Aqueous chlorination of sulfamethazine and

FOR APPROVAL ONLY

Received: 1 November 2017

Revised: 18 March 2018

Accepted: 4 April 2018

DOI: 10.1002/jms.4191 Journal of

MASS SPECTROMETRY

RESEARCH ARTICLE

Aqueous chlorination of sulfamethazine and sulfamethoxypyridazine: Kinetics and transformation products identification | Aurélien Trivella3,4 Rania Nassar1,2,3,4 | Ahmad Rifai2 Samia Mokh2 | Mohamad Al‐Iskandarani1,2

|

Patrick Mazellier3,4

|

1

Faculty of Public Health I, Lebanese University, Hadath, Lebanon

Abstract

2

Sulfonamides (SNs) are synthetic antimicrobial agents. These substances are continu-

Laboratory for Analysis of Organic Compound (LACO), Lebanese Atomic Energy Commission (LAEC), Lebanese National Council for Scientific Research (CNRSL), 11‐ 8281, Riad El Solh, Beirut 1107 2260, Lebanon

3

Univ. Bordeaux, UMR EPOC CNRS 5805, LPTC, Talence F‐33405, France

4

CNRS, EPOC, UMR 5805, LPTC, Talence F‐33400, France

Correspondence Mohamad Al‐Iskandarani, Faculty of Public Health I, Lebanese University, Hadath, Lebanon. Email: [email protected]

ally introduced into the environment, and they may spread and maintain bacterial resistance in the different compartments. The chlorination of 2 SNs, namely, sulfamethazine (SMT) and sulfamethoxypyridazine (SMP), was investigated to study their reactivity with chlorine at typical concentrations for water treatment conditions. Experiments conducted in purified water show an acceleration of SMT and SMP degradation of a factor 1.5 by comparison to drinking water matrix. This difference is due to pH variation and competitive reactions between SNs and mineral and organic compounds, with chlorine in drinking water. In the presence of an excess of chlorine (6.7 μmol·L−1) in ultrapure water at pH 7.2, second‐order degradation rate constants were equal to 4.5 × 102M−1·s−1 and 5.2 × 102M−1·s−1 for SMT and SMP, respectively. The structures of transformation products were investigated by liquid chromatography tandem mass spectrometry analyses with equimolar concentrations between chlorine and SNs. SO2 elimination, cyclization, and electrophilic substitutions were the main pathways of by‐products formation. Moreover, the toxicity of the proposed structures was predicted by using toxicity estimation software tool program. The results indicated that most by‐products may present developmental toxicity. KEY W ORDS

antibiotics, by‐products, chlorination, sulfamethazine, sulfamethoxypyridazine, sulfonamides

1

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humans through the food chain and drinking water.5,6 Sulfamethazine

I N T RO D U CT I O N

and SMP are amphoteric polar substances that are readily soluble in Sulfamethazine (SMT) and sulfamethoxypyridazine (SMP) are 2 antibi-

water (1.5 and 3.3 g·L−1, respectively). Therefore, they possess high

otics that belong to the pharmaceutically important group of

migration ability in environmental water matrices.7 Sulfonamides were

heterocyclic SNs, which are widely used in human and veterinary med-

detected in surface water and groundwater at concentration levels6,8,9

1

icine to treat infections. They act against the gram‐negative and

ranged from 10−9 to 10−6 g·L−1.

gram‐positive bacteria by inhibiting bacterial folic acid synthesis and 2,3

stopping the DNA replication.

They have been introduced into the

In water treatment plants (WTPs), UV radiations, ozonation, and chlorination are used because of their efficiency for disinfection.10,11

environment and are suspected to spread bacterial resistance. This

However, free chlorine including HOCl and ClO− species is the most

problem is one of the major concerns due to the presence of antibiotic

used due to its low cost and high activity for oxidation.12-14

4

residues into the environment. Furthermore, this class of antibiotics is

Chlorination is mainly applied for water disinfection but rarely

considered potentially toxic for aquatic organisms and eventually to

used for oxidation of micropollutants because the reaction can

J Mass Spectrom. 2018;1–10.

wileyonlinelibrary.com/journal/jms

Copyright © 2018 John Wiley & Sons, Ltd.

