Identification of low molecular weight organic acids by ion chromatography/hybrid quadrupole time-of-flight mass spectrometry during Uniblu-A ozonation

Article abstract of DOI:10.1002/rcm.6429

RATIONALE: The balance of organic nitrogen and sulfur during ozonation of organic pollutants often shows a lack of complete mineralization. It follows that polar and ionic byproducts are likely to be present that are difficult to identify by liquid chromatography/mass spectrometry (LC/MS). METHODS: The structural elucidation of low molecular weight organic acids arising from Uniblu-OH ozonation has been investigated by ion chromatography/electrospray tandem mass spectrometry (IC/ESIMS/MS) employing a quadrupole timeofflight mass spectrometer. Unequivocal elemental composition of the byproducts was determined by a combination of mass accuracy and high spectral accuracy. RESULTS: The employed identification strategy was demonstrated to be a powerful method of unequivocally assigning a single chemical composition to each identified compound. The exact mass measurements of [M-H]^- ions allowed the elemental formulae and related structures of eighteen byproducts to be determined confidently. The main degradation pathways were found to be decarboxylation and oxidation. The experimental procedure allowed the identification of both nitrogen- and sulfur-containing organic acid by-products arising from Uniblu-OH ozonation. CONCLUSIONS: The obtained results are of environmental relevance for the balance of organic nitrogen and sulfur during the ozonation of organic pollutants due to the lack of complete mineralization of the compounds containing these atoms. Copyright 2012 John Wiley & Sons, Ltd. Copyright

Full text of DOI:10.1002/rcm.6429

Research Article
Received: 7 August 2012
Revised: 27 September 2012
Accepted: 28 September 2012
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2013, 27, 187–199
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6429
Identification of low molecular weight organic acids by ion
chromatography/hybrid quadrupole time-of-flight mass
spectrometry during Uniblu-A ozonation
*
Apollonia Amorisco, Vito Locaputo, Carlo Pastore and Giuseppe Mascolo
Istituto di Ricerca Sulle Acque, Consiglio Nazionale delle Ricerche, Viale F. De Blasio 5, 70132 Bari, Italy
RATIONALE: The balance of organic nitrogen and sulfur during ozonation of organic pollutants often shows a lack of
complete mineralization. It follows that polar and ionic by-products are likely to be present that are difficult to identify
by liquid chromatography/mass spectrometry (LC/MS).
METHODS: The structural elucidation of low molecular weight organic acids arising from Uniblu-OH ozonation
has been investigated by ion chromatography/electrospray tandem mass spectrometry (IC/ESI-MS/MS) employing a
quadrupole time-of-flight mass spectrometer. Unequivocal elemental composition of the by-products was determined
by a combination of mass accuracy and high spectral accuracy.
RESULTS: The employed identification strategy was demonstrated to be a powerful method of unequivocally assigning a
single chemical composition to each identified compound. The exact mass measurements of [M–H]– ions allowed the
elemental formulae and related structures of eighteen by-products to be determined confidently. The main degradation
pathways were found to be decarboxylation and oxidation. The experimental procedure allowed the identification of
both nitrogen- and sulfur-containing organic acid by-products arising from Uniblu-OH ozonation.
CONCLUSIONS: The obtained results are of environmental relevance for the balance of organic nitrogen and sulfur
during the ozonation of organic pollutants due to the lack of complete mineralization of the compounds containing these
atoms. Copyright © 2012 John Wiley & Sons, Ltd.
It is known that the removal of organic pollutants during
wastewater and drinking water treatment can be success-
fully achieved by powerful chemical methods, such as ozone
treatment alone or ozone combined with UV or hydrogen
peroxide.[1] The latter methods are known as advanced
oxidation processes (AOPs) and are based on the generation
of hydroxyl radicals (•OH), which, in turn, are able to
oxidize contaminants in a non-selective manner.[2–7] AOPs
are not employed at high dosages due to the high opera-
tional costs. This leads, in addition to the complete removal
of parent organic pollutants, to the formation of several
degradation products, which may be more toxic than the
parent compounds.[8,9] It follows that the identification of
the chemical structures of degradation products is a key
issue for the environmental understanding of the investi-
gated treatment process. As AOPs are oxidative processes,
the resulting degradation by-products are more polar
than the parent compounds, and liquid chromatography/
electrospray ionization mass spectrometry (LC/ESI-MS),
often employing accurate mass measurement, is a suitable
technique for their analysis.[10,11] The degradation products
formed in the early stage of AOPs are transient compounds
and they are further degraded, leading to the formation of
low molecular weight carbonyl compounds which, in turn,
are finally degraded to low molecular weight organic acids.
Previous investigation showed that during the ozonation of
dyestuffs, low molecular weight carbonyl compounds and
organic acids identified only on the basis of authentic
standards failed to account for the total organic carbon
present in the ozonated aqueous solution.[12] It follows that
other low molecular weight compounds are present that
are not detectable in reversed-phase LC. Ion chromatography
(IC) can, however, also be conveniently interfaced to ESI-MS
and combined with accurate mass measurement, both in single
and tandem MS, for the identification of unknown low
molecular weight organic acids.
Such an approach was used herein to identify the final
degradation product during the ozonation of hydrolyzed
Uniblu-A (Uniblu-OH) at long contact times. Uniblu-A is a
representative reactive dyestuff based on the anthraquinone
structure used in cellulose fiber dyeing. Due to their fused
aromatic structure these compounds are more resistant to
biodegradation than azo-based ones.[13] The recovery of
residual dye from spent baths is not possible because the
fixation reaction onto fibers leads to the formation of the
hydrolyzed dyestuff. As the fixation rate is usually below
90% the resulting spent waters are of no further use and they
should be disposed of properly. Therefore, the efficient
removal of residual hydrolyzed dye from wastewater is of
environmental relevance.
* Correspondence to: G. Mascolo, Istituto di Ricerca Sulle
Acque, Consiglio Nazionale delle Ricerche, Viale F. De
Blasio 5, 70132 Bari, Italy.
