Identification of antimycotic drugs transformation products upon UV exposure

Journal of Hazardous Materials 289 (2015) 72–82

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Identification of antimycotic drugs transformation products upon UV exposure Jorge Casado, Isaac Rodríguez ∗ , María Ramil, Rafael Cela Departamento de Química Analítica, Nutrición y Bromatología, Instituto de Investigación y Análisis Alimentario (IIAA), Universidad de Santiago de Compostela, Santiago de Compostela 15782, Spain

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• Evaluation of antimycotic drugs UV stabilities in model supports.

• Simultaneous detection of precursor

tion from their scan, accurate MS/MS spectra. • Directed search of identified transformation products in sand and soil samples. • Preliminary toxicity estimations.

Cleavage

Cleavage

drugs and transformation products.

• Transformation products identifica-

KTZ-TP6

KTZ-TP1

Reducve de-chlorinaon

KTZ-TP3

Reducve de-chlorinaon

Hydroxylaon

Cleavage

KTZ-TP4

KTZ

KTZ-TP2

Intra-molecular Cyclizaon

+ KTZ-TP5

KTZ-TP5

Degradaon routes of Ketoconazole (KTZ) upon exposure of model supports to 254 nm light

a r t i c l e

i n f o

Article history: Received 31 October 2014 Received in revised form 13 January 2015 Accepted 11 February 2015 Available online 17 February 2015 Keywords: Antimycotic drugs UV degradation Transformation products Liquid chromatography–quadrupole time of flight mass spectrometry

a b s t r a c t The reactivity of three imidazolic, environmental persistent antimycotic drugs (clotrimazole, CTZ; ketoconazole, KTZ; and miconazole, MCZ) upon exposure to ultraviolet (UV) radiation is discussed. First, precursor compounds were immobilized in a silicone support which was further exposed to UV light at two different wavelengths: 254 and 365 nm. After solvent desorption, degradation kinetics of the precursor pharmaceuticals, identification of the arising transformation products (TPs) and evaluation of their time-course were investigated by liquid chromatography (LC) with quadrupole time-of-flight (QTOF) mass spectrometry (MS) detection. The three antimycotics displayed similar stabilities when exposed to 254 nm light; however, CTZ was significantly more stable than MCZ and KTZ when irradiated with the 365 nm lamp. TPs identified in silicone supports resulted from de-chlorination, cleavage, intra-molecular cyclization and hydroxylation reactions. Many of these species were also detected when exposing other solid matrices, such as sand and agricultural soil, previously spiked with target compounds, to UV light. The 50% estimated lethal concentration, calculated using the 48-h Daphnia magna test, for the two main TPs of CTZ and MCZ, at both wavelengths, were lower than those corresponding to the precursor drugs. © 2015 Elsevier B.V. All rights reserved.

1. Introduction ∗ Corresponding author. Tel.: +34 881814387; fax: +34 881814468. E-mail address: [email protected] (I. Rodríguez). http://dx.doi.org/10.1016/j.jhazmat.2015.02.031 0304-3894/© 2015 Elsevier B.V. All rights reserved.

If the comprehension of the persistence and/or the degradability of emerging pollutants in the environment constitutes a valuable