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ET AL.

produce biologically active transformation products.15 Thus, it is nec-

system at different reaction time.19 The same conditions were used

essary to study the reactivity of micropollutants towards chlorine.

for samples prepared in drinking water (pH = 7.7). On the other hand,

Prior studies reported that chlorine can easily react with some antibi-

the inhibiting effect of Na2S2O3 towards the chlorination reaction was

otics like sulfamethoxazole, ciprofloxacin, enrofloxacin, and tetracy-

tested. In this aim, 2 samples containing SNs and Na2S2O3 were pre-

cline. Degradation reactions followed second‐order kinetics.16

pared. Chlorine was added only in one of them. Sulfonamides area

Chlorination of antibiotics can lead to the formation of intermediates

obtained by HPLC‐UV analysis was the same for both samples. The

with increased toxicity relative to the parent compound.5 Several

determination of rate constants of reaction was based on a previous

authors reported the chlorination by‐products identification of sulfon-

study of el Najjar et al15 on the chlorination of levofloxacin at

amides.1,17,18 Recently, de Jesus Gaffney et al5 have shown the effi-

pH 7.2; el Najjar et al15 explained that in the presence of a large

ciency of free chlorine on the oxidation of 6 sulfonamides under

excess of chlorine, the reaction is of apparent first‐order with respect

common conditions used in drinking WTPs, and they identified struc-

to sulfonamide concentration:

tures of by‐products. To date, no kinetics studies were reported on chlorination of SMP, and no data described their transformation products.

d½SNs ¼ −k·½chlorine·½SNs; dt

(1)

d½SNs ¼ −kapp ½SNs; dt

(2)

Aims of the present study were to assess SMT and SMP fates during chlorination, to evaluate rate constants of reaction in different water matrices and to proceed to structural identification of degradation products by high‐performance liquid chromatography (HPLC) coupled to a tandem mass spectrometer (LC‐MS/MS).

where [SNs] and [chlorine] are the initial concentrations (mol·L−1), respectively, in SNs and chlorine, k is the second‐order rate constant

2 2.1

EXPERIMENTAL SECTION

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|

of chlorination reaction (L·mol−1·s−1), and kapp is the apparent first‐ order rate constant (s−1). The reaction order relative to chlorine was then examined in UP

Chemicals

water at pH 7.2 by varying the chlorine concentration (6.7 ≤ [chlo-

High purity standards of SMT (≥99%) and SMP (≥97%) were pur-

rine] ≤ 40.5 μmol·L−1) and monitoring the disappearance of SNs. The

chased from Sigma‐Aldrich. Chlorine stock solutions were prepared

second‐order rate constant was calculated by dividing the measured

by dilution from commercial 5% sodium hypochlorite solution and

apparent first‐order rate constant by the initial concentration of

standardized by iodometric titration. Organic solvents (methanol and

chlorine.

acetonitrile) and DPD N,N‐diethyl‐p‐phenylenediamine reagent were

For the structural identification of transformation by‐products,

supplied by Sigma‐Aldrich. Phosphate buffer, Na2S2O3, and colorimet-

chlorination experiments were performed in 100 mL of UP water at

ric agents were supplied by Prolabo.

pH 7.2. The initial concentrations of SNs and free chlorine were 36

Chlorine and antibiotic stock solutions were prepared in purified water (ultrapure [UP]) obtained from Sigma‐Aldrich (with impu-

and 40 μmol·L−1, respectively. Unquenched samples were analyzed at different chlorination time by using LC‐MS/MS.

rity < 10−4%). Drinking water was sampled from the WTP of Dbayeh‐Lebanon and filtered with a 0.45‐μm membrane prior to