E-mail: giuseppe.mascolo@ba.irsa.cnr.it
Rapid Commun. Mass Spectrom. 2013, 27, 187–199
Copyright © 2012 John Wiley & Sons, Ltd.

A. Amorisco et al.
It was previously shown that ozonation of Uniblu-OH led
to the formation of intermediate by-products more polar than
the parent compound.[12] Their further degradation led to the
formation of low molecular carbonyl compounds, aldheydes
and ketones,[14] which, in turn, as a result of extensive
oxidation of the Uniblu-OH, disappeared leading probably
to hydroxylated organic acids.
pattern can be used as a tool to help identify unknowns on
various mass spectrometer types. In this case the concept
of spectral accuracy was employed to further enhance
the formula determination using a high-resolution time-of-
flight instrument.
Several methods, e.g. gas chromatography (GC),[15,16] capil-
lary electrophoresis (CE),[17,18] LC,[19,20] and various kinds of
ion chromatography, such as ion-exchange chromatography
and ion-exclusion chromatography,[21,22] are suitable for the
analysis of polyhydroxy organic acids. In particular, ion
chromatography with conductivity detection (IC-CD) has
been employed in many applications.[23–25]
EXPERIMENTAL
Chemicals
Uniblu-A (95% purity; Sigma-Aldrich, Milan, Italy) was used
without further purification. Uniblu-OH was obtained by
hydrolyzing an aqueous solution of Uniblu-A (500 mg/L) at
pH 12 and 50 ꢀC for 1 h (hydrolysis yield >90%),[14] as shown
in Scheme 1.
Water used for ion chromatography as well as for preparing
all standard aqueous solutions (18.2 MΩcm, organic carbon
content ≤4 mg/L) was obtained from a Milli-Q Gradient A-10
system (Millipore, Billerica, MA, USA). NaOH used for IC
was from J.T. Baker (Deventer, The Netherlands). All other
reagents were analytical grade and purchased from VWR
(Milan, Italy) or Carlo Erba (Milan, Italy).
The identification of final ozonation by-products should
be possible by coupling an IC system, equipped with a
membrane ion suppressor, and ESI-MS using accurate mass
measurement, both in single and tandem MS, using quadrupole
time-of-flight mass spectrometry (QqTOF-MS), although it is
not always possible to differentiate isomers. In IC/ESI-MS a
membrane ion suppressor is successfully used to minimize
background spectral interferences and to enhance the
[M–H]– ion signal. Therefore, it is possible to combine
the advantages of the unique selectivity offered by IC with the
specificity and structural elucidation capability of MS.[26–29]
IC/MS has previously been successfully used for the detec-
tion of inorganic and organic species at low detection
limits.[30,31] However, only a few studies have focused on
its use for the analysis of low molecular mass mono- and
dicarboxylic acids.[32–34] Specifically, in IC/ESI-MS the use
of a membrane ion suppressor has been proved to effectively
remove sodium ions from the mobile phase that cause
the formation in the MS interface of uncharged species (i.e.
organic/inorganic anion with a sodium counter-ion). These
neutral species reduce transfer efficiency from the atmospheric
part of the interface to the vacuum region causing, in turn, a
decrease in the MS detection sensitivity. Therefore, the ion
suppressor makes it possible to combine the advantages of
the unique selectivity offered by IC with the specificity and
structural elucidation capability of MS.[31,35,36]
Ozonation experiments
Uniblu-OH aqueous solution (500 mL), after adjusting the pH
to 7 by the addition of HCl, was placed into a Normag reactor
(Hofheim am Taunus, Germany). Ozone was produced by a
502 ozonator (Fisher, Meckenheim, Germany) fed with oxygen.
The oxygen/ozone mixture (flow rate of 1 L/min, 16 mg/L)
was split to allow only 0.1 L/min to be bubbled into the
Normag reactor. The ozone output was monitored before
each experiment by determining (by titration with sodium
thiosulfate) the amount of free iodine liberated from a potassium
iodide solution. Samples (10 mL) were withdrawn from the
reaction mixtures at scheduled times and the residual ozone
was stripped from them by purging with air.
IC/ESI-QqTOFMS and IC/ESI-QqTOFMS/MS analysis
The objective of this investigation was the identification of
low molecular weight aliphatic carboxylic acids, formed as
end products during the ozonation of hydrolysed Uniblu-A
(Uniblu-OH). The assignment of chemical structures was
made possible by combining high-resolution mass spectro-
metry data, obtained in single and in tandem MS mode, with
the information contained in high-resolution mass spectra
about the isotopic distribution of ions, defined as spectral
accuracy.[37,38] Several reports have focused on how isotope
Data acquisition
Determinations were carried out by a GS50 chromatography
system (Dionex-Thermo Fisher Scientific, Sunnyvale, CA,
USA) equipped with an AS50 autosampler, an ED50 conduc-
tivity detector and an ASRS-ultra suppressor, operated at
100 mA in external water mode. Samples, injected via a
25 mL loop, were eluted at a flow rate of 0.5 mL/min through
an analytical IonPac AS-11 column (250 mm  2 mm; Dionex)
O
O
NH2
NH
O
O
NH2
NH
SO3Na
SO3Na
OH-
SO2CH=CH2
SO2CH2CH2OH
Uniblu-A
Uniblu-OH
Scheme 1. Uniblu-A hydrolysis employed to obtain Uniblu-OH.
