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Systematic suspect screening and identification of sulfonamide antibiotic

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Anal Bioanal Chem
DOI 10.1007/s00216-015-8748-5
RESEARCH PAPER
Systematic suspect screening and identification of sulfonamide
antibiotic transformation products in the aquatic environment
Marius Majewsky 1 & Thomas Glauner 2 & Harald Horn 1,3
Received: 4 March 2015 / Revised: 19 April 2015 / Accepted: 28 April 2015
# Springer-Verlag Berlin Heidelberg 2015
Abstract The formation of environmental sulfonamide trans-
formation products (TPs) has only been investigated for very
selected compounds, and their transformation pathways are
incompletely elucidated. Given their homologous series, it is
likely that similar transformation reactions occur for all sul-
fonamides. It has recently been demonstrated for sulfameth-
oxazole (SMX) that its derivative TPs retain antibiotic activity.
Unfortunately, TP reference standards are often not available
and a number of TPs are still unknown. Therefore, in the
present study, a generic scheme was developed to predict 29
potential TPs of sulfonamide antibiotics via identification of
the major transformation and breakdown reactions. Mass
shifts were calculated for each transformation allowing for a
suspect screening using LC/QTOF. Based on the structural
elucidation of the parent sulfonamide product ion spectra, five
characteristic product ions could be predicted for each of 15
derivative TPs. The predictions were confirmed with refer-
ence standards of SMX TPs as well as with data of TPs of
further sulfonamides reported in literature. In a final step,
samples of activated sludge biotransformation experiments
Electronic supplementary material The online version of this article
(doi:10.1007/s00216-015-8748-5) contains supplementary material,
which is available to authorized users.
* Marius Majewsky
marius.majewsky@kit.edu
1
Karlsruhe Institute of Technology (KIT), Engler-Bunte-Institut,
Chair of Water Chemistry and Water Technology, Engler-Bunte-Ring
1, 76131 Karlsruhe, Germany
2
Agilent Technologies Sales & Services GmbH, Hewlett-Packard-Str.
8, 76337 Waldbronn, Germany
3
DVGW Research Laboratories, Water Chemistry and Water
Technology, Engler-Bunte-Ring 9, 76131 Karlsruhe, Germany
were screened for suspect TPs of two sulfonamides. In total,
13 ecotoxicologically relevant TPs could be tentatively iden-
tified by use of the product ion predictions and LC/QTOF.
Keywords Sulfa drugs . Biotransformation . Metabolites .
Intermediates . Aquatic systems
Introduction
Sulfonamide antibiotics are widely used in human and veter-
inary medicine, and their ubiquitous occurrence in wastewater
and surface waters has been well documented in litera-
ture [13, 24, 4]. There is in particular growing concern
regarding their potential to alter the composition of en-
vironmental bacterial communities [17] and to cause or
to promote antibiotic resistance in bacteria and patho-
gens [3]. While in the past risk assessment often has
only addressed the parent compounds, a focus in envi-
ronmental chemistry lately shifted to the formation, fate,
and activity of their transformation products (TPs) in
the aquatic environment. A recent study revealed the
importance to consider the acetyl-TP of the most prom-
inent sulfonamide, sulfamethoxazole (SMX), with re-
spect to its back-transformation potential [5]. Further,
it was shown that a number of SMX TPs, which feature
the sulfonamide toxicophore, exhibit similar or even in-
creased antibacterial activity compared to that of the
parent compound [20].
Frequently, fate assessment of TPs proves to be dif-
ficult since the manifold TPs first have to be identified
and second have to be synthesized or elaborately isolat-
ed and purified if not commercially available for both
analysis and fate studies. Although notable progress has
been made using accurate mass tandem mass