2.3

|

LC‐UV and LC‐MS/MS analyses

HPLC analysis. Physicochemical properties of this water are described Concentration of SNs compounds was quantified as a function of

in Table S1.

chlorination time by liquid chromatography (Agilent 1100 series)

2.2

|

Chlorination experiments

coupled with a UV detector (LC‐UV). Absorbance of SMT and SMP was recorded at 260 nm. A Nucleosil C18 column (5 μm‐100 Å,

Kinetic studies were conducted in a 500‐mL Pyrex glass sealed reactor

250 mm × 4.6 mm) was used for SMT and SMP analyses. The elution

surrounded by an aluminum foil to avoid light effect and maintained

of SNs was performed in isocratic mode. The eluent was constituted

under magnetic stirring. Experiments were performed in triplicate at

of 65% of methanol and 35% of UP water, and the flow rate was set

25°C in UP water at pH 7.2 using phosphate buffer1,17 (1 mmol·L−1)

to 1 mL·min−1; 20 μL of sample was injected.

and in drinking water with an initial concentration of chlorine of

The structural identification of transformation by‐products was

6.7 μmol·L−1. Chlorination kinetics started by mixing the chlorine stock

performed with an HPLC system (Agilent 1200, Agilent Technologies,

solutions to SMT (pKa1 = 2.8, pKa2 = 7.0) or SMP (pKa1 = 2.2,

USA) coupled to an Agilent 6410 triple quadrupole mass spectrometer

pKa2 = 7.3) at a concentration of 0.3 μmol·L−1. At scheduled time,

(LC‐MS/MS). The same separation conditions as above were used

1.0 mL of solution was transferred into vials containing 100 μL of

except a flow rate of 0.6 mL·min−1. The mass spectrometer was

−1

sodium thiosulfate (1 mmol·L ) to quench the chlorination reaction.

equipped with an electrospray ionization source and operated in

In the meantime of experiments, the variation of chlorine concentra-

positive mode. The following conditions were used: source tempera-

tion (measured by DPD procedure) was less than 5%, and no pH

ture, 450°C; capillary voltage, 3000 V. The collision energy was varied

variation was observed. Samples were analyzed using HPLC‐UV. For

from 5 to 30 V to obtain the fragmentation patterns when performing

checking the feasibility of using Na2S2O3 for quenching the chlorine

product ion scans. Nitrogen was used as collision and nebulization

action, unquenched samples were directly injected into the HPLC‐UV

gases at 30 and 40 psi, respectively.

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Computer‐aided toxicity prediction

Toxicity estimation software tool (TEST) is an Environmental Protection Agency (US EPA) online available predictive system with quantitative structure‐activity relationship (QSAR) mathematical models. The TEST has different toxicity endpoints used to predict chemical toxicity values from the physical properties of the molecular structure. The TEST model uses a simple linear function of molecular descriptors such as the octanol‐water partition coefficient, the molecular weight, or the number of benzene rings. Toxicity ¼ ax1 þ bx2 þ c;

3

water were determined from the slope of the linear time‐course plot of ln([SNs]t/[SNs]0) showed in Figure 1. Sulfamethazine degradation rate constants were, respectively, (3.0 ± 0.3) × 10−3 and (1.8 ± 0.2) × 10−3 s−1 in UP and drinking waters (Figure 1A). The value of kapp obtained in chlorination experiment of SMT is threefold higher than that obtained by Chamberlain and Adams17 due to the difference in initial experiment conditions. Indeed, the kinetic experiment of this latest research was performed in distilled water at pH 7.6 and in presence of 0.1 mg·L−1 of chlorine. Subsequently, the concentration of HOCl decreased with the decrease of initial chlorine concentration and

the

increase

of

pH.