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Copyright © 2012 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2013, 27, 187–199

Identification of organic acid by-products by QqTOF
equipped with a IonPac AG-11 guard-column (50 mm  2 mm)
with the following gradient: from 10/0/90 (NaOH 5 mM/
NaOH 100 mM/water), held for 2.5 min, to 100/0/0 in
3.5 min, then to 50/50/0 in 12 min, held for 5 min. The flow
from the conductivity detector of the IC system was split
1:1, by means of a zero dead volume T-piece, to allow one-half
to enter the interface of the mass spectrometer. The optimiza-
tion of the ESI-MS interface was performed by using only the
IonPac AG-11 guard column in isocratic mode (0/20/80 of
NaOH 0.5 mM/NaOH 100 mM/water) at a flow rate of
0.5 mL/min. These analytical conditions were carefully chosen
in order to elute all the organic acids within 2 min and, conse-
quently, to run several injections during a MS acquisition for
optimizing each ESI interface parameter. Ten mL of a standard
solution of organic acids (1 mg/L) was injected through a
7125-Ti Rheodyne valve.
equivalents (DBEs) was set between –0.5 and 5.0. The profile
mass range, which determines the relative mass spectral range
used for the isotope profile comparison and the spectral
accuracy calculation, was set between –0.5 and 2.5. The
calibration range was centered to the approximate m/z value
of the monoisotopic peak and was 1 m/z unit wide. The spectral
accuracy was calculated as (1 – RMSE) * 100 where RMSE is the
fit error between the calibrated and theoretical spectra.
RESULTS AND DISCUSSION
A detailed screening of the degradation products formed
during Uniblu-OH ozonation was carried out. The intermediate
degradation products were identified employing conventional
reversed-phase HPLC/MS.[12] At reaction times longer than
15 min such compounds disappeared completely. Several low
molecular weight aldehydes and ketones were identified
employing derivatization with 2,4-dinitrophenyl hydrazine
(DNPH).[14] Once again, these compounds disappeared after
time but the total organic carbon content of the aqueous solu-
tion did not reduce consistently. This suggested that a number
of very polar degradation products had been formed as a result
of extensive oxidation. Such compounds are likely to be low
molecular weight organic acids. This is also consistent with
the acidic pH (3.7) of the reaction mixture at longer ozonation
times. Ozonated samples were also analyzed for organic acids
on the basis of authentic standards (oxalic and formic acids).[12]
Preliminary evidence on other minor peaks, not identified for
lack of authentic standards, suggested the presence of several
novel acids. In addition, on the basis of the low extent of both
nitrogen and sulfur mineralization during ozonation, it was
reasonable to assume the presence of very polar sulfur- and
nitrogen-containing by-products, presumably ionic, and there-
fore not detectable by reverse phase HPLC.
IC interfaced to ESI-QqTOFMS was employed for the
identification of such final by-products allowing 18
unknown by-products to be identified on the basis of
accurate mass measurements of the [M–H]– ions (Table 1).
It should be noted that the mass-measured [M–H]– ions
reported in Table 1 often do have not a single unequivocally
assigned elemental composition due to the employed
QqTOFMS instrumentation (accurate mass error 5 ppm).
The number of candidates could be reduced by limiting the
possible elements as well as by applying other chemical
constraints. In the present investigation it was only possible
to set –0.5 ≤ DBE ≤ 5.0 and to include C, H, N, O and S
for elemental composition, consistent with Uniblu-OH
ozonation at a long reaction time. In order for the elemental
composition of each by-product to be unequivocally
assigned, spectral accuracy was also employed. Spectral
accuracy is the measure of similarity between the entire ion
isotope pattern spectrum and the theoretical one. Several
papers about the calculation of theoretical isotope distribu-
tions and on convolution of a peak shape function have
been published.[39–41] The results obtained using spectral
accuracy are also listed in Table 1. It should be noted that,
despite the limited instrumental mass accuracy, the employ-
ment of spectral accuracy allowed a single elemental compo-
sition to be obtained for most of the by-products although
the mass accuracy threshold was set to 10 ppm. These
results are consistent with the theoretical considerations of
A QSTAR QqTOFMS/MS system (AB Sciex, Framingham,
MA, USA) equipped with a TurboIonSpray source operated
in negative ion mode was used throughout this work.
High-purity nitrogen gas was used as both the curtain and
the collision gas, while high-purity air was used as the
nebulizer and auxiliary heated gas. The TurboIonSpray
interface conditions were: nebulizer voltage, –4200 V;
declustering potential, –40 V; focusing potential, –120 V;
nebulizer gas flow rate, 1 L/min; curtain gas flow rate,
0.66 L/min; and auxiliary gas (air) delivered by a turbo
ꢀ
heated probe, flow rate 5.5 L/min at 450 C. Accurate mass
measurements (four decimal places) were carried out at a mass
resolution higher than 5000 (full width at half maximum) by
obtaining averaged spectra from chromatographic peaks and
then recalibrating them using the [M–H]– ions of a five-acids
mix injected post-column: C2H3O–2 at m/z 59.0139, C2HO3– at
m/z 72.9931, C2H2ClO–2 at m/z 92.9749, C8H7O–3 at m/z
151.0395 and C10H10O3Cl at m/z 213.0324 with its characteristic
fragment ion at m/z 126.9956. The product ion mass measure-
ments were carried out by fragmenting the target precursor
[M–H]– ions at an optimized collision voltage (CV) and at a
collision gas pressure of 4 mTorr. Each averaged spectrum
was recalibrated using, as a lock mass, the precursor ion mass
obtained in single MS mode. The collision energy (CE) was
optimized for each compound in order to obtain, where
possible, spectra showing acceptable signal-to-noise (S/N)
ratios in the MS/MS spectra, with both the [M–H]– ion
and the greatest number of product ions of high enough
abundance for accurate mass determination.
Data analysis
All MS data handling was performed using Analyst QS
software (AB Sciex). All mass spectral data acquired by
QqTOFMS were exported as ASCII data and analyzed by
sCLIPS (self Calibrated Line-shape Isotope Profile search)
through Mass Works (version 2.0; Cerno Bioscience, Danbury,
CT, USA). sCLIPS is a formula determination tool that performs
a peak-shape-only calibration and matches calibrated experi-
mental isotope pattern against possible theoretical ones
using the spectral accuracy as discussed later. The mass
tolerance for the sCLIPS searches was 10 ppm. The elemental
number was restricted to include C, H, O, N, and S. The
formula constraints were set by the software on the basis
of chemical rules, i.e. C, H, O ≥ 1, S ≥ 0, and N ≥ 0 or 1,
following the nitrogen rule. The number of double-bond
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A. Amorisco et al.