J. Casado et al. / Journal of Hazardous Materials 289 (2015) 72–82

knowledge, understanding their degradation routes, identifying the generated transformation products (TPs) and monitoring their time-course, is even a more challenging issue. Despite the dramatic advances in instrumental techniques, and particularly in liquid chromatography (LC) followed by high resolution mass spectrometry (MS), identification of TPs, arising from a given precursor, in a complex real-life matrix, e.g. agricultural soil, still represents a paramount task. Two of the major difficulties in such studies are: (1) being able to recover a non-target compound from the matrix and (2) correlating the decrease in the signal of the precursor pollutant with the presence of new features in LC–MS chromatograms. An effective, although expensive and not always possible, solution to the above problem is to label the precursor species with a nonstable, radioactive isotope [1,2]. Another possibility is to divide the study in two steps. First, TPs are identified in a model support (glass surface, wax film), impregnated with the precursor pollutant, from where precursor and potential TPs can be easily recovered. Thereafter, a target search of previously identified TPs is carried out in the extracts from real-life matrices [3,4]. In a previous study, we have demonstrated that technical grade silicone materials can be used as supports to investigate the transformation routes of selected pesticides under exposure to UV light, in laboratory experiments, and to outdoors environmental conditions [5]. Silicone-based materials behave more as a solvent than as a solid sorbent (analytes are equally distributed in the whole volume of the support and not only in the surface) [6]; therefore, they retain the precursor compounds and the arising TPs more effectively than other model supports [3] proposed to investigate the photochemical transformation routes of chemical compounds. Also, the sorbed species can be easily recovered with an organic solvent, without an excessive matrix background, even when technical grade silicone polymers are employed [6]. Antimycotic drugs are recognised as environmental relevant pollutants since (1) they are designed to disrupt the enzymatic system of fungi [7,8] and are suspected to disturb the endocrine system of other organisms, such as amphibians [9], (2) they are poorly removed during conventional sewage treatments [10,11] and, (3) the most lipophilic species are concentrated in sludge at sewage treatment plants (STPs) [7,12]. Particularly, the highly prescribed, low-polar drugs clotrimazole (CTZ), ketoconazole (KTZ) and miconazole (MCZ) might reach relevant concentrations, up to 1000 ng g−1 , in sludge of STPs [10,11,13–16]. From sludge, they can be incorporated into agricultural soils as part of fertiliser preparations [17,18]. When present in soil, the UV components of solar radiation might contribute to partial degradation, or even complete mineralization, of the fraction of these pollutants in the upper soil layer. To the best of our knowledge, no data have been published regarding the transformation routes of CTZ and MCZ upon exposure to UV radiation, neither under laboratory nor in real-life, environmental conditions. As regards KTZ, Staub et al. [19] reported the formation of two de-chlorinated derivatives after exposure of solutions, and personal care compounds, containing this drug to UV sources. The UV degradation reactions of any of these three lipophilic pharmaceuticals in solid surfaces, the formation of potential TPs and the evaluation of their relative toxicities have not been explored yet. The aim of this investigation was to obtain information regarding the stability and potential TPs arising from CTZ, KTZ and MCZ upon exposure to UV light in laboratory studies. To accomplish this goal, precursor compounds were loaded in silicone supports which were further exposed to the selected UV radiation (254 and 365 nm). After solvent desorption, the organic extracts were analysed using a LC hybrid quadrupole time-of-flight (QTOF) MS system to simultaneously follow their degradation and detect potential TPs. The chemical structures of identified TPs were proposed from their accurate product ion (MS/MS) scan spectra.

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Additional experiments were performed exposing spiked sand and agriculture soil samples to UV light in order to establish whether same TPs can be identified, or not. 2. Experimental 2.1. Standards, solvents and supports CTZ (100%), KTZ (98%) and (±)-MCZ nitrate salt (100%) were obtained from Sigma (Milwaukee, WI, USA) and dissolved in methanol. Acetonitrile and methanol, HPLC-grade purity solvents, and ammonium acetate (99%) were supplied by Merck (Darmstadt, Germany). Ultrapure water was obtained from a Milli-Q Millipore (Billerica, MA, USA) system. Technical grade silicone, in a hollow tube format (2 mm i.d., 3 mm o.d.), was acquired at Goodfellow (Bad Nauheim, Germany). The tubular polymer was cut in 1 cm-length pieces and preconditioned by immersion in acetonitrile and sonication for 30 min. Thereafter, the silicone support pieces were dried at room temperature, for 5–10 min, and stored in glass vessels until being loaded with the selected antimycotic drug. Antimycotic drugs were incorporated in the silicone supports using 10 mL ultrapure water solutions, spiked with the selected compound at 1 ␮g mL−1 . These solutions were stirred with a PTFE covered stir bar, for 14 h, in presence of 20 silicone supports turning freely within the sample. The antimycotic loaded silicone pieces, were dried with a lint-free tissue and kept in amber vessels, at −20 ◦ C, until being used in degradation experiments. Each support was used for a sole test. Thus, a new batch of supports was loaded with the corresponding substances for each degradation experiment, ensuring that all pieces contained the same amount of each precursor pharmaceutical. Since the presence of salts may affect the transformation routes of a substance, they were not added to sampling vessels. Acid washed silicon dioxide (sand), 50–70 mesh (0.3-0.2 mm) particle size, was purchased at Sigma. Agricultural soil (total organic carbon 5.3%) was obtained from a corn field in the Northwest of Spain, sieved and the fraction below 0.3 mm used in this study. Spiked sand and soil samples were prepared mixing a given amount of the solid matrix with a standard solution of the selected compound, prepared in methanol. The slurry was homogenized, using a glass stirrer, and left in the hood for solvent evaporation. Spiked samples were then stored at −20 ◦ C until being used. 2.2. Degradation experiments Degradation experiments were performed using two different UV sources, a low pressure 8 W Hg lamp (Philips reference G8T5) emitting at 254 nm, and a 8 W black light blue fluorescence lamp (Philips reference F8T5/BLB) with maximum emission wavelength at 365 nm. Both sources provided nominal emission intensities of 2 mW cm−2 at a distance of 5 cm. The setup of UV degradation experiments using loaded silicone supports was described in detail elsewhere [5]. In brief, a batch of supports was inserted through a PTFE wrapped wire, which was set parallel to the UV lamp, at a distance of 5 cm. Some supports (3–4 units) were used as control (zero time experiments), being desorbed without UV exposure. Also, 3 units were protected from UV radiation, with aluminium foil, and desorbed at the end of each exposure series (dark control experiments). The rest of silicone pieces were retired at different times. Analytes (precursor drugs and their potential TPs) were recovered from the supports soaking them in 0.5 mL of acetonitrile, inside a glass insert (0.6 mL capacity) for 10 min. Thereafter, the support was removed and the extract injected directly in the LC–MS/MS instrument (5 ␮L injection volume). Variability of