In

case

of

SMP,

kapp

were

(3.3 ± 0.2) × 10−3 s−1 and (2.3 ± 0.3) × 10−3 s−1 in UP and drinking

where x1 and x2 are independent descriptor variables and a, b, and c

waters, respectively (Figure 1B). The degradation of the 2 SNs com-

are fitted parameters. Models for assessing toxicity solely from molec-

pounds is about 1.5‐fold faster in UP water than in drinking water

ular structure are based on information‐rich structural descriptors that

matrix certainly due to the presence of organic and inorganic com-

quantify transport, bulk, and electronic attributes of a chemical struc-

pounds that enter in competition for the chlorination reaction11 and

ture. Besides molecular weight, the QSAR model uses size‐corrected

the difference of pH between both waters.

E‐values for quantification of molecular bulk. The size‐corrected

Figure 2 shows the reaction order corresponding to chlorine

E‐values are computed from a rescaled count of valence electrons.

assessed by studying the variation of kapp under varying chlorine

Electrotopological state values (Evalues) as numerical quantifiers of

concentrations. Second‐order rate constant of SMT degradation

molecular structure encode information about the electron content

(kHOCl/SMT) is enclosed between 2.9 × 102M−1·s−1 and 4.5 × 102M−1·s−1,

(valence, sigma, pi, and lone pair), topology, and environment of an

while kHOCl/SMP is ranging from 2.9 × 102M−1·s−1 to 5.2 × 102M−1·s−1

atom or a group of atoms in a molecule. The predicted toxicity is esti-

(Table S2). The values of apparent first‐order and second‐order rate

mated by taking an average of the predicted toxicities from the above

constants of SMT and SMP are close due to the similarity of

QSAR methods20-22 provided that the predictions are within the

structures.

respective applicability domains. We were mainly interested in the oral rat LD50, fathead minnow LC50 during 96 hours, the development toxicity, and the mutagenicity results.23,24 In previous study,25,26 oral rat LD50 of SMT was determined to be ranged from 2000 to −1

3.2

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Chlorination pathways of SMT and SMP

10 000 mg·kg , while the SMP one was ranged from 1700 to

Sulfamethazine and SMP have structural similarities (Figure 3), espe-

4500 mg·kg−1.

cially the 4‐amino‐benzenesulfonamide part that is present in both structures. 4,6‐Dimethylpyridin‐2‐yl and 6‐methoxypyridazin‐3‐yl cor-

3

RESULTS AND DISCUSSION

|

respond to the second part of SMT and SMP structures, respectively. Therefore, same degradation pathway was expected after the attack

3.1

|

Kinetics data

of chlorine. Scheme 1 showed the chlorination pathways of SMT and SMP. First, the presence of nitrogen in the aromatic ring of SMT

Chlorination reactions in UP water and drinking water exhibit an

and SMP led to the formation of chloramine or chlorammonium, as

apparent first order with respect to the SNs concentration. Apparent

intermediates, able to change the reactivity of the 2 molecules to

first‐order rate constants (kapp) obtained in UP water and drinking

obtain the first generation of by‐products. Second, the successive

(A)

(B)

FIGURE 1 Apparent first‐order kinetics plot of (A) sulfamethazine (SMT) and (B) sulfamethoxypyridazine (SMP) chlorination in ultrapure (UP) (Δ, pH 7.2) and drinking (●, pH 7.8) waters ([SNs]0 = 0.3 μM, [chlorine]0 = 6.7 μM, room temperature). SNs, sulfonamides