Table 1. Possible elemental composition for the unknown [M–H]– ions calculated by mass accuracy and by spectral accuracy
within error of 10 ppm, without atoms constraints (C ≥ 1, H ≥ 1, O ≥ 1, N ≥ 0, S ≥ 0) and with –0.5 ≤ DBE ≤ 5.0. The correct
match is ranked as number 1 by Spectral accuracy. DBE: double-bond equivalent, RMSE: fit error between the calibrated
and theoretical spectrum
By-product
number
Measured mass,
Elemental
composition
Calculated
mass (m/z)
Error
(ppm)
Elemental
composition
Spectral
accuracy
[M–H]–(m/z)
DBE
RMSE
1
2
3
138.9704
140.9860
196.9758
C2H3O5S–
C2H5O5S–
C4H5O7S–
C5H9O2S–3
C4H5O8S–
CH5N6OS–3
C5H9O3S–3
CH5N6OS–3
C4H5O8S–
C6H9O3S–3
C2HN4O–9
C5H5O8S–
C6H9O3S–3
C2HN4O–9
C5H5O8S–
C4H4NO6S–
C4H4NO6S–
C5H8NOS–3
C4H4NO6S–
C3H2NO–4
C2H2NO–5
C3HO–5
138.9707
140.9863
196.9761
196.9770
212.9711
212.9692
212.9719
212.9692
212.9711
224.9719
224.9749
224.9711
224.9719
224.9749
224.9711
177.9816
193.9765
193.9773
193.9765
115.9989
119.9938
116.9829
133.0142
149.0100
149.0092
161.0100
161.0092
179.0197
179.0205
192.9990
192.9998
–1.9
–2.2
–1.8
–6.2
–3.6
4.9
–7.6
0.3
–8.3
6.1
1.5
0.5
2.5
1.5
2.5
2.5
1.5
2.5
2.5
2.5
4.5
3.5
2.5
4.5
3.5
3.5
3.5
2.5
3.5
3.5
2.5
3.5
2.5
1.5
2.5
2.5
3.5
2.5
1.5
3.5
2.5
C2H3O5S–
C2H5O5S–
C4H5O7S–
95.3373
95.4909
95.6101
14
8
2
4
212.9703
C4H5O8S–
96.2591
95.1599
0
1
CH5N6OS–3
5
6
212.9693
224.9733
C4H5O8S–
98.0143
0
1
0
1
C5H5O8S–
98.2773
95.0093
–7.1
9.9
C2HN4O–9
7
224.9732
5.6
C5H5O8S–
98.1916
0
7.6
9.5
8
9
177.9800
193.9769
–8.8
2.1
C4H4NO6S–
C4H4NO6S–
95.1120
99.0034
0
0
–2.3
–8.1
–9.7
0.4
–0.4
1.9
–0.2
5.6
1.0
6.4
10
11
12
13
14
15
193.9749
115.9978
119.9939
116.9829
133.0145
149.0100
C4H4NO6S–
C3H2NO–4
C2H2NO–5
C3HO–5
99.0130
95.4047
95.0147
98.0959
98.6007
98.6302
0
1
1
3
0
1
C4H5O–5
C4H5O–5
C5H9OS–2
C4H5O–6
C4H5O–6
16
17
18
161.0102
179.0196
192.9994
C6H9OS–2
C5H5O–6
C5H5O–6
C5H7O–7
C5H5O–8
97.9233
98.3357
95.7336
0
0
0
C5H7O–7
–0.7
–5.5
2.1
C6H11O2S–2
C5H5O–8
C6H9O3S–2
–2.4
Kind and Fiehn who mentioned that the use of isotopic
abundance information (i.e., spectral error) can eliminate
95% of the false candidates.[42]
which spectral accuracy is able to distinguish between the
[M–H]– ions of two possible compounds with similar
absolute mass accuracy error. A spectral accuracy value of
99.0% was assigned to the ion of elemental composition
C4H4NO6S–. Once elemental compositions had been obtained
the chemical structure of the by-products was assessed by
obtaining accurate MS/MS spectra using, as a lock mass,
the precursor ion mass obtained in single MS mode. The
accurate mass measurements of both the [M–H]– ions and
their main product ions are listed in Table 2.
The effectiveness of spectral accuracy is particularly
evident for by-products 6 and 7 that being isomers both
have the elemental composition for their [M–H]– ions of
C5H5O8S–. Specifically, despite their low mass accuracies
(9.9 and 9.3 ppm), the observed isotope patterns achieved
a spectral accuracy of better than 98%. The above reported
elemental composition was associated to their [M–H]– ions
using the same s-CLIPS parameters. The comparison between
measured and theoretical isotopic pattern profiles for by-
product 6 is reported in Supplementary Fig. S1 (Supporting
Information). It should also be noted that although a second
possible elemental composition (C2HN4O–9) for the [M–H]–
ion of by-product 6 resulted from the application of spectral
accuracy, this was not chemically reasonable due to the low
number of hydrogen atoms and high number of oxygen
atoms. There was a similar situation for by-product 4 (Table 1)
where the second elemental composition obtained employing
spectral accuracy was not chemically consistent for an
oxidized sulfur-containing compound, as each sulfur atom
usually requires at least two oxygen atoms. An excellent
result was obtained for by-product 9 at m/z 193.9769 for
By-products 1 and 2 show [M–H]– ions at m/z 138.9704
and 140.9860, consistent with the elemental compositions
C2H3O5S– and C2H5O5S– (errors –1.9 and –2.2 ppm, respec-
tively). Their MS/MS spectra (Figs. 1(a) and 1(b)) show
product ions at m/z 79.9563 and 79.9582 for by-products 1
and 2, respectively, which were attributed to the radical
anion SO–3. (errors –13.3 and 10.5 ppm), suggesting that they
are sulfur-containing degradation products. Fragmentation
of the [M–H]– ion of by-product 1 also displayed the
presence of a carboxylic group in the parent compound
due to the product ion at m/z 94.9815 assigned to CH3O3S–
(error 6.9 ppm). These experimental results suggested that
by-product 1 was a carboxymethanesulfonate, probably
formed by further oxidation of the carbonyl group of the
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Identification of organic acid by-products by QqTOF
Table 2. Chemical structures of identified by-products, accurate mass measurements and elemental compositions of [M–H]– ions
as well as their product ions detected using IC/QqTOFMS and IC/QqTOFMS/MS analysis. CV: collision voltage
Measured [M–H]–
mass (m/z)
CV
(V)
Major
ions (m/z)
Elemental
composition
Calculated
mass (m/z)
Error
(ppm)
By-product
(1)
(2)
138.9704
20
138.9704
94.9815
79.9563
C2H3O5S–
CH3O3S–
SO–3.