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Table 1 Performance of the LC–ESI(+)–QTOF–MS system for precursor antimycotic drugs detection. Reported data correspond to the C18 LC column, operating the system in the single MS mode. Compound

CTZ

KTZ

MCZ

Formula Retention time (min) MS quantification ion (Da) Linearity evaluation (1–1000 ng mL−1 , 10 levels) Slope ± standard deviation Intercept ± standard deviation Determination coeficient (R2 ) LOQs (ng mL−1 )

C22 H17 ClN2 27.7 277.0788

C26 H28 Cl2 N4 O4 26.6 531.1560

C18 H14 Cl4 N2 O 29.2 414.9933

14177 ± 220 2256 ± 1790 0.9999 0.5

9754 ± 200 2044 ± 1670 0.9992 0.5

5564 ± 120 2044 ± 1670 0.9991 0.8

extraction and desorption steps remained below 10% for the three precursor antimycotic drugs involved in this study. Regarding exposure of sand and agricultural soil to UV radiation, experiments were conducted spreading a layer of spiked (20 ␮g g−1 ) sample (ca. 0.2–0.3 cm depth, 4 cm wide) over aluminium foil at a distance of 5 cm from the longitudinal axis of the lamp. After a given time, a fraction (0.5 g) of the solid matrices was soaked with 10 mL of methanol for 30 min [11,13,14]. Extracts were filtered, using 0.22 ␮m syringe filters, and injected in the LC–QTOF–MS system, without any further clean-up. 2.3. Determination conditions The determination of targeted compounds and their TPs was simultaneously performed with a LC–ESI–QTOF–MS instrument acquired from Agilent Technologies (Wilmington, DE, USA). The LC system was an Agilent 1200 Series, comprised of the following modules: a vacuum degasser unit, two isocratic high-pressure mixing pumps, an autosampler and a chromatographic oven. The QTOF mass spectrometer was an Agilent 6520 model, equipped with a Dual-Spray ESI source and a hexapole collision cell situated between the quadrupole and the TOF analysers. Substances were separated in a Zorbax Eclipse XDB C18 column (100 mm × 2 mm, 3.5 ␮m), acquired from Agilent Technologies, under gradient programme and at a constant flow, 0.2 mL min−1 . The column, connected to the binary pump after a C18 (4 mm × 2 mm) guard cartridge, from Phenomenex (Torrance, CA, USA), was thermostated at 30 ◦ C within the chromatographic oven. Some separations were also undertaken in a Zorbax Eclipse SB-phenyl column (100 mm × 2.1 mm, 3.5 ␮m), aiming an improvement in the separation of certain TPs which co-eluted when using the C18 -type column. In both cases, the mobile phases consisted of water (A) and methanol (B), both containing 5 mM of ammonium acetate, and the gradient programme was as follows: 0–3 min, 5% B; 23 min, 100% B; 24–31 min, 100% B; 32 min, 5%; 33–40 min, 5% B. Compounds were determined operating the ionisation source in the positive mode (ESI+). Source parameters were adopted from a previous work dealing with the quantification of antimycotic drugs in environmental matrices with the same instrument [20]. The ESI source counted with a secondary nebuliser, which was constantly infusing a mass reference solution (Agilent calibration solution A). This way, recalibration of the mass axis was continuously performed considering the ions 121.0509 and 922.0098, and the accuracy of m/z assignations was guaranteed. Regarding the QTOF

