transformation products by liquid chromatography–tandem mass spectrometry

Journal of Chromatography A, 1333 (2014) 87–98

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Degradation of fluoroquinolone antibiotics and identification of metabolites/transformation products by liquid chromatography–tandem mass spectrometry夽 Alexandra S. Maia a,b , Ana R. Ribeiro a,b,c , Catarina L. Amorim b,c , Juliana C. Barreiro d , Quezia B. Cass d , Paula M.L. Castro b , Maria Elizabeth Tiritan a,c,∗ a

CESPU, Instituto de Investigac¸ão e Formac¸ão Avanc¸ada em Ciências e Tecnologias da Saúde, Rua Central de Gandra, 1317, 4585-116 Gandra PRD, Portugal CBQF - Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa/Porto, Rua Dr. António Bernardino Almeida, 4200-072 Porto, Portugal c Centro de Química Medicinal da Universidade do Porto (CEQUIMED-UP), Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal d Departamento de Química, Universidade Federal de São Carlos, CP 676, 13560-970 São Carlos, SP, Brasil b

a r t i c l e

i n f o

Article history: Received 30 July 2013 Received in revised form 22 October 2013 Accepted 24 January 2014 Available online 31 January 2014 Keywords: Biodegradation Fluoroquinolones Transformation products

a b s t r a c t Antibiotics are a therapeutic class widely found in environmental matrices and extensively studied due to its persistence and implications for multi-resistant bacteria development. This work presents an integrated approach of analytical multi-techniques on assessing biodegradation of fluorinated antibiotics at a laboratory-scale microcosmos to follow removal and formation of intermediate compounds. Degradation of four fluoroquinolone antibiotics, namely Ofloxacin (OFL), Norfloxacin (NOR), Ciprofloxacin (CPF) and Moxifloxacin (MOX), at 10 mg L−1 using a mixed bacterial culture, was assessed for 60 days. The assays were followed by a developed and validated analytical method of LC with fluorescence detection (LC–FD) using a Luna Pentafluorophenyl (2) 3 ␮m column. The validated method demonstrated good selectivity, linearity (r2 > 0.999), intra-day and inter-day precisions (RSD < 2.74%) and accuracy. The quantification limits were 5 ␮g L−1 for OFL, NOR and CPF and 20 ␮g L−1 for MOX. The optimized conditions allowed picturing metabolites/transformation products formation and accumulation during the process, stating an incomplete mineralization, also shown by fluoride release. OFL and MOX presented the highest (98.3%) and the lowest (80.5%) extent of degradation after 19 days of assay, respectively. A representative number of samples was selected and analyzed by LC–MS/MS with triple quadrupole and the molecular formulas were confirmed by a quadruple time of flight analyzer (QqTOF). Most of the intermediates were already described as biodegradation and/or photodegradation products in different conditions; however unknown metabolites were also identified. The microbial consortium, even when exposed to high levels of FQ, presented high percentages of degradation, never reported before for these compounds. © 2014 Elsevier B.V. All rights reserved.

1. Introduction In the last couple of decades the presence of active pharmaceutical ingredients (API) in different environmental compartments has been an evolving subject in the environmental science field [1,2]. They are named as “pseudo-persistent” pollutants [3] as

夽 Presented at the 39th International Symposium on High-Performance LiquidPhase Separations and Related Techniques, Amsterdam, Netherlands, 16–20 June 2013. ∗ Corresponding author at: CESPU, Instituto de Investigac¸ão e Formac¸ão Avanc¸ada em Ciências e Tecnologias da Saúde, Rua Central de Gandra, 1317, 4585-116 Gandra PRD, Portugal. Tel.: +351 224 157 204; fax: +351 224 157 100. E-mail addresses: [email protected], [email protected] (M.E. Tiritan). 0021-9673/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2014.01.069

their transformation vs. elimination rate is balanced by their uninterrupted input into the environment [4]. Wastewater treatment plants (WWTP) represent a critical spot of contribution to the increase of pharmaceuticals in the environment since they are not designed with the ability to completely eliminate all organic compounds at low levels of concentration [5,6]. A raising number of reports spotted trace levels of API in WWTP effluents [7–9], surface and ground waters [10–12] and even in drinking water supplies [13]. Antibiotics are an extensively studied class of API due to the development of multi-resistant bacteria and their high frequency in aquatic environmental compartments [14–16]. The levels reported are usually within the ng L−1 to ␮g L−1 range, however reports of effluents from pharmaceutical manufacturing facilities located in India and China presented higher levels, reaching concentrations up