spectrometry for TP identification, the search for TPs -
especially in complex matrices - remains challenging if
transformation pathways are unknown. Often, the lack
of data on the environmental occurrence and fate of
TPs is because they are not available, are expensive,
or are simply unknown to this day. As a result, analyt-
ical data, such as mass spectra, are only available for
the parent compounds and some selected TPs but are
largely lacking for a wide range of TPs.
Prediction or deduction of TPs by theoretical approaches is
still very challenging. In particular, the prediction of biological
TPs is often incomplete, among others, due to the complexity
of enzyme-based biotransformation reactions [12] and the
wide range of microbial mechanisms [16]. To date, more than
15 first-daughter TPs of SMX alone have been experimentally
identified originating from biotransformation (references are
given in Table 1 and Table S1 (Electronic Supplementary
Material)). Given that approximately 20 sulfonamides are in
constant use, it is realistic to assume that a large number of
sulfonamide TPs are present in the aquatic environment. Due
to the difficulty of identifying these TPs, their environmental
occurrence and effects are largely unexplored. For these rea-
sons, in this approach, we aim to develop a general theoretical
scheme, which allows to predict and to identify suspect TPs of
all sulfa drugs with homologous structure. Although most
likely not all biotic and abiotic transformation reactions are
necessarily valid for all sulfonamides in equal measure, there
is much to suggest that their homologous structure exhibits
similar reactive sites and thus undergoes similar
transformations.
In preliminary work, TPs of SMX were exemplarily
classified regarding their antibacterial activity for SMX
[20]. Based upon this, the present study aims at identi-
fying the major characteristic environmental transforma-
tion reactions leading to ecotoxicologically relevant sul-
fonamide TPs to allow a targeted suspect screening and
tentative identification using MS/MS experiments. The
approach uses the mass shifts from the parent com-
pound to the according TPs to calculate the molecular
masses and m/z of the TP precursor ions. By structural
elucidation of the typical sulfonamide parent com-
pounds’ product ion spectra, a generalized scheme al-
lows to predict five characteristic product ions applica-
ble for (tentative) identification for each suspected de-
rivative TP of any sulfonamide. In a final step, the
scheme is verified by (i) TP reference standards, (ii)
literature data where available, and the (iii) screening
and subsequent tentative identification of TPs in bio-
transformation batch tests for two arbitrarily chosen sul-
fonamides using accurate mass LC/QTOF. The objective
was to derive a systematic workflow for a targeted
screening of sulfonamide TPs in monitoring and degra-
dation studies (Fig. 1).
M. Majewsky et al.
Material and methods
Chemicals
All reagents and solvents were HPLC or LC/MS grade. Ace-
tonitrile and methanol were purchased from Baker
(Mallinckrodt Baker, Deventer, Netherlands). Ultrapure water
was produced using a MilliQ Integral system equipped with a
0.22-μm point-of-use membrane filter cartridge (EMD
Millipore, Billerica, MA, USA). Acetic acid was from Fluka
(Fluka AG, Buchs, Switzerland). Sulfamethoxazole (CAS
723-46-6) was purchased from Sigma-Aldrich. N4-Acetyl-sul-
famethoxazole (CAS 21312-10-7), 4-nitroso-
sulfamethoxazole (CAS 131549-85-4, purity >90 %), and
N4-hydroxy-sulfamethoxazole (CAS 114438-33-4) were pur-
chased from Toronto Chemicals (Toronto, ON, Canada). NO-
sulfamethoxazole was stored at −20 °C. Sulfadiazine (CAS
68-35-9) and sulfapyridine (CAS 144-83-2) were purchased
from Dr. Ehrenstorfer GmbH (Augsburg, Germany). N4-OH-
acetyl-sulfamethoxazole, 4-NO2-sulfamethoxazole (29699-
89-6), and 4-OH-sulfamethoxazole were synthesized and con-
firmed by NMR and HRMS as described in Majewsky
et al. [20]. Accurate mass calculations and structures
were done using ChemSketch software (ACD/Labs,
http://www.acdlabs.com).
Analytical method
To record the accurate mass product ion spectra of SMX TPs,
a 1290 Infinity UHPLC system was coupled to a 6550 quad-
rupole time-of-flight LC/MS system (both Agilent Technolo-
gies, Waldbronn, Germany). Chromatographic separation was
performed at 35 °C with a flow rate of 0.5 mL min−1 using a
Zorbax Eclipse Plus C-18 (100×2.1 mm, 1.8 μm) separation
column from Agilent Technologies. Mobile phase A consisted
of 0.1 % acetic acid in ultrapure water, and mobile phase B
contained 0.1 % acetic acid in a water/acetonitrile mixture
(5/95, v/v). Injection volumes were 0.1 μL for the reference
standards and 1 to 20 μL for the samples from the activated
sludge experiments. The total run time of the chromatographic
run was 15 min. A gradient was run from 10 to 50 % mobile
phase B within 10 min and to 95 % B within the next 0.1 min.
After a hold time of 2 min at 95 % B, starting conditions of
10 % B were adjusted within 0.1 min and were kept for an-
other 2.8 min for column equilibration. The QTOF was oper-
ated in the 2-GHz extended dynamic range with positive and
negative ionization. The acquisition rate in both MS and MS/
MS modes was five scans per second, and a mass range of 50
to 1200 amu was recorded. Precursor masses for the proton-
ated or deprotonated SMX TPs were specified as target
masses, and fixed collision energies of 20 and 40 eV were
used for collisional induced dissociation. The settings of the
Agilent Jet Stream ionization source were as follows: gas
Systematic suspect screening and identification of sulfonamide antibiotic transformation products