hybrid analyser, it worked in the 2 GHz Extended Dynamic Range resolution mode (mass resolution 5000 at m/z values of 118.0862 to 13,000 at m/z values of 1521.9715). The Mass Hunter Workstation software (Agilent) was used to control the LC–ESI–QTOF–MS system and to process the recorded data, while the Mass Profiler software (Agilent) was used to find similarities and differences amongst chromatograms acquired for extracts obtained at different reaction times and control experiments. Degradation kinetics of precursor drugs were followed in the single MS mode from extracted ion chromatograms (EIC, mass window 20 ppm) corresponding to the [M + H]+ ion, case of KTZ and MCZ, and the [M–C3 H3 N2 ]+ ion for CTZ. As previously explained, CTZ is prone to in-source fragmentations, rendering an intense ion resulting from removal of the imidazole moiety [21]. In fact, with the fragmentor voltage considered in this work (160 V), the [M + H]+ ion was hardly observed in the MS spectrum of CTZ. Table 1 summarizes some features of the LC–ESI(+)–MS procedure for precursor antimycotic drugs determination. Reported data correspond to the C18 -type LC column using an injection volume of 5 ␮L. Retention times and precursor ions of TPs were identified using the Mass Profiler software, considering LC–MS chromatograms corresponding to different exposure times for silicone extracts [22]. Thereafter, their ion product scan (MS/MS spectra were recorded at a rate of 2.5 spectra s−1 ), in a range of m/z values between 50 and 600 units, fixing a time window of 2 min around the retention time of each analyte, and considering collision energies in the range from 5 to 40 eV. Extracts from solid matrices (spiked soil and soil samples) were explored for the presence of those TPs previously identified in model experiments with silicone supports.

2.4. Ecotoxicity assessment Predictions about the toxicity of the selected antimycotic drugs, as well as, their identified TPs were done with the Toxicity Estimation Software Tool (TEST) [23], developed by the US Environmental Protection Agency (EPA). This software calculates the toxicity of an organic compound from physical characteristics of its structure, basing the results on mathematical models called Quantitative Structure Activity Relationships (QSARs) [24]. The 48-h Daphnia magna 50% lethal concentration (LC50 ) test was selected because it is one of the most popular ecotoxicological endpoints. The chosen way to do the estimation was consensus methodology, which consists on the average value of five different calculation approaches [25].

Table 2 Half-life (t1/2 ) values calculated after exposure of silicone supports to 254 and 365 nm light. Antimycotic drug

CTZ KTZ MCZ

254 nm

365 nm

t1/2 (min)

R2

Time interval (min)

t1/2 (h)

R2

Time interval (h)

10.2 8.5 15.9

0.9903 0.9923 0.9773

0–30 0–30 0–30

92.6 9.7 40.8

0.9783 0.9838 0.9870

0–100 0–50 0–50

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3. Results and discussion 3.1. Degradation kinetics Table 2 compiles the t1/2 values calculated for antimycotic drugs, previously incorporated in silicone supports, using UV light of two different wavelengths. Exposure times up to 30 min were used with the 254 nm lamp, more than 4 days (100 h) were considered for the fluorescence lamp emitting at 365 nm. A minimum of 9 different exposures times were investigated in each series of experiments. For all compounds, the differences between the average responses for zero time (n = 4 replicates) and dark control experiments (n = 3 replicates) accounted for less than 10%, pointing out to negligible thermal degradation due to heat emitted from lamps. The natural logarithmic plots of their peak areas versus time fitted reasonable with a first-order kinetics (linear trend), with determination coefficients (R2 ) above 0.97 for the three precursor compounds. The calculated t1/2 values (defined as t1/2 = ln (0.5)/k, with k representing the slope of logarithmic plots) at 254 nm remained below 20 min, with CTZ and KTZ displaying similar stabilities. On the other hand, at 365 nm, which can be considered as representative of the UV fraction of solar radiation reaching the surface of earth, the calculated t1/2 values differed up to 10 times, with CTZ and KTZ remaining as the most stable and the most labile species, respectively. 3.2. Transformation products detection The detection of the TPs was performed using the Find by Molecular Feature function of the Mass Hunter software followed by comparison of molecular features data files (obtained using the above function and corresponding to experiments at different reaction times and zero time control) with the Mass Profiler software package to highlight peaks with changing intensities depending of the irradiation time. [25]. Fig. 1 depicts the EIC chromatograms for CTZ and its TPs, using an extraction window of 20 ppm, before and after 15 min of exposure to 254 nm light. As can be observed, while the signal of precursor compound decreases, peaks corresponding to TPs arise. The empirical formula of detected TPs was generated by the Mass Hunter software, taking into account the exact mass of a given ion (usually, although not always, the [M + H]+ species) in the full scan MS spectrum of its chromatographic peak, the spacing between the cluster of signals which surround this ion and their