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to mg L−1 [17,18]. Pharmaceutical industries do not represent the main source of pharmaceuticals in the environment, as indicated in the environmental risk assessment study for wastewater and river water [19], however in some cases a direct association has been reported [17,18,20]. According to the 2010 European Centre for Disease Prevention and Control report on antimicrobial consumption in Europe, the consumption proportion of quinolone antibacterials regarding systemic use ranged from 2.5% in the United Kingdom to 13.3% in Portugal. Ciprofloxacin (CPF) accounted for most of the consumption of quinolone antibacterials [21]. Considering the 2010 European Medicines Agency report on veterinary antimicrobial agents’ sales in 19 countries, the major antimicrobial classes sold, as oral solutions, were tetracyclines (24%) and fluoroquinolones (FQ) (20%). FQ accounted for 2.2% of the total sales of antimicrobial veterinary medicine products [22]. The presence of FQ in many environmental matrices has been described, including marine aquaculture samples in Pearl River Delta, South China, in concentrations between 1.88 and 11.20 ng g −1 (dry weight) [23]; sea water in Hong Kong, China [24] and also in drinking water ranging from 1.0 to 679.7 ng L−1 , in a study performed in Guangzhou, China [13]. Recently, a study described the occurrence of several FQ and the presence of FQ-resistant genes in bacterial species in surface waters [25]. In general, FQ are described to be resistant to hydrolysis [26,27] and readily adsorbed to particulate matter [28], exhibiting WWTP removal efficiencies over 80% [29] or around and above 50% [15,28,30,31]. Along with adsorption to particulate matter, photodegradation and biodegradation are reported as the most important removal processes for elimination of API in WWTP [32]. However, photodegradation and biodegradation tend to produce metabolites that are rarely quantified in monitoring studies of WWTP effluents. This work describes the biodegradation of four important FQ antibiotics: ofloxacin (OFL), norfloxacin (NOR), CPF and moxifloxacin (MOX), by a microbial consortium able to degrade fluorinated aromatic compounds. An integrated approach was used to identify the metabolites/transformation products originated in the biodegradation studies. FQ primary degradation was followed by the validated LC–FD method and the metabolites/transformation products were identified by LC–MS/MS with a triple quadrupole analyzer (TQD) and confirmed by a time of flight (TOF) couple to a quadrupole analyzer (QqTOF). Most of the intermediates were already described as biodegradation and/or photodegradation products in different conditions. QqTOF analysis provided relevant data with new molecular formulas, which allowed the proposal of structures of two new metabolites, not described before.

2.2. Chromatographic conditions A Shimadzu UFLC Prominence System equipped with two Pumps LC-20AD, an Autosampler SIL-20AC, a column oven CTO20AC, a Degasser DGU-20A5, a System Controller CBM-20A and a LC Solution, Version 1.24 SP1 (Shimadzu) was used to follow the primary degradation. The fluorescence detector coupled to the LC System was a Shimadzu RF-10AXL. The column was a ˚ particle size 3 ␮m, Luna Pentafluorophenyl (2), pore size 100 A, 150 mm × 4.6 mm, a modified reverse phase column from Phenomenex, with pentafluorophenyl groups bound to silica surface that offer high aromatic selectivity due to highly electronegative fluorine atoms on the periphery of each phenyl ring. The optimized mobile phase consisted in an isocratic mixture of 0.1% triethylamine solution acidified to pH 2.2 adjusted with trifluoroacetic acid (eluent A) and ethanol (eluent B) 64:36 (v/v) at a flow rate of 0.6 mL min−1 . The injection volume was 10 ␮L and the column oven temperature was maintained at 38 ◦ C. The fluorescence detector was set to an excitation wavelength of 290 nm and an emission wavelength of 460 nm. A Waters ACQUITY® UPLC® (Waters Corporation, Milford, MA, USA) coupled to a Waters TQD mass spectrometer (electrospray ionization mode ESI-tandem quadrupole) was used to analyze selected samples. Data acquisition software was MassLynx V 4.1 with MetaboLynx XS option (Waters). MS detection settings of Waters TQD mass spectrometer were as follows: source temperature 140 ◦ C, desolvation temperature 350 ◦ C, desolvation gas flow rate 900 L h−1 , cone gas flow 50 L h−1 , capillary voltage 3.00 kV, and cone voltage 30 V. The data were obtained in a positive ionization scan mode ranging from 50 to 1000 m/z in time 0.5 s intervals for the metabolite detection using MetaboLynx XS. After the metabolite detection, MS/MS spectra (daughter scans) were obtained using 15 eV and 25 eV collision energy. For each metabolite/transformation produts two MRM transitions were optimized using the most intense fragments of the MS/MS spectra. The chromatographic separations were held on a Water Acquity HSS T3 column, particle size 1.8 ␮m, 150 mm × 2.1 mm. The injection volume was 5 ␮L with a flow rate of 0.4 mL min−1 . Mobile phase consisted in water 0.1% formic acid/acetonitrile 0.1% formic acid. Elution was performed with gradient 99/1 to 0/100 in 14 min (v/v). The identity of the metabolites/transformation products was confirmed by high resolution mass spectrometry with a Dionex RSLC LC-system (Bruker Daltonics, Germany) equipped with an ESI source, coupled to a Bruker QqTOF maXis impact mass spectrometer. Identification was performed by 80–1300 m/z full scan and auto MS/MS and bbCID at 5 Hz. The chromatographic condition was the same used for TQD analysis except for the injection volume (2–5 ␮L). Data acquisition software was DataAnalysis with MetaboliteTools (Bruker).