Table 1 Potential transformation reactions of sulfonamide antibiotics observed in this study and in literature, environmental processes
for which the reaction was observed, mass shifts as compared to the parent compound, references, and availability of reference standards

+C2H3O T
[5]

4
+C2H3O2 T

[19]

4
+C2H3O2 [26] I

T
[15]

[9]
[7]

T I
[26]

[18]
T
−NH2
[10]

+O2H2 [15]

+O2H2
−NH2

−H2 [5]

+O2 T
−H2
[22]

[22]

−SO2 T

[27]
[9]
T

[9]

4
+C6H8O6 [26]

4
+C6H10O5
[9]

a
for any sulfonamide antibiotic

temperature, 160 °C; gas flow, 18 L min−1; nebulizer pressure,
35 psi (241 kPa); sheath gas temperature, 375 °C; sheath gas
flow, 12 L min−1, capillary voltage, 3500 V; and nozzle volt-
age, 1000 V. The quadrupole for precursor isolation was op-
erated with a mass resolution of 1.2 amu (FWHM). Stock
solutions of the reference compounds were prepared in aceto-
nitrile at a concentration of 1 μg mL−1.
Batch reactors with activated sludge
Biotransformation experiments were run to generate sulfon-
amide TPs for testing of the prediction scheme. Activated
sludge was collected from the nitrification tank of the Karls-
ruhe wastewater treatment plant Neureut (450,000 population
equivalents). Four hundred-milliliter washing flasks were