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relative intensities [22,25]. These three parameters are combined to calculate a global score (0–100), which represents how well the empirical MS spectrum fits the theoretical one of the candidate TP, with a value of 100 representing a perfect match. Additionally to empirical formulae, calculated and empirical masses, Table 3 summarises some additional relevant data of TPs, such as their retention times, in two different LC columns, and the number of double bond equivalents (DBE), which is useful to predict re-arrangements and formation of new cycles in the structures of precursor pharmaceuticals. The acronym used in each case is the corresponding to the precursor substance, followed by “TP” (transformation product) and the elution order number in the C18 LC column. In some cases, two separated peaks were noticed with the same exact mass. In these situations, the same code was assigned to both species, unless significant differences are observed in their MS/MS spectra. 3.3. Transformation product structures elucidation 3.3.1. Clotrimazole Four TPs were identified upon exposure of CTZ loaded supports to UV light (Table 3). CTZ–TP4 appeared only when using 254 nm light and it could not be chromatographically resolved from the signal of CTZ, with any of the tested LC columns; however, this TP was not detected neither in zero time (Fig. 1) nor in dark control experiments. The most intense signal in the MS spectra of CTZ–TP4 appeared at 243.1168 Da (theoretical mass), versus 277.0788 Da for CTZ. Likely, CTZ–TP4 arises from the substitution of chlorine by hydrogen. As occurred with the precursor drug, the its [M + H]+ ion was not detected in the MS spectrum, but that resulting from the removal of the imidazolic moiety [M–C3 H3 N2 ]+ . Regarding the MS/MS spectrum of CTZ–TP4, a single intense product ion, corresponding to the C13 H9 + species (nominal mass 165 Da) was noticed, see Fig. S1. The base signals in the MS spectra of CTZ–TP1 to CTZ–TP3 are assumed to correspond to their respective [M + H]+ ions, Table 3. Their MS/MS spectra and proposed structures are compiled in Fig. 2. As observed, CTZ–TP2 and CTZ–TP3 share the same parent ion; however, they displayed different MS/MS spectra. In the first case, MS/MS transitions reflect a first removal of a C6 H6 moiety (309 > 231) followed by elimination of HCN (231 > 204) (Fig. 2A). On the other hand, the MS/MS spectrum of CTZ–TP3 contains common fragments to those existing in the spectrum of the CTZ (Fig. S1),

Fig. 1. Extracted ion chromatograms (EIC) for CTZ ([M–C3 H3 N2 ]+ ion) and the most intense ion in the ESI(+)–MS spectra of its transformation products. Dashed line, zero time experiment. Solid line, 15 min exposure to 254 nm light. Chromatograms acquired using the C18 column.

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Table 3 Retention times, experimental and theoretical mass of the parent ion, proposed empirical formula for this ion, score and number of double bond equivalents (DBE) for each TP. Precursor

Transformation product

Retention time (min) C18 column

Phenyl column

Experimental [M + H]+

Theoretical [M + H]+

Score

Proposed formula

DBEb

CTZ C22 H17 ClN2

CTZ–TP1 CTZ–TP2 CTZ–TP3 CTZ–TP4

26.8 26.8 27.0 27.8

25.4 26.4 26.7 27.2

270.1279 309.1380 309.1384 243.1180a

270.1277 309.1386 309.1386 243.1168a

99.2 91.0 98.0 99.4

C20 H15 N C22 H16 N2 C22 H16 N2 C22 H18 N2

14 16 16 15

KTZ C26 H28 Cl2 N4 O4

KTZ–TP1 KTZ–TP2 KTZ–TP3 KTZ–TP4 KTZ–TP5 KTZ–TP6

18.8, 20.2 19.2 22.0 22.7 25.3, 25.8 25.5, 26.0

21.3, 21.7 20.2, 20.6 20.8, 21.5 23.2 27.0, 27.7 27.2, 27.8

295.0823 311.0777 183.0205 329.0454 495.1784 497.1938

295.0844 311.0793 183.0207 329.0453 495.1794 497.1950

98.1 95.4 99.8 99.6 99.6 80.2

C14 H15 ClN2 O3 C14 H15 ClN2 O4 C9 H7 ClO2 C14 H14 Cl2 N2 O3 C26 H27 ClN4 O4 C26 H29 ClN4 O4

8 8 6 8 15 14

MCZ C18 H14 Cl4 N2 O

MCZ–TP1 MCZ–TP2 MCZ–TP3

27.9 28.2 28.3

26.6, 26.7 26.9, 27.0 27.1, 27.2

379.0143 381.0301 381.0296

379.0166 381.0323 381.0323

99.9 81.8 80.6

C18 H13 Cl3 N2 O C18 H15 Cl3 N2 O C18 H15 Cl3 N2 O

12 11 11

a b

Mass corresponding to the [M–C3 H3 N2 ]+ ion. Double bond equivalents.