138.9707
94.9808
79.9574
–1.9
6.9
–13.3
140.9860
196.9758
20
20
140.9860
122.9793
94.9816
79.9582
C2H5O5S–
C2H3O4S–
CH3O3S–
SO–3.
140.9863
122.9758
94.9808
79.9574
–2.2
28.8
8.0
10.5
(3)
A
196.9758
178.9585
152.9847
134.9793
80.9661
C4H5O7S–
C4H3O6S–
C3H5O5S–
C3H3O4S–
HSO–3
196.9761
178.9656
152.9863
134.9758
80.9652
–1.8
–39.6
10.6
26.3
11.3
71.0167
C3H3O–2
71.0139
40.1
B
C
(4)
212.9703
212.9693
224.9733
10
10
10
212.9703
168.9782
150.9669
96.9471
C4H5O8S–
C3H5O6S–
C3H3O5S–
HSO–4
212.9711
168.9812
150.9707
96.9601
–3.6
–17.9
–25.2
–134
(5)
(6)
212.9693
168.9832
120.9626
C4H5O8S–
C3H5O6S–
C2HO4S–
212.9711
168.9812
120.9601
–8.3
11.6
20.6
224.9733
180.9802
142.9946
110.9784
96.9623
C5H5O8S–
C4H5O6S–
C5H3O–5.
CH3O4S–
HSO–4
224.9711
180.9812
142.9986
110.9758
96.9601
9.9
–5.7
–27.9
23.8
22.7
(Continues)
Rapid Commun. Mass Spectrom. 2013, 27, 187–199
Copyright © 2012 John Wiley & Sons, Ltd.
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A. Amorisco et al.
Table 2. (Continued)
Measured [M–H]–
mass (m/z)
CV
(V)
Major
ions (m/z)
Elemental
composition
Calculated
mass (m/z)
Error
(ppm)
By-product
(7)
224.9732
224.9732
152.9861
145.0146
134.9774
110.9770
106.9830
96.9579
C5H5O8S–
C3H5O5S–
C5H5O–5
224.9711
152.9863
145.0142
134.9758
110.9758
106.9808
96.9601
9.5
–1.4
2.4
12.2
11.2
20.2
–22.7
10
20
C3H3O4S–
CH3O4S–
C2H3O3S–
HSO–4
(8)
(9)
177.9800
193.9769
177.9800
159.9744
96.0104
80.9668
C4H4NO5S–
C4H2NO4S–
C4H2NO–2
HSO–3
177.9816
159.9710
96.0091
80.9652
–8.8
21.2
13.5
19.9
20
193.9769
149.9865
113.0090
106.9815
80.9637
C4H4NO6S–
C3H4NO4S–
C4H3NO–3.
C2H3O3S–
HSO–3
193.9765
149.9867
113.0118
106.9808
80.9651
2.1
–1.0
–25.1
6.2
–18.4
(10)
(11)
193.9749
115.9978
20
20
193.9749
175.9639
119.9876
112.0060
80.9648
C4H4NO6S–
C4H2NO5S–
C3H4O3S–.
C4H2NO–3
HSO–3
193.9765
175.9659
119.9887
112.004
–8.1
–11.5
–8.9
17.7
80.9652
–4.8
115.9975
72.0099
C3H2NO–4
C2H2NO–2
115.9989
72.0091
–9.7
11.1
(12)
(13)
119.9939
116.9829
20
10
119.9939
89.9968
C2H2NO–5
C2H2O–4.
119.9938
89.9959
0.4
10.5
116.9829
72.9906
C3HO–5
C2HO–3
116.9829
72.9931
–0.4
–34.5
(14)
(15)
133.0145
149.0100
20
20
133.0145
115.0026
89.0247
72.9931
71.0142
59.0144
C4H5O–5
C4H3O–4
C3H5O–3
C2HO–3
133.0142
115.0037
89.0244
72.9931
71.0139
59.0139
–1.9
–9.4
3.2
–0.2
4.9
9.3
C3H3O–2
C2H3O–2
149.0100
130.9991
105.0185
87.0078
C4H5O–6
C4H3O–5
C3H5O–4
C3H3O–3
149.0092
130.9986
105.0193
87.0088
5.6
3.8
–7.9
–11.1
(Continues)
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Copyright © 2012 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2013, 27, 187–199

Identification of organic acid by-products by QqTOF
Table 2. (Continued)
Measured [M–H]–
CV
(V)
Major
ions (m/z)
Elemental
composition
Calculated
mass (m/z)
Error
(ppm)
By-product
mass (m/z)
(16)
A
161.0102
20
161.0102
142.9980
117.0191
99.0093
C5H5O–6
C5H3O–5
C4H5O–4
C4H3O–3
161.0092
142.9986
117.0193
99.0088
–6.4
–4.2
–2.0
5.4
B
(17)
179.0196
192.9994
20
20
179.0196
161.0095
133.0142
103.0028
99.0078
89.0234
71.0128
59.0126
C5H7O–7
C5H5O–6
C4H5O–5
C3H3O–4
C4H3O–3
C3H5O–3
C3H3O–2
C2H3O–2
179.0197
161.0092
133.0142
103.0037
99.0088
89.0244
71.0139
59.0139
–0.7
2.1
–0.3
–8.6
–9.8
–11.4
–15.5
–21.2
(18)
192.9994
149.0094
121.0140
105.0190
77.0236
C5H5O–8
C4H5O–6
C3H5O–5
C3H5O–4
C2H5O–3
192.9990
149.0092
121.0142
105.0193
77.0244
2.1
1.6
–2.0
–3.2
–10.6
previously detected 2-oxoethanesulfonic acid.[14] The
product ion mass spectrum of deprotonated by-product 2
(Fig. 1(b)) revealed an ion at m/z 122.9793 with elemental
composition of C2H3O4S–. It was formed through loss of
water from the [M–H]– ion, indicating the probable presence
of a hydroxyl group in by-product 2. This compound was a
1,2-dihydroxyethanesulfonate, formed by cleavage of the
Uniblu-OH 2-hydroxyethylsulfonyl group and hydroxylation
at the 1-position. All the information obtained from the
product ion spectra of by-products 1 and 2 is rationalized in
the fragmentation pathways depicted in Supplementary
Fig. S2 (Supporting Information) and Fig. 2, respectively.