Fig. 1 Workflow used in this
study to identify active
transformation products of
sulfonamide antibiotics;
ecotoxicological classification of
transformation products was done
before [20]
used as batch reactors and aerated with continuously operating
aquarium pumps to ensure aerobic conditions. pH was mea-
sured in the beginning of the experiment and was 7±0.4 and
was not further controlled. Room temperature was T=20 °C.
Sulfapyridine and sulfadiazine were spiked to a final concen-
tration of 10 mg L−1 to enable the detection of TPs without
pre-concentration. Primary substrate consisting of yeast ex-
tract, tryptone, ammonium chloride, and sodium dihydrogen
phosphate monohydrate was added as CNP source (C:N:P
ratio of approximately 100:5:1) at regular intervals to sustain
microbial activity. Experiments were run over 20 days, and
samples were filtered using 0.45-μm cellulose acetate syringe
filters. Samples were subsequently frozen at −20 °C until
analysis. Primarily, the batch experiments were not designed
to mimic realistic conditions of municipal wastewater treat-
ment. Emphasis has been put on the generation of TPs, on
which the proposed identification scheme can be tested. For
that reason, experimental simplifications have been accepted
with regard to the operation and feeding of the batch reactor as
described. The extrapolation of degradation rates, formation,
and formation rates to full-scale treatment is therefore subject-
ed to these restrictions.
Results and discussion
Transformation product classification
The TPs of sulfonamides can generally be classified into (i)
products that undergo transformation (here BTraPs^), such as
hydroxylation, acetylation, or nitration, where the parent is not
decomposed, and (ii) breakdown products (here BBrePs^)
with or without transformation resulting from parent
M. Majewsky et al.
compound cleavage. In principle, transformations can also
occur after cleavage. A general scheme abstracting the possi-
ble transformation is shown in Fig. 2.
The BrePs formed from the 4-aminobenzenesulfonamide
moiety are identical for all sulfonamide antibiotics since this
moiety is the pharmacophore and therefore present in all sul-
fonamides. These BrePs are aniline, benzenesulfonamide, and
sulfanilamide. There are a variety of further common BrePs
formed via hydrolysis or hydroxylation such as 4-
aminophenol, sulfanilic acid, or benzenesulfonic acid. More-
over, the compound-specific R moiety, alone and transformed,
belongs to the BrePs. Overall, we compiled 13 first-daughter
BrePs possible for each sulfonamide, which are listed in
Table S1 (Electronic Supplementary Material). Ring cleavage
has generally not been considered. For the majority of the
BrePs, reference standards are available and therefore they
can be easily included in targeted analytical methods. Apart
from that, most of them have been documented in literature [5,
6, 25] and, thus, they should not be discussed here again.
However, what is more important - contrary to TraPs - BrePs
have been shown to lose their antibacterial (bacteriostatic)
activity [20] and therewith also their potential to cause antibi-
otic resistance of microbes. For these reasons, focus will be on
the identification of TraPs in the following sections.
Transformation reactions
Based on the TraPs reported in literature, most of them for
SMX, nine major transformation reactions for first-daughter
TraPs could be identified. These include acetylation,
glucuronidation, glucosidation, hydroxylation, nitrosation, ni-
tration, deamination, formylation, and desulfonation
(Table 1). As this study focuses on natural environmental
Systematic suspect screening and identification of sulfonamide antibiotic transformation products
Fig. 2 Homolog structure,
generic scheme, and denotation of
sulfonamide transformation and
breakdown products; SA
sulfonamides, BSA
benzenesulfonamide; for SMX:
R=3-amino-5-methylisoxazole
processes including human metabolism, transformation reac-
tions of technical treatment processes such as chlorination or
advanced oxidation processes were not included. The main
reaction sites for these transformations are the primary N4
amino group in para position, the N1 of the amide moiety,
the substituted and unsubstituted sites of the benzene moiety
as well as substance-specific reactive sites at the R moiety.
Acetylation, glucosidation, and glucuronidation mainly occur
at electron-rich nucleophilic sites such as the N4 or the N1 site.
Hydroxylation was reported to occur at the N1, but mostly at
the N4 site leading to the hydroxylamine derivative, which is
well investigated in medicinal context [7]. N4-Hydroxylation
can be the initial step for further oxidation steps resulting in
the nitroso and nitro derivatives. The latter as well as deami-
nated SMX have recently been detected in karst spring sam-
ples [22]. The authors observed deamination during denitrifi-
cation and another study showed that this TraP can also be
formed during photolysis [5]. Further, aromatic and heterocy-
clic moieties are prone to mono- or dihydroxylation. Ipso sub-
stitution of the para amino group by a hydroxy group leads to
4-OH-SA. The latter was proposed to be an intermediate of the
microbial SMX biotransformation pathway (Gauthier et al.
2011) but has not been analytically confirmed yet. Theoreti-
cally, deamination with subsequent hydroxylation at the ortho
or meta position is also possible and might only be distin-
guished by chromatographic retention from 4-OH-SAs, but
Fig. 3 Hypothesized structures, calculated m/z, measured m/z, and the according most likely elemental formula of the five selected characteristic product
ions of sulfonamide homologs in ESI+ mode during MS/MS experiments
not using MS/MS. Desulfonation was reported several times
[8, 5], but it is not fully clear to date if it is formed by envi-
ronmental processes or if the desulfonated TraP is generated in
the ion source [27]. Further, binding of a formyl group to the
N4 amino group was reported for sulfamethazine by García-
Galán et al. [9]. It should be noted that further combinations of
transformation reactions at multiple binding sites are
possible. Table 1, which gives an overview of the trans-
formation reactions for TraPs, reveals that all TraPs can
be or are likely to be formed by environmental process-
es. It is therefore difficult to exclusively assign the
mentioned reactions to one single process including hu-
man metabolism.
Precursor calculation of sulfonamide TraPs
Given the transformations in Table 1 and hypothesizing that
these transformation reactions can equally occur for any other
SA homolog, generic equations can be formulated to calculate
the molecular mass of the TraPs and, thus, the m/z precursor
ions for suspect screening using accurate mass or triple-quad
MS/MS experiments. The molecular mass of the suspect
TraPs before ionization can be calculated by
 
M ½SATraP  ¼ M SAp þ ΔM ð1Þ
 M. Majewsky et al.

