mass Spectrom. 2012, 23 (1), 179–185.

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