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FIGURE 2 Evolution of apparent first‐order rate constants of sulfonamides chlorination as a function of initial chlorine concentration in ultrapure water at pH 7.2 and room temperature. SMP, sulfamethoxypyridazine; SMT, sulfamethazine of SMP where the electrophilic attack was on the tertiary amine of SMP pyridinium ring. These 2 electrophilic substitutions were followed by an elimination of 7 center of HCl and proton loss to give the proposed structures of SMT‐5 and SMP‐2. The loss of SO2 is one of the main pathways of SNs degradation.5 In the case of SMT, the loss of SO2 led to obtain the proposed structure of SMT‐2. Furthermore, an electrophilic attack of chlorine on tertiary amine of SMT structure led to obtain a cationic unstable intermediate, which gave FIGURE 3 Molecular structures of sulfamethazine (SMT) and sulfamethoxypyridazine (SMP) [Colour figure can be viewed at wileyonlinelibrary.com]

the proposed structure of SMT‐3 after elimination of HCl and H+. The electrophilic attack of chlorine on tertiary and not on secondary amine is due to the steric hindrance caused by the presence of sulfur dioxide and the 2 conjugated rings. The last degradation pathway was

electrophilic attack of chlorine led the formation of the second gener-

not observed in the case of SMP degradation. On the other hand, a

ation of by‐products.

direct substitution of chlorine on sulfur cuts the molecules of SMT

In fact, the electrophilic substitution on primary amine of SMT

and SMP into 2 moieties. The first one was proposed to be

structure led to obtain primary chlorammonium followed by the for-

4‐aminobenzenesulfonyl chloride, and it was not detected by LC‐MS

mation of SMT‐4. The latest pathway was not observed in the case

analysis.

The

second

moieties

labeled

SMT‐1

and

SMP‐1

SCHEME 1 Chlorination pathways of sulfamethazine (SMT) and sulfamethoxypyridazine (SMP) [Colour figure can be viewed at wileyonlinelibrary.com]

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corresponded to pyrimidine and pyridazine groups attached to NH2 of SMT and SMP, respectively,

5

The structure of SMT‐1 product (m/z 124, Figure 4) is supported by results obtained by García‐Galán et al8 and Nassar et al.27 This compound is known as the 2‐amino‐4,6‐dimethylpyrimidine. SMT‐1 losts 4,6‐dimethylpyrimidin‐2‐amine, NH3 (−17), and CH2CHCH3

3.3 | Fragmentation pathways of SMT and SMP by MS/MS The fragmentation spectra of SMT and SMP (Figures S1 and S2) showed 3 common ions corresponding to aniline, ethenesulfonamide, and 4‐sulfonylaniline ions with m/z 92, 108, and 156, respectively. The other ions can be divided into 3 groups, and each one contains 2 ions with close values of m/z. The ions with m/z 124 (SMT spectra) and 126 (SMP spectra) were proposed to be 4,6‐dimethylpyrimidin‐2‐ amine and 6‐methoxypyridazin‐3‐amine, respectively. They were obtained after elimination of 4‐aminobenzenesulfonamide. The ions with m/z 186 (SMT spectra) and 188 (SMP spectra) were proposed to be N‐(4,6‐dimethylpyrimidin‐2‐yl) sulfonic amide and N‐(6‐methoxypyridazin‐3‐yl) sulfonic amide, and they were obtained after elimination of aniline ring. Finally, the loss of SO2 followed by the elimination of H2 in 7 center led to the formation of fragment ions with m/z 213 and 215 showed in SMT and SMP fragmentation spectra, respectively. The fragmentation pathway of SMT and SMP was shown in Scheme 2.

(−42) to obtain the 3‐amino‐6‐iminocyclohexa‐2,4‐dien‐1‐ylium (m/z

108),

dimethylated

pyrimidine

ring

(m/z

107),

and

1‐ethylidyneguanidinium (m/z 82). The elimination of NH3 was followed by the loss of CHCCH3 (−40) to give N‐cyanoethanaminium (m/z 67). SMT‐2 (m/z 215) and SMT‐3 (m/z 213) are both formed after release of SO2 from SMT. SMT‐2 losts NH3 (−17), CH2CHCH3 (−42), and CHCCH3 (−40) to give the ions of 4‐((4,6‐dimethylpyrimidin‐ 2‐yl)amino)benzene‐1‐ylium

(m/z

198),

1‐(4‐aminophenyl)‐3‐

ethylidyneguanidinium (m/z 173), and (Z)‐5‐(but‐1‐en‐3‐yn‐1‐yl)‐N‐ ethylidyne‐1H‐imidazol‐2‐aminium

(m/z

158).