Table 2 Verification of the five predicted characteristic product ion spectra of sulfonamide transformation products; predicted product ions are in line
with the TraP heading; transformation at the R moiety is specially marked

Compounds ESI 4-Aminobenzene-based product ions R-based product ions References

Sulfamethoxazole (SMX) + 92.0495 108.0444 156.0114 99.0553 161.0015 This study QTOF
4-Hydroxy-SMX + 93.0335 109.0284 156.9954 99.0558 161.0021
Measured + 93.0339 109.0286 156.9957 99.0557 161.0015 This study QTOF
Mass deviation [ppm] −0.4 −0.2 −0.3 0.1 0.6
Measured + 93 109 157 99 Gauthier et al. [10]
N -Hydroxy-SMX
4
+ 108.0444 124.0393 172.0063 99.0558 161.0021
Measured + 108.0446 124.0391 172.0063 99.0557 161.0018 This study QTOF
Mass deviation [ppm] −0.2 0.2 0.0 0.1 0.3
x-Hydroxy-SMX + 108.0444 124.0393 172.0063 99.0558 161.0021
Measured + 108.0 171.9 98.9 161.3 Hu et al. [15]
x-Hydroxy-SMX (OH at R) + 92.0495 108.0444 156.0114 115.0508 176.9970
Measured + Signala 156.0 115.0 Hu et al. [15]
4-Nitroso-SMX − 106.0287 122.0237 169.9906 97.0407 159.9943
Measured − 106.0299 122.0246 169.9915 This study QTOF
Mass deviation [ppm] −1.2 −0.9 −0.9 – –
4-Nitro-SMX − 122.0248 138.01967 185.9856 97.0407 159.9943
Measured − 122.0234 138.0426 185.9858 97.0399 161.0019 This study QTOF
Mass deviation [ppm] 1.4 −23 −0.2 0.8 −1007.6
Measured − 138 186 Nödler et al. [22]
N4-Acetyl-SMX + 134.0600 150.0550 198.0219 99.0558 161.0021
Measured + 134.0599 150.0549 198.0221 99.0553 161.0014 This study QTOF
Mass deviation [ppm] 0.1 0.1 −0.2 0.5 0.7
N -Hydroxy-acetyl-SMX
4
+ 150.0550 166.0499 214.0169 99.0558 161.0021
Measured + 150.0550 166.0499 214.0169 99.0553 161.0016 This study QTOF
Mass deviation [ppm] 0.0 0.0 0.0 0.5 0.5
Desamino-SMX − 77.0386 93.0335 141.0005 97.0407 159.9943
Measured − 77 141 Nödler et al. [22]
Sulfadiazine (SDZ) + 92.0495 108.0444 156.0114 96.0562 158.0024
4-Hydroxy-SDZ (OH at R) + 92.0495 108.0444 156.0114 112.0511 173.9973
5-Hydroxy-SDZ (OH at R) + 92.0495 108.0444 156.0114 112.0511 173.9973
Measured + 92.0494 108.0443 156.0112 112.0504 173.9964 Lamshöft et al. [18]
Mass deviation [ppm] 0.1 0.1 0.2 0.7 0.9
Measured + 92 108 156 112 174 Pfeifer et al. [23]
N4-Formyl-SDZ + 120.0444 136.0393 184.0063 96.0562 158.0024
Measured + 120 184 96 158 Lamshöft et al. [18]
N -Acetyl-SDZ
4
+ 134.0600 150.0550 198.0219 96.0562 158.0024
Measured + 134 158 198 96 Lamshöft et al. [18];
Pfeifer et al. [23]
N4-Acetyl-4-hydroxy-SDZ (OH at R) + 134.0600 150.0550 198.0219 112.0511 173.9973
Measured + 134 198 112 174 Lamshöft et al. [18]
Sulfamethazine + 92.0495 108.0444 156.0114 124.0875 186.0337
N4-SMZ-glucoside + 254.1023 270.0972 318.0642 124.0875 186.0337
Measured + 254.0971 Signala 318.0627 124.0860 186.0368 García-Galán et al. [9]
Mass deviation [ppm] 5.2 – 1.5 1.5 −3.1
N -Formyl-SMZ
4
+ 120.0444 136.0393 184.0063 124.0875 186.0337
Measured + Signala 136.0482 Signala 124.0892 186.0349 García-Galán et al. [9]
N4-Hydroxyl-SMZ + 108.0444 124.0393 172.0063 124.0875 186.0337
Systematic suspect screening and identification of sulfonamide antibiotic transformation products