with the first MS/MS transition corresponding to the removal of the imidazolic ring (309 > 241) (Fig. 2B). Both transformation products contain 16 DBE, this is one more than the precursor CTZ. Probably, they arise from CTZ de-chlorination followed by formation of

a five-member cycle. In case of CTZ–TP3, this new cycle involves two benzene rings; whereas, for CTZ–TP2 the new bond is established between the de-chlorinated benzene and the imidazole ring. As further explained for MCZ and KTZ, two possible isomers, dif-

Fig. 2. MS/MS spectra and proposed structures for CTZ intramolecular cyclization transformation products.

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Fig. 3. MS/MS spectra and chemical structures of KTZ and its transformation products.

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fering in the atom of carbon of the imidazolic ring linked to the de-chlorinated carbon, might be generated (Fig. 2A); however, a single peak was noticed with both tested LC columns for CTZ–TP2. CTZ–TP2 rendered a less intense response than CTZ–TP3 and it was only observed when using the 254 nm source In case of CTZ–TP1, the cyclization reaction involves the removal of the C2 H2 N moiety resulting in a four-cycle species, with only one atom of nitrogen and 14 DBE (Fig. 2C). Likely, CTZ–TP1 is formed from CTZ–TP2. LC–ESI(+)–MS chromatograms corresponding to CTZ degradation experiments were also explored for the [M + H]+ ion of 2-chlorophenyl-diphenyl methanol, reported as the main TP of CTZ in soils through biodegradation processes [1]; however, no peak was noticed for this species.

3.3.2. Ketoconazole Since KTZ has a more complicated structure than CTZ, its degradation paths are more tangled. The proposed structures for its TPs together with their MS/MS spectra are presented in Fig. 3. KTZ–TP6 and KTZ–TP5 are the result of a de-chlorination reaction, followed, in the latter case, by an intra-molecular cyclization. For both TPs, two possible isomers can be generated. These isomers could be partially resolved with both LC columns used in this study (Table 3). For KTZ–TP6 there is one possible structure with the atom of chlorine in orto and the other in para. The MS/MS spectrum for the earlier eluting isomer in the phenyl LC column is shown in Fig. 3B. Main MS/MS transitions were analogues to those corresponding to KTZ (Fig. 3A). KTP–TP6 isomers have been previously reported by Staub in experiments performed with aqueous solutions [19]. KTZ–TP5 isomers

Fig. 4. MS/MS spectra and chemical structures of MCZ and its transformation products.

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are supposed to be generated by de-chlorination of the orto-carbon in the phenyl ring, followed by cyclization with one of the two ␣C atoms of the imidazole moiety. The products ions at 453, 275 and 219 Da (nominal masses) observed in the MS/MS spectrum of KTZ–TP5 (Fig. 3C) confirm the existence of this new cycle. Another starting point for the KTZ degradation chain is the cleavage of the ether bond, between the secondary carbon and the phenyl group, giving rise to KTZ–TP4 (Fig. 3D). This TP retains the two Cl atoms but if one of them is lost, KTZ–TP1 is generated. There are two possible isomers for this compound depending whether chlorine remains in orto or para positions (Fig. 3E). Alternatively to de-chlorination, a hydroxyl (OH) group can enter in the position of the lost chlorine, giving place to KTZ–TP2 (Fig. 3F), with its corresponding two isomers again, which could be separated only with the phenyl LC column. The MS/MS spectra for these three TPs (KTZ–TP4, KTZ–TP1 and KTZ–TP2) follow the same fragmentation route, with the first transition reflecting the opening of the dioxane ring. In the case of the KTZ–TP4 a di-chlorotoluene (m/z 158.9763) can be observed, while a mono-chlorotoluene (m/z 125.0153) and a hydroxychlorotoluene (m/z 141.0102) appear for KTZ–TP1 and KTZ–TP2, respectively (Fig. 3D–F).