After

multiple

rearrangements, the pseudo molecular ion of SMT‐2 gave the ion of 1‐ethylidyne‐3‐methylguanidinium (m/z 99). The ions at m/z 108, 107, and 92 presented the 2 parts of the molecules of 3‐amino‐6‐ iminocyclohexa‐2,4‐dien‐1‐ylium, dimethylated pyridine ring, and 4‐aminobenzene‐1‐ylium. All fragment structures proposed were presented in Figure 5. SMT‐3 arises after elimination of NH3 (−17), HCN (−27), and CH3CN (−41) to give 2,4‐dimethylbenzo[4,5]imidazo[1,2‐a]pyrimidin‐ 7‐ylium (m/z 196), 4‐amino‐2‐(3,5‐dimethyl‐1H‐pyrazol‐1‐yl)benzene (m/z 186), and 1‐ylium‐6‐amino‐1‐methyl‐3H‐azeto[1,2‐a]benzo[d]

3.4

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By‐products identification

imidazol‐8‐ium (m/z 172). The elimination of NH3 was followed by the loss of CH3CN to give the ion of 1‐methylazeto[1,2‐a]benzo[d]

Chlorination of SMT and SMP leads to 5 (SMT‐1 to SMT‐5) (Figure S3)

imidazol‐6‐ylium (m/z 155). The 2 ions at m/z 107 and 92 were also

and 2 (SMP‐1 and SMP‐2) (Figure S4) transformation products,

observed, corresponding to dimethylated pyrimidine ring and

respectively. Table 1 gathers structural information recorded by

4‐aminobenzene‐1‐ylium (Figure 6).

LC‐MS/MS.

SMT‐4 (m/z 313) and SMT‐5 (m/z 247) were identified as primary

Fragmentation spectra of SMT transformation products allow to

chloramines. The elimination of the N‐chlorocyclobuta‐1,3‐dien‐1‐

observe 2 ions at m/z 107 and 108. These ions correspond to the

amine gave the ion at m/z 211, while SMT‐5 losts the chlorine radical

substituted pyrimidine ring. The second one has also been observed

to give the ion at m/z 212. In other hand, SMT‐4 losts the

in the fragmentation spectrum of SMT, which suggests that no chem-

N‐chloroaniline to give the ion at m/z 186. The fragmentation pattern

ical change occurred in the corresponding part of the compound.

of SMT‐4 (Figure 7) showed also the presence of ions at m/z 142 and

SCHEME 2 Tandem mass spectrometry fragmentation pathways of sulfamethazine (SMT) and sulfamethoxypyridazine (SMP) [Colour figure can be viewed at wileyonlinelibrary.com]

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TABLE 1

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ET AL.

LC‐MS/MS ESI+ analysis of SMT and SMP and of their degradation products

Name

m/z (RT)

Proposed Chemical Formula

Fragments (Relative Intensity in %)

SMT

279.2 (7.2)

C12H15N4O2S

279.2 (10), 213.2 (15), 186.1 (100),156.0 (45), 108.0 (60)

SMT‐1

124.3 (4.4)

C6H10N3

124.3 (100), 107.3 (70), 82.3 (30), 66.9 (25)

SMT‐2

215.4 (4.7)

C12H15N4

215.4 (40), 198.3 (15), 173.2 (40), 158.2 (25), 108.1 (10), 98.9 (100)

SMT‐3

213.3 (4.8)

C12H13N4

213.3 (70), 196.0 (100), 186.1 (15), 172.0 (10), 155.3 (60) 107.1 (15), 92.4 (10)

SMT‐4

313.1 (13.6)

C12H14ClN4O2S

313.1 (20), 211.1 (8), 186.2 (100), 142.1 (35), 123.9 (70)