Table 2 (continued)

Compounds ESI 4-Aminobenzene-based product ions R-based product ions References

Measured + 108.0415 124.0893 186.3b García-Galán et al. [9]

Mass deviation [ppm] 2.9 – – −1.8 –
Desulfonated-SMZ + 92.0495 107.0604 No logicc No logicc No logicc
Measured + 92.0465 107.0572 García-Galán et al. [9]
Mass deviation [ppm] 3.0 3.2 – – –
Desaminated-SMZ + 77.0386 93.0335 141.0005 124.0875 186.0337
Measured + 77.0410 141.0052 124.0789 186.0538 García-Galán et al. [9];
Mass deviation [ppm] −2.4 – −4.7 8.6 −20.1
Measured + 77 141 186 [2]
N -Acetyl-SMZ
4
+ 134.06004 150.05495 198.02194 124.0789 186.0538
Measured + 198 124 186 [2]

ESI electrospray ionization mode

a
Signal is observable in the PIS but m/z data was not given by the authors
b
Measured using HPLC-QqLIT
c
Generic prediction of only two product ions possible

where M[SATraP] = molecular mass of the sulfonamide
TraP, M[SA p] = molecular mass of the parent sulfon-
amide, and ΔM=the mass change of the transformation
as given in Table 1.
Using the mass shifts, 16 suspect TraPs can be computed
for every sulfonamide homolog. The accurate mass, elemental
composition, and isotopic patterns can give a first indication,
however, identification with a high degree of certainty can
only be achieved by MS/MS fragmentation and reference
standards. In contrast to the structurally simple and small-
molecular BrePs, reference standards are rarely available for
the suspected TraPs, and a systematic scheme for tentative
qualitative identification is lacking. For that reason, the prod-
uct ion spectra (PIS) of sulfonamides were elucidated to derive
another logic to predict characteristic product ions for the
TraPs in analogy to the theoretical predictions carried out for
the precursors.
PIS of sulfonamide parent compounds
Sulfonamide antibiotics were reported to show three common
characteristic product ions, namely m/z 92, 108, and 156
(ESI+) with high intensities ([11, 14]; Agilent
MassHunter Database [1]) (Fig. 3) on triple-quadrupole
MS and QTOF systems. A comparison with mass spec-
tra from the MassBank data base [21] also shows these
product ions using other MS/MS systems. This addition-
ally argues for the eligibility and representativeness of
these product ions. As these product ions are all origi-
nating from the 4-aminobenzenesulfonamide moiety,
they can be observed for all sulfonamide homologs.
On the contrary, product ions containing the mostly
heterocyclic R moiety are specific for each sulfonamide.
There are certainly further intensive product ions pres-
ent, and this can only be an incomplete list. Here, we
selected five typical intensive product ions, of which
three originate from the aminobenzenesulfonamide moi-
ety and two from the R moiety. This can later on assist
in specifying the location of the transformation on the
molecule. The proposed product ion structures were
cross-checked with the elemental formulae revealed by
LC/QTOF measurements and further references ([11];
Agilent MassHunter Database [1]).
Prediction of product ions and structural information
of sulfonamide TraPs
Based on the structural elucidation of the five product ion
spectra of the parent compounds, we hypothesize that the
mass shifts of a given transformation (Table 1) must be also
observable in the characteristic m/z of the product ions as it is
valid for the precursors. Here we can transfer the equation
formulated before (Eq. 1) to the prediction of the product ions
(Eq. 2). If the transformation takes place at the aminobenezene
moiety, i.e., at the para amino group or at one of the
aromatic sites, the m/z of the product ions originating
from this moiety shifts by the ΔM given in Table 1 with
consideration for the proton of the respective ionization
mode, while the product ions of R remain unchanged.
In the same way, m/z shifting occurs for the R-based
product ions if the transformation has occurred on the
R moiety, while the three typical aromatic product ions
92.0495, 108.0444, and 156.0114 must be observable. Further,
all product ions can shift if transformations occur on both the 4-