By-product 3 showed a [M–H]– ion at m/z 196.9758 consistent
with elemental composition C4H5O7S– (error –1.8 ppm) for
which three possible chemical structures can be proposed
(Table 2). Its product ion mass spectrum (Supplementary Fig.
S3, Supporting Information) revealed an ion at m/z 80.9661
attributed to HSO–3 (error 11.3 ppm) suggesting this compound
to be another sulfur-containing by-product. Examination of the
product ion spectrum reveals other minor ions at m/z 178.9585,
152.9847 and 134.9793 with elemental compositions C4H3O6S–,
C3H5O5S– and C3H3O4S–, respectively. The first two ions can be
rationalized through loss of water and carbon dioxide from the
precursor ion, respectively. Both these product ions led to the
formation of the third product ion (m/z 134.9793) through
further loss of CO2 and water, respectively. Finally, a product
ion at m/z 71.0167, assigned to C3H3O–2 (error 40.1 ppm), can
be rationalized by loss of sulfurous acid from the above
discussed product ion at m/z 153. The high errors associated
with some of the product ions of by-product 3 are reasonable,
due to their low signal intensity. The product ion spectrum of
deprotonated by-product 3 did not allow us to unequivocally
assign its structure, there being three possible structures:
3-carboxy-3-oxopropane-1-sulfonate, substituted with
a
hydroxyl group at position 1 (A) or 2 (B) and 3-carboxy-3-
hydroxy-1-oxopropane-1-sulfonate (C). All three structures
are consistent with the fragmentation pattern outlined in
Supplementary Fig. S4 (Supporting Information).
By-products 4 and 5 are two closely eluting isomers having
[M–H]– ions at m/z 212.9703 and 212.9693. On the basis of both
spectral accuracy (Table 1) and mass accuracy (Table 2), their
elemental composition was assigned as C4H5O8S– (errors –3.6
and –8.3 ppm, respectively) suggesting that these by-products
were the result of a hydroxylation on by-product 3. Interest-
ingly, the product ion spectrum of deprotonated by-product
5 (Table 2) showed a diagnostic ion at m/z 120.9626, not
observed for by-product 4, with elemental composition of
C2HO4S– (error 20.6 ppm). Its formation was rationalized by
consecutive loss of water and formaldehyde from the ion at
m/z 168.9832 assigned to C3H5O6S– (error 11.6 ppm). This
Rapid Commun. Mass Spectrom. 2013, 27, 187–199
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A. Amorisco et al.
Figure 1. Calibrated product ion spectra of [M–H]– ions of (a) by-product 1, (b) by-product 2, (c) by-
product 9, and (d) by-product 10. [M–H]– ions at m/z 138.9707, 140.9863 and 193.9765 were used as lock
masses. CV = 20 V.
compounds were sulfur-containing by-products as demon-
strated by the presence of product ions at m/z 96.9623 and
96.9579 assigned to HSO–4 (errors 22.7 and –22.7 ppm, respec-
tively). The ions at m/z 110.9784 and 110.9770 were consistent
with the elemental composition CH3O4S– (errors 23.8 and
11.2 ppm, respectively). The presence of such a product ion
made it probable that both compounds had a hydroxyl
group at the a-position of the sulfur group. Two other ions
in the product ion spectrum of deprotonated by-product 6
(Supplementary Fig. S7(a), Supporting Information) at m/z
180.9892 and 142.9946 with elemental compositions
C4H5O6S– and C5H3O–5 (errors –5.7 and –27.9 ppm, respec-
tively) suggested neutral loss of CO2 and H2SO3, respectively,
Figure 2. Proposed fragmentation pathway of [M–H]– ion of
by-product 2 from IC/QqTOFMS/MS data at CV = 20 V.
Accurate mass measurements of product ions are reported
in Table 2.
from the [M–H]– ion. The product ion spectrum of deproto-
nated by-product 7 (Supplementary Fig. S7(b), Supporting
information) showed a base peak at m/z 152.9861 with ele-
mental composition C3H5O5S– (error –1.4 ppm) probably
arising from homolytic carbon–carbon cleavage between the
carbonyl groups in positions 3 and 4 of the [M–H]– ion. This
product ion, in turn, gave rise to ions at m/z 134.9774 and then
at m/z 106.9830 through consecutive losses of water and CO.
On the basis of the identified product ions of deprotonated
by-products 6 and 7, the fragmentation patterns outlined
in Supplementary Figs. S7(c) and S7(d) (Supporting
Information) were proposed.
finding led to by-product 5 being unequivocally assigned
as 3-carboxy-1,2-dihydroxy-3-oxopropane-1-sulfonate. The
structure of by-product 4 was proposed to be 3-carboxy-2,
3-dihydroxy-1-oxopropane-1-sulfonate. The identified
product ions of these structures were consistent with the
fragmentation patterns outlined in Supplementary Figs. S5
and S6 (Supporting Information).
By-products 6 and 7 were two closely eluting isomers, which
showed [M–H]– ions at m/z 224.9733 and 224.9732. The
elemental composition of both ions was determined to be
C5H5O8S– based on mass accuracy (errors –9.9 and –9.5 ppm,
respectively) and spectral accuracy (Table 2). Their fragmenta-
tion patterns showed significant differences, allowing the
different chemical structures to be determined. Both
By-products 8–12 all showed even-mass [M–H]– ions
suggesting that they were nitrogen-containing compounds.