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The final photo-degradation stage we found for KTZ is the formation of KTZ–TP3 (Fig. 3G). This species, which presents the lowest intensity LC–MS chromatographic peak, is formed after the loss of a big part of the molecule and chlorine substitution by hydrogen. It can arise from KTZ–TP1 and KTZ–TP6. The proposed pathways to explain the formation of above TPs from KTZ are provided as Supplementary information, Fig. S2. Dechlorinated with or without further intra-molecular cyclization and ether cleavage TPs (KTZ–TPs 1, 4–6) were formed at 254 and 365 nm; whereas, KTZ–TPs 2 and 3 were only noticed when using the 254 nm lamp. 3.3.3. Miconazole For MCZ, three transformation products were found. MCZ–TP2 and MCZ–TP3 have the same empirical formula (C18 H15 Cl3 N2 O), which differs from that of MCZ in the substitution of chlorine by hydrogen (Table 3). Using the C18 column, only two highly overlapped chromatographic peaks were obtained for the [M + H]+ ion corresponding to the above empirical formula. However, the phenyl LC column rendered 4 peaks for the same molecular ion, with two different MS/MS spectra. These spectra differed in the presence of a

Fig. 5. Time course of TPs identified in silicone supports loaded with antimycotic drugs and exposed at two different UV sources.

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di-chlorinated toluene product ion ([C7 H5 Cl2 + ] for MCZ–TP2), also observed in the MS/MS spectrum of MCZ, or a mono-chlorinated one ([C7 H6 Cl+ ] for MCZ–TP3) (Fig. 4A–C). Thus, reductive dechlorination (chlorine substitution by hydrogen) occurs in both benzene rings. The other transformation product, MCZ–TP1, is formed after an intra-molecular cyclization following a de-chlorination reaction. This reaction happens between one ␣-C atom from the imidazole ring and the de-chlorinated carbon in orto (Fig. 4D). This way, two structural isomers can be formed again, depending on the ␣-C atom of the imidazole ring that reacted. In fact, two not-baselineresolved peaks (26.6 and 26.7 min) could be detected with the phenyl-type LC column. Conversely to MCZ–TP2 and MCZ–TP3, which were only observed when using the 254 nm source, both isomers of MCZ–TP1 were noticed at the two investigated wavelengths. As it happens with KTZ, the molecule of MCZ contains an ether bond; however, no TPs generated from hydrolysis of the ether link could be observed for the latter species. 3.4. Time-course of TPs The temporal evolution of identified TPs is depicted in Fig. 5. In case of isomeric species, with same MS/MS spectra, the sum of their responses is used. The two different time scales (minutes and hours) were selected considering the t1/2 values of the precursor drugs at 254 and 365 nm. The Y axis represents the normalised responses versus those obtained for the precursor antimycotic drug at zero time (n = 3 replicates). Those percentages (normalized responses) cannot be used as direct estimations of transformation yields since ESI(+) ionisation efficiencies might differ significantly amongst compounds; however, they serves to evaluate the temporal stability of TPs and also to compare the relative extension of their formation at the two investigated wavelengths. Except in case of KTZ–TP6, the TPs arising from chlorine substitution by hydrogen (reductive de-chlorination) were not detected when using the 365 nm lamp. Also, the relative responses of the TPs generated at 365 nm were significantly lower than at 254 nm. The exception was the cleavage species KTZ–TP4, formed in a larger extend under irradiation at 365 nm. Also, this compound seems to be relatively stable in comparison to KTZ. In fact, the t1/2 for the precursor drug at 365 nm remained below 10 h (Table 2), whereas the response for KTZ–TP4 reached a maximum at 30 h and in the next 70 h decreased only in a 50% (Fig. 5). Conversely to this behaviour, MCZ–TP1 seems

to be less stable at 365 nm than the precursor drug MCZ. The calculated t1/2 for the latter compound was 40.8 h and the response measured for MCZ–TP1 decreased steady after 6 h (Fig. 5). In agreement with the longest t1/2 of CTZ at 365 nm, the response of its main TP (CTZ–TP3) started to decrease only after 80 h. 3.5. Transformation products in other matrices Once TPs generated in the model silicone supports were characterised, further assays were carried out with spiked sand and soil samples. The objective of these experiments was to check whether same TPs could be identified, or not. In this case, it is obvious that only the fraction of molecules from each drug in contact with the upper surface layer of the solid matrices receives the UV radiation proceeding from the lamp. Thus, degradation kinetics are expected to be significantly slower than those occurring in silicone supports. Also, the presence of organic matter in the agriculture soil might affect the elimination of spiked antimycotic species. Table 4compiles data about the identified TPs in each of the three matrices at the longest considered exposure times (30 min and 100 h for 254 and 365 nm irradiation, respectively) and the remaining percentage of the precursor drug. For both wavelengths, the remaining percentage of the precursor drug increased in the following order: silicone < sand < soil. The most intense TPs identified in silicone supports were also noticed in sand and soil samples exposed at 254 nm light. However, when using the 365 nm source, KTZ was the only species undergoing a significant removal after 100 h of irradiation of the spiked soil sample. On the other hand, for this matrix, CTZ and MCZ did not undergo any significant removal after irradiation at 365 nm for 100 h. 3.6. Predicted ecotoxicity A preliminary assessment of the toxicity of the transformation compounds was undertaken with the EPA predictive TEST software following the procedure described in Section 2.4. The results of this study are presented in Table 5. There are listed the LC50 values, which represent the concentration required to kill one half of the population, and the ratio between the LC50 for each antimycotic drug and the corresponding transformation substance. Thus, if this ratio is higher than one indicates that the TP is more toxic than the corresponding precursor, and vice versa. For example, CTZ–TP3, which is the main TP of CTZ at both investigated wavelengths (Fig. 5) and has been detected in the model support and spiked solid