aminobenzene and the R moiety, as shown, e.g., for N4-acetyl-
4-hydroxy-sulfadiazine ([18], Table 2). The proposed scheme
accounts for all three possibilities.
pTP ¼ pp þ ΔM ð2Þ
where pTP =the m/z of the product ion of the sulfonamide TraP,
pp =product ion of the parent sulfonamide, and ΔM=the mass
change given in Table 1.
To verify the predicted product ions, experimental data for
accurate mass MS/MS spectra were obtained from LC/QTOF
experiments for SMX and six of its TraPs modified at the para
position, for which reference standards were available. PIS
were recorded at three different collision energies (10, 20,
and 40 V) to yield different intensities of the five product ions.
For the first time, PIS are presented for 4-OH-SMX and N4-
hydroxyl-acetyl-SMX, which both have been suggested as
TraPs of SMX [10, 19]. Additionally, supplementary PIS re-
ported in literature for TraPs of SA other than SMX were
Fig. 4 Extracted ion chromatograms (EIC) and Q/TOF mass spectra of
two sulfapyridine TraPs, (a) hydroxy-N4-acetyl-sulfapyridine and (b) N4-
formyl-sulfapyridine, and two sulfadiazine TraPs, (c) 4-OH-sulfadiazine
and (d) 4-nitro-sulfadiazine; the characteristic product ions predicted by
M. Majewsky et al.
evaluated and compared to the prediction scheme for further
verification. As can be seen from Table 2, all measured m/z
matched the predicted m/z for the listed TraPs. Mass devia-
tions between predicted and measured product ions (accurate
m/z only) were mostly below 1 ppm except for NO2-SMX and
in most instances below 5 ppm for literature data. However,
not all of the five predicted product ions were always detected,
for instance, no R-based product ions could be observed for
NO-SMX (Table 2). This is probably linked to the instrument
type and operation. Using a triple-quad LC-MS/MS, we could
clearly detect the nominal m/z of the predicted R-based prod-
uct ions.
An exception is the desulfonated TraP. The loss of SO2
from the middle of the molecule significantly changes its frag-
mentation behavior. Hence, oxygen rearrangement from SO2
to the benzene ring (Fig. 3) is no longer possible. Therefore,
only two product ions could be predicted, of which one is the
aminobenzene moiety (m/z 92.0495, ESI+) and the other is 1,
4-diaminebenzene (m/z 107.0604, ESI+). The other product
the presented scheme are given at the upper edge of the mass spectra
plots; chromatograms and mass spectra of the other TraPs observed can
be found in the Electronic Supplementary Material
Systematic suspect screening and identification of sulfonamide antibiotic transformation products
ions incorporate fragments of the compound-specific R moie-
ty, whose fragmentation behavior cannot be predicted. For
instance, fragmentation of desulfonated sulfamethazine results
in demethylation of R [9], while the R of most other SA do not
feature methyl groups.
The prediction scheme was implemented in an Excel
spreadsheet and is available free of charge from the authors.
It calculates 29 suspect TPs for each of 22 sulfonamide anti-
biotics. These suspect TPs include 13 BrePs and 16 TraPs and
for each of 15 of the latter, five characteristic product ions are
predicted. The various positions of the transformation can
accordingly be selected.
TraP identification in activated sludge batch experiments
To experimentally test the proposed scheme for the identifica-
tion of sulfonamide TraPs, two sulfonamides, sulfapyridine
(SPY) and sulfadiazine (SDZ), were chosen for biotransfor-
mation batch tests. While for SDZ some selected TraPs have
already been reported, less information is available about the
environmental TraPs of SPY. Samples of the activated sludge
Fig. 5 Extracted ion chromatogram and mass spectra of m/z 266.0594; a) hydroxylated SPY with OH located at the heterocyclic R moiety; b)
hydroxylated SPY with OH located at the aminobenzene moiety
batch reactors were screened for the suspected TraPs predicted
for each compound using accurate mass LC/QTOF. Target m/z
were fragmented at different collision energies, and the obtain-
ed PIS were then compared to the five predicted characteristic
product ions for tentative identification. In total, 13 TraPs
could be tentatively identified by matching the predicted and
measured product ions. These were for SDZ: 4-OH-SDZ (at
aminobenzene moiety), NO2-SDZ, N4-formyl-SDZ, OH-
SDZ, desamino-SDZ, N4-acetyl-SDZ, and desulfo-SDZ. The
latter has been described as an artifact of in-source fragmen-
tation [27]. In this study, it was observed to co-elute with SDZ
at the same retention time. However, the significantly higher
intensity of the desulfo-SDZ signal in the degradation exper-
iment compared to the signal coming from the in-source frag-
mentation of an SDZ reference standard indicates that desulfo-
SDZ is (also) formed as a TP in the biodegradation
experiment.
Apart from that, for SPY, N4-formyl-SPY, OH-N4-acetyl-
SPY, desamino-SPY, OH-SPY with two different locations of
the OH group, and 4-OH-SPY could be identified. To the best
of our knowledge, the formation of NO2-SDZ as well as the