The product ion spectra of deprotonated by-products 8–10
revealed the presence of an ion at m/z 81 whose accurate mass
was consistent with the elemental composition HSO–3, as
already found for by-product 3. By-products 9 and 10 showed
[M–H]– ions with the same elemental composition of
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Rapid Commun. Mass Spectrom. 2013, 27, 187–199

Identification of organic acid by-products by QqTOF
C4H4NO6S– (errors 2.1 and –8.1 ppm, respectively). However,
the fragmentation patterns of these ions showed significant
differences (Figs. 2(c) and 2(d)). Specifically, the product ion
spectrum of deprotonated by-product 9 showed a base peak
at m/z 149.9865 with elemental composition C3H4NO4S–
(error –1.0 ppm). This is consistent with CO2 loss, indicating
the presence of a monocarboxylic acid group. A close exami-
nation of this product ion spectrum revealed another minor
odd-electron ion at m/z 113.0090, corresponding to the radical
be correlated with by-product 8 by a further oxidation of
the terminal carbon. Based on the identified product ions of
by-products 9 and 10 the fragmentation patterns outlined in
Figs. 3(a) and 3(b) were proposed.
Table 1 also shows that by-products 13–18 yielded product
ions consistent with consecutive neutral loss of 44 and 18 Da,
indicating the presence of a carboxylic acid and a hydroxyl
group, respectively. Therefore, the structures of these com-
pounds were consistent with mono- and polyhydroxylated
dicarboxylic acids of low molecular weight. By-product 13
with its [M–H]– ion at m/z 116.9829, assigned to C3HO–5 (error
–0.4 ppm), was previously identified as the corresponding
DPNH derivative, derivatized at the oxo position.[14] By-
product 14 showed a [M–H]– ion at m/z 133.0145 with
elemental composition C4H5O–5 (error –1.9 ppm) whose
product ion spectrum (Fig. 4) showed an intense ion at m/z
115.0026 and a minor one at m/z 89.0247 with elemental
compositions C4H3O–4 and C3H5O3– (errors –9.4 and
3.2 ppm, respectively). These ions were formed by competi-
tive loss of water and CO2, respectively, and both underwent
further fragmentation leading to acrylate and acetate
ions through CO2 and formaldehyde loss. In addition, the
ion at m/z 72.9931, assigned to deprotonated glyoxylic acid
(error –0.2 ppm), could be rationalized by C2H4O2 loss
through cleavage at the 2-3 position on the [M–H]– ion.
This information allowed us to assign by-product 14 to
2-hydroxysuccinic acid whose fragmentation pathway is
depicted in Fig. 5. On the basis of exact mass measurement
data, and confirmed by high spectral accuracy (better than
98%), by-products 15–17 were assigned as listed in Table 2.
In particular, by-product 15 showed a [M–H]– ion at
m/z 149.0100 with elemental composition C4H5O6–
(error 5.6 ppm), consistent with the conjugated form of 2,3-
dihydroxysuccinic acid. This suggested that the compound
was formed by a further hydroxylation of by-product 14.
The product ion spectrum of deprotonated by-product 16
did not allow us to distinguish between two possible
dicarboxylic acid structures: 3-hydroxy-2-oxopentanedioic
acid and 2-hydroxy-4-oxopentanedioic acid. By-product 17
showed a [M–H]– ion at m/z 179.0196 with elemental compo-
sition C5H7O–7 (error -0.7 ppm), assigned as deprotonated
2,3,4-trihydroxypentanedioic acid.
–
•
ion with an elemental composition of C4H3NO3 , which
–
•
derives from elimination of the HSO3 radical ion from
the [M–H]– ion. This by-product was assigned as 3-carboxy-
3-nitrosoprop-1-ene-1-sulfonate. The product ion mass
spectrum of deprotonated by-product 10 revealed an ion at
m/z 175.9639 and an odd-electron ion at m/z 119.9876, consis-
tent with elemental compositions C4H2NO5S– and C3H4O3S–.
(errors –11.5 and –8.9 ppm), respectively. The former ion was
derived through the loss of water, while the latter derived
from consecutive elimination of carbon dioxide and an NO.
radical. Both these product ions suggest the presence of
a monocarboxylic acid. In addition, the base peak of the
product ion spectrum was at m/z 112.0060 with elemental
composition C4H2NO–3 (error 17.7 ppm) probably arising
from loss of H2SO3 from the [M–H]– ion and further
migration of the NO group to the terminal carbon. The
structure was thus assigned as 3-carboxy-3-nitrosoprop-1-
ene-2-sulfonate, with the nitro group probably located at the
b-position of the carboxylic acid group. This compound can
a
115.0026
55
50
45
b
71.0142
by-product 14
40
35
30
25
133.0142
20
15
10
5
72.9931
89.0247
90
59.0144
60
0
50
70
80
100 110 120 130 140 150
m/z
Figure 3. Proposed fragmentation pathway of [M–H]– ions
of by-product 9 (a) and 10 (b) from IC/QqTOFMS/MS data
at CV = 20 V. Accurate mass measurements of product ions
are reported in Table 2.
Figure 4. Calibrated product ion spectrum of [M–H]– ion of
by-product 14 at CV = 20 V. [M–H]– ion at m/z 133.0142 was
used as a lock mass.
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A. Amorisco et al.
6e+5
4e+5
2e+5
0
by-product 1
by-product 2
by-product 3
by-product 11
by-product 13
by-product 15
0
50
100
150
200
ozonation time (min)
by-product 7
by-product 8
by-product 9
by-product 10
by-product 12
by-product 18
30000
20000
10000
0
Figure 5. Proposed fragmentation pathway of [M–H]– ion of
by-product 14 from IC/QqTOFMS/MS data at CV = 20 V.
Accurate mass measurements of product ions are reported
in Table 2.