2. Material and methods 2.1. Chemicals and standards preparations

2.3. Fluoride release

Antibiotics OFL, NOR and CPF standards were purchased from Sigma Aldrich. MOX standard was donated by Bayer. All the standards presented a purity degree above 98%. The ethanol HPLC grade was purchased from Merck. Triethylamine with ≥ 99% purity was obtained from Sigma–Aldrich. Acetic acid and trifluoroacetic acid were purchased by Panreac and Acros Organics, respectively. Ultrapure water was supplied by a Milli-Q water system. OFL, NOR and CPF stock solutions were prepared at 1000 mg L−1 in water:acetic acid 10% (50:50, v/v). MOX stock solution was prepared at 1000 mg L−1 in water. These solutions were stored at −20 ◦ C in amber bottles. The working solution of the four FQ was obtained by a dilution of the stock solutions in ultra-pure water to a concentration of 50 mg L−1 and prepared weekly.

The measurement of fluoride released into the culture supernatant was performed using an ion-selective electrode (CH-8902 Mettler-Toledo GmbH). A calibration curve was carried out by using newly prepared standard solutions of sodium fluoride in minimal mineral medium (MM) in six concentration levels: 0.01 mM, 0.05 mM, 0.10 mM, 0.50 mM, 1.00 mM, and 5.00 mM. This method was adapted from previous studies of the work group [33]. 2.4. LC–FD method validation parameters The method was validated according to International Conference on Harmonization Guidelines [34], considering the following parameters: selectivity, linearity and range of application,

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accuracy and precision, recovery, detection limit (DL) and quantification limit (QL). Selectivity was assessed by comparing chromatograms of standard mixture prepared in ethanol with those prepared in MM in the presence of the microbial consortium. Linearity was evaluated with seven different concentrations (n = 3) with standard solutions diluted in MM: 0.5, 2.0, 4.0, 6.0, 8.0, 10.0, and 12.0 mg L−1 . The curves were obtained by linear regression corresponding to the correlation between the peak area and the nominal concentration. Accuracy, intra- and inter-day precision were estimated using three quality control samples prepared at three different concentrations (1.0, 5.0 and 11.0 mg L−1 ), all in triplicate. Intra- and inter-day precisions were expressed as the relative standard deviation (RSD) of the replicate measurements. The percentage of recovery was obtained by comparing the peak area ratio of the supernatant of the centrifuged FQ from the biodegradation assays to those prepared at the same concentrations in the solvent. DL and QL were calculated by the signal/noise ratio. The determination of the signal-to-noise ratio was performed by comparing measured signals from samples with known low concentrations of the analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. A signal-to-noise ratio of 3:1 was considered for estimating the DL and a ratio of 10:1 for estimating the QL [34] of each compound. 2.5. Biodegradation assay A combination of three bacterial strains, namely Labrys portucalensis F11, Rhodococcus sp. FP1 and Rhodococcus sp. S2, was used in the biodegradation assays. These bacterial strains, previously isolated in the laboratory, are capable to degrade a range of fluoroaromatic compounds, such as fluorobenzene, fluorophenols and 4-fluorocinnamic acid [35–37]. Studies were carried out using MM, with the following composition per liter: Na2 HPO4 ·2H2 O, 2.67 g; KH2 PO4 , 1.40 g; MgSO4 ·7H2 O, 0.20 g and (NH4 )2 SO4 , 0.5 g and 10 mL of a trace elements solution with the following composition per liter: NaOH, 2.0 g; Na2 EDTA2 ·2H2 O, 12.0 g; FeSO4 ·7H2 O 2.0 g; CaCl2 , 1.0 g; Na2 SO4 , 10.0 g; ZnSO4 , 0.4 g; MnSO4 ·4H2 O, 0.4 g; CuSO4 ·