SPY TraPs except for the hydroxylated SPY are reported for
the first time. Four PIS examples are given in Fig. 4; the
remaining spectra can be found in the Electronic Supplemen-
tary Material.
In the sulfapyridine batch test samples, two peaks emerged
with m/z 266.0594 [ESI+] in the extracted ion chromatograms
(EIC) suggested to be hydroxylated SPY. Fragmentation of
both peaks revealed different fragmentation patterns. The
PIS of the first peak showed all five predicted product ions
of OH-SPY (Fig. 5b) and, thus, could be tentatively identified
as SPY hydroxylated at the aminobenzene moiety. The PIS of
the second peak (Fig. 5a) indicates an unchanged
aminobenzene moiety due to the occurrence of typical frag-
ments m/z 92.0495, 108.0444, 156.0114, but the predicted m/z
173.0021 could not be observed. Further, the scheme predict-
ed m/z 111.0558, but only m/z 110.0480 occurred. Both differ
by exactly the mass of one proton (1.0078 Da), and therefore,
this difference is probably due to the localization of the ioni-
zation. Hence, we hypothesize that this compound is SPY
hydroxylated at the R moiety. This difference was also ob-
served for the R product ion of some other TPs such as 4-
NO2-SDZ (Fig. 4d), desamino-SPY (Figure S2, Electronic
Supplementary Material), or desamino-SDZ (Figure S8, Elec-
tronic Supplementary Material). Hence, the difference of one
proton for this product ion must be kept in mind when using
the scheme. A further general limitation is the possible change
of the fragmentation behavior. Even though in our study the
five product ions were accurately predicted for both our own
reference standards and data from literature, deviations from
the generic fragmentation behavior through other transforma-
tions cannot be fully excluded, e.g., as shown for
desulfonation.
The identification of sulfonamide TraPs likely to exhibit
antibacterial activity is a further step towards the assessment
of the total antibiotic stress exerted by all sulfonamides and
sulfonamide derivatives present in the aquatic environment.
The proposed scheme calculates 29 suspect TPs for each of 22
sulfonamide homologs. For each of 15 TraPs, five character-
istic product ions were predicted and confirmed with mea-
sured data. These five product ions in combination with iso-
topic patterns and accurate mass determination allow to iden-
tify the targeted suspect TraPs with a very high degree of
certainty using triple-quad or QTOF systems. The scheme
can also easily be extended to include further transformation
reactions of environmental interest such as chlorination or
ozonation.
Reference standards of TraPs are merely available, and
therefore, this approach can provide a preceding tentative
identification. This was exemplarily demonstrated in activated
sludge batch tests, in which 13 ecotoxicological relevant
TraPs of SDZ and SPY could be tentatively identified. Surely,
a definite certainty and quantification can only be achieved
with reference standards, which have to be synthesized. To
M. Majewsky et al.
date, only very selected sulfonamide TraPs are considered in
regular wastewater and surface water monitoring programs.
Even though they probably occur in the nanogram-per-liter
range, it is necessary to assure that their remaining cumulative
antibacterial activity does not pose an environmental risk, in
particular in terms of antibiotic resistance promotion.
Conflict of interest The authors declare that they have no competing
interests.
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