SMT‐5

247.2 (20.1)

SMP

281.2 (5.4)

C11H13N4O3S

281.2 (100), 156.0 (80), 126.2 (40), 107.9 (15)

SMP‐1

126.3 (4.2)

C5H8N3O

126.3 (30), 110.9 (100), 83.1 (10), 54.2 (25)

SMP‐2

249.2 (18.3)

C11H10ClN4O

249.2 (15), 213.2 (100), 199.2 (20), 161.9 (28), 135.2 (58), 107.2 (42)

247.2 (15), 212.2 (100), 197.2 (5), 171.9 (15), 159.2 (20), 134.3 (18), 108.1 (25)

Abbreviations: ESI, electrospray ionization; LC‐MS/MS, liquid chromatography tandem mass spectrometry; RT, retention time (min); SMP, sulfamethoxypyridazine; SMT, sulfamethazine.

FIGURE 4 Fragmentation spectrum of sulfamethazine (SMT)‐1 recorded using a collision energy of 20 eV. ESI, electrospray ionization [Colour figure can be viewed at wileyonlinelibrary.com]


124 corresponding to N1‐chlorobenzene‐1,4‐diamine and 2‐amino‐

Sulfamethoxypyridazine chlorination lead to the formation of

4,6‐dimethylpyrimidin‐1‐ium, respectively. SMT‐5 losts NHCl radical

SMP‐1 (m/z 126) and SMP‐2 (m/z 249) transformation products.

to give the ion at m/z 197. The ion at m/z 212 losts CH3CCH and

Figure 9 shows the SMP‐1 fragmentation pattern; a fragment at m/z

NHCCHCH to give the ions at m/z 172 and 159, respectively. The last

111 with high intensity was observed corresponding to a mass loss of

one losts CHC˙ and CHCCN to give the ions at m/z 134 and 108,

15 (CH3). The 2 ions at m/z 83 and 54 were obtained after elimination

respectively (Figure 8).

of CH2CHNH2 and 2,3‐dihydro‐1,3,4‐oxadiazole, respectively. This

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product with the same fragmentation spectra was also observed by

ions at m/z 213 and 199, respectively. In addition, the ions at m/z 162

Nassar et al27 after photolysis of SMP. The fragmentation spectrum pre-

and 135 correspond to (E)‐N‐(4‐aminophenyl)‐N′‐methyleneformo-

sented in Figure 10 showed that SMP‐2 losts HCl and ˙NHCl to give the

hydrazonamide and N‐(4‐aminophenyl)formimidamide, respectively.

8

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FIGURE 9 Fragmentation spectrum of sulfamethoxypyridazine (SMP)‐1 recorded using a collision energy of 20 eV. ESI, electrospray ionization [Colour figure can be viewed at wileyonlinelibrary.com]

FIGURE 10 Fragmentation spectrum of sulfamethoxypyridazine (SMP)‐2 recorded using a collision energy of 20 eV. ESI, electrospray ionization [Colour figure can be viewed at wileyonlinelibrary.com]

Finally, 2 structures were proposed for the ion at m/z 107: the 3‐amino‐

Furthermore, relative to de Jesus Gaffney et al study, the sulfonyl

6‐iminocyclohexa‐2,4‐dien‐1‐ylium and the 7‐oxa‐2,3‐diazabicyclo

amido‐nitrogen moiety of SNs does not play an important role in chlo-

[4.2.0]octa‐1,5‐dien‐3‐yn‐3‐ium.

rination reactions. For example, the product at m/z 215 was found by

Sulfamethazine and SMP present similar structures. To compare

de Jesus Gaffney et al, after SMT chlorination. The MS/MS fragmen-

their behavior during chlorination reaction, Figure 11 shows the

tation of this ion gave ions at m/z 108 and 198 corresponding to the

evolution of SMT, SMP, and their by‐product areas as a function of

fragment loss of SO2(CH3)(NHCH3) and SO2(ph‐NH2)(NHCHNH),

chlorination time. It shows that SMT‐1, SMT‐4, SMT‐5, SMP‐1, and

respectively. In our study, the MS/MS fragmentation of the ion at

SMP‐2 were the major by‐products generated during chlorination of

m/z 215 gave ion at m/z 108 corresponding to the loss of pyrimidinyl

SMT and SMP and their structures identified above allowed to con-

group.