The last identified compound, by-product 18, showed a
[M–H]– ion at m/z 192.9994, with elemental composition
C5H5O–8 (error 2.1 ppm), whose product ion spectrum
(Supplementary Fig. S8, Supporting Information) showed
major ions at m/z 149.0094 and 105.0190 with elemental
compositions C4H5O6– and C3H5O–4 (errors 1.6 and –3.2 ppm,
respectively). These ions were formed by consecutive losses of
CO2 from the [M–H]– ion, indicating that this by-product was
a dicarboxylic acid. A product ion at m/z 121.0140 of elemental
composition C3H5O–5 (error –2.0 ppm) was derived by CO
elimination from m/z 149. This finding allowed the oxo group
to be located at the 4-position. The structure of this compound
was assigned as 2,2,3-trihydroxy-4-oxopentanedioic acid.
Interestingly, the absence of any water loss in the product ion
spectrum could be explained by the fact that all positions on
the aliphatic chain are locked and thus the dehydration
pathway was not possible. The information obtained from
the product ion spectrum was rationalized in the fragmenta-
tion pathway depicted in Supplementary Fig. S9 (Supporting
Information).
0
50
100
150
200
ozonation time (min)
Figure 6. Formation/degradation profiles of selected
identified carboxylic acids (extracted ion peak area from
IC/ESI-QqTOFMS) during ozonation of Uniblu-OH.
identified over a long ozonation time, could help in
understanding the reaction pathway depicted in Fig. 7.
The identified organic acids (Table 1) arose from further
degradation of low molecular weight carbonyl compounds,
namely keto-acids and aliphatic aldehydes detected in the
early stage of the ozonation process.[14] Although complex
competitive and consecutive reactions occurred during ozona-
tion, it was possible to describe a degradation pathway on the
basis of reasonable chemistry, supported by some observed
correlations between the by-products belonging to the same
class. After the formation of the sulfur-containing by-products
1–7 and by-products 16 and 17, the degradation proceeded
further with the decarboxylation of by-product 16 (A) and 16
(B) followed by oxidation of the terminal carbon of the formed
intermediate, leading to by-product 14. This oxidation process
was based on hydrogen homolytic abstraction by a hydroxyl
radical leading to a peroxy radical which, in turn, was
transformed into a hydroperoxide. By-product 13 was formed
from by-product 17 by means of the same oxidation route.
It followed that three of the detected by-products all led
to the formation of a single compound, 2-oxomalonic acid
(by-product 13). This was also consistent with the formation/
decomposition profiles of the detected by-products. By-
product 13 was the lowest molecular weight dicarboxylic acid
detected and it persisted in high abundance at long reaction
times (Fig. 6). The finding that 2-oxomalonic acid slowly
disappeared at longer ozonation time suggested that it was
Formation mechanism of identified low molecular weight
organic acids
The formation/degradation of identified organic acids was
monitored over a long ozonation time in order to gain
insights into their formation mechanism, and selected
measured profiles are depicted in Fig. 6. The identified
by-products were consistent with further oxidation and
decarboxylation of the previously identified carbonyl by-
products.[14] From Fig. 6 it can be seen that by-product 12
was always formed at ozonation times longer than 90 min,
suggesting that it was an end by-product. A careful analysis
of the measured profiles shown in Fig. 6, including possible
structures of low molecular weight carboxylic acids
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Rapid Commun. Mass Spectrom. 2013, 27, 187–199

Identification of organic acid by-products by QqTOF
Figure 7. Proposed degradation pathway for Uniblu-OH ozonation leading to low
molecular weight organic acids.
degraded to oxalic and/or formic acid, according to previous
investigation employing authentic standards.[12] The same
aforementioned mechanisms including decarboxylation and
subsequent oxidation can be applied to all the sulfur- and
nitrogen-containing by-products 1–12 in order to explain
the formation of their lower molecular weight homologues.
Sulfur-containing by-products were still persistent at longer
ozonation time confirming that the sulfur present in Uniblu-
OH did not undergo complete mineralization. Finally, by-
product 12 was still formed at longer ozonation time (Fig. 6)
and therefore was another end product. This confirmed the
finding that the organic nitrogen compound, Uniblu-OH,
was not completely mineralized.
the sCLIPS algorithm allowed the unequivocal identification
of 18 low molecular weight organic acids as a result of
extensive oxidation of the parent organic pollutant. Excellent
spectral accuracy of better than or close to 95% was found for
most of the identified compounds. Most of the by-products
gave rise in the MS collision cell to either single or
double CO2 and water loss, consistent with assignment to
polyhydroxylated carboxylic acids. Some of identified
organic acids were shown to be both sulfur- and nitrogen-
containing carbonyl by-products. Due to the complexity of
reactions involved during Uniblu-OH ozonation it was
possible to assess the presence of correlations between
different identified by-products, such as polyhydroxylated
carboxylic acids. These compounds in turn decompose
to known low molecular weight organic acids through
decarboxylation and further oxidation and then undergo
complete mineralization. However, sulfur- and nitrogen-
containing compounds still remain at longer ozonation
times. This result is of environmental interest due to the
potential toxicity of such compounds.
CONCLUSIONS
The coupling of ion chromatography (IC) and electrospray
ionization mass spectrometry (ESI-MS), proved to be a
powerful technique for the identification of low molecular
weight organic acids formed as final by-products of Uniblu-OH
degradation by the ozonation process, an oxidation method
widely employed to degrade recalcitrant organic pollutants
in industrial wastewater. Organic acid identification was
possible due to the employment of a NaOH gradient and
a membrane ion suppressor that minimized the background
spectral interferences and enhanced the signals of the [M–H]–
ions. The combination of mass accuracy, MS/MS information
and high spectral accuracy was demonstrated to be a
powerful method for unequivocally assigning a single
chemical composition to each identified compound. Specifi-
cally, the employment of the spectral accuracy concept using
SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article.
Acknowledgements
This work was partially supported by Apulia Region by
funding from the Rela-Valbior Project within the Scientific
Research Framework Program 2007-2013.
Rapid Commun. Mass Spectrom. 2013, 27, 187–199
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A. Amorisco et al.
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