Table 4 Residual responses of precursor pharmaceuticals after the considered exposure time (data as percentage) and summary of identified TPs in three different matrices. Transformation product

CTZ CTZ–TP1 CTZ–TP2 CTZ–TP3 CTZ–TP4 KTZ KTZ–TP1 KTZ–TP2 KTZ–TP3 KTZ–TP4 KTZ–TP5 KTZ–TP6 MCZ MCZ–TP1 MCZ–TP2 MCZ–TP3 nd, not detected.

254 nm (0.5 h)

365 nm (100 h)

Silicone

Sand

Soil

Silicone

Sand

Soil

14 ± 1% x x x x 5 ± 0.7% x x x x x x 23 ± 2% x x x

79 ± 7% x nd x nd 50 ± 11% x x nd x x x 77 ± 6% x x x

97 ± 1% x nd x nd 69 ± 12% nd x nd x x x 90 ± 7% x x x

50 ± 4% x nd x nd <1% x nd nd x x x 21 ± 2% x nd nd

83 ± 4% x nd x nd 38 ± 8% x nd nd x x x 71 ± 2% x nd nd

>99% nd nd nd nd 45 ± 4% x nd nd x nd x >99% nd nd nd

J. Casado et al. / Journal of Hazardous Materials 289 (2015) 72–82 Table 5 Estimated 50% lethal concentrations (LC50 , ␮M) for the 48-h D. magna test, and ratios of the value for the corresponding precursor drug and its TPs. Compound

LC50 ␮M

Drug/TP ratio

CTZ CTZ–TP1 CTZ–TP2 CTZ–TP3 CTZ–TP4 KTZ KTZ–TP1 KTZ–TP2 KTZ–TP3 KTZ–TP4 KTZ–TP5 KTZ–TP6 MCZ MCZ–TP1 MCZ–TP2 MCZ–TP3

1.17 1.07 0.40 0.40 3.80 0.55 5.01 5.50 16.22 2.29 2.51 2.34 0.17 0.08 0.20 0.20

– 1.10 2.95 2.95 0.31 – 0.11 0.10 0.03 0.24 0.22 0.23 – 2.14 0.81 0.83

samples (except in case of agriculture soil exposed at 365 nm light), is three times more toxic than CTZ as happens with the isomeric form CTZ–TP2. CTZ–TP1 is as toxic as CTZ and CTZ–TP4 is three times less toxic. In the case of KTZ, all TPs are less toxic than their precursor and for MCZ, MCZ–TP1 is two-fold more toxic than the precursor antimycotic drug, while MCZ–TP2 and MCZ–TP3 display similar estimated LC50 values. 4. Conclusions CTZ, KTZ and MCZ can be largely degraded under UV exposure, with removal kinetics being much faster at 254 nm radiation than at 365 nm. At the latter wavelength, CTZ resulted by far more stable than the other two antimycotics. Silicone tubes can retain the generated transformation products, matching most of them with those formed in sand and agricultural soil. However, degradation rates are matrix dependant, with increased stabilities when passing from silicone supports to sand and soil. LC–QTOF–MS provides valuable information to identify the structures of generated TPs through accurate, scan product ion spectra, acquired using different collision energies. Nevertheless, chromatographic separation of TPs with different structures but same empirical formulae, and thus common [M + H]+ ions, is mandatory to obtain their pure MS/MS spectra. Two TP generated from the CTZ and another one from the MCZ are estimated to be more toxic than their precursors, the three species are generated through intra-molecular cyclization following a de-chlorination step. The rest of TPs, including all the substances generated from KTZ, are less toxic than their precursors. Reductive de-chlorination and intra-molecular cyclization, usually involving the imidazole ring, after de-chlorination were common transformation routes of the three precursor drugs. On the other hand, cleavage of the ether bond represented a major transformation via for KTZ, not observed in the case of MCZ. Acknowledgements This study has been financially supported by the Spanish Government, Xunta de Galicia and E.U. FEDER funds (projects CTQ2012-33080 and EM14/004). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat. 2015.02.031.

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