clude that their formation pathways were similar. In fact, the aniline group and the sulfonyl amido‐nitrogen moiety are very reactive. SO2 extrusion, cycle recombination, and electrophilic substitution constitute the major pathways of transformation products. These pathways

3.5 | In silico toxicity prediction of SMT and SMP by‐products

proposed in our study are different with those proposed by de Jesus

In silico simulation calculations were performed by TEST to investigate

Gaffney et al,5 who studied the reaction of 6 sulfonamides with free

the toxicity of SMT, SMP, and their by‐products. In silico toxicity pro-

chlorine. The major transformation products identified by de Jesus

gram was validated by the US Environmental Agency. This program

Gaffney et al were formed after cleavage of pyrimidinyl group and

takes under consideration the toxicity of all similar structure of the

subsequent C‐chlorination and chlorination of the aniline group.

compound drawn in the system.19 In this study, only the significant

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chlorination pathways were proposed for the formation of these by‐ products. The toxicity prediction by usingTEST program has proved that SMT and SMP and their by‐products presented a developmental toxicity and mutagenicity except 2 compounds that constitute a potential risk for human health. The oral rat LD50 concentrations were less than 1000 mg·kg−1 for most generated compounds. The proposed structures of SMT and SMP by‐products could be confirmed by LC/ESIQTOFMS to help researchers confirm their toxicity. ORCID Ahmad Rifai

http://orcid.org/0000-0002-5749-924X

Mohamad Al‐Iskandarani

http://orcid.org/0000-0003-2558-2716

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FIGURE 11 Evolution of areas of sulfamethazine (SMT), sulfamethoxypyridazine (SMP), and their by‐products as a function of chlorination time results were reported like oral rat (LD50), mutagenicity, and developmental toxicity. For the last 2 results, TEST gives only a positive or negative result for the proposed structure. The result showed that SMT presented an oral rat concentration equal to 6000 mg·kg−1, which is in agreement with the literature. The by‐products SMT‐1, SMT‐2, and SMT‐3 presented an oral rat concentration between 139 and 921 mg·kg−1, while for SMT‐4 and SMT‐5, no oral rat LD50 was detected. Sulfamethoxypyridazine and SMP‐1 presented oral rat concentrations of 3405 and 756.46 mg·kg−1, respectively, while no oral rat was detected for SMP‐2. On the other hand, SMT, SMP, and their by‐products were predicted to present a developmental toxicity and to be mutagenic except SMT‐4 and SMT‐5.

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The chlorination kinetics of SMT and SMP were studied in UP water at pH 7.2 and 25°C. Then the experiments were performed using a real drinking water matrix to evaluate their fate during water treatment. Sulfamethazine and SMP reacted rapidly with chlorine during chlorination processes commonly used in water treatment systems. The chlorination kinetics of these compounds in real drinking water matrix were lower for a same dose of chlorine due to differences in matrix (real water vs purified water). Identification of chlorination by‐ products of SMT and SMP was achieved by LC‐MS/MS analysis. Under our experimental conditions, several by‐products were observed during chlorination and persisted after 3 hours of chlorination, and different

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SUPPORTI NG INFORMATION Additional supporting information may be found online in the Supporting Information section at the end of the article.

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How to cite this article: Nassar R, Rifai A, Trivella A, Mazellier

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P, Mokh S, Al‐Iskandarani M. Aqueous chlorination of sulfamation products identification. J Mass Spectrom. 2018:1–10. https://doi.org/10.1002/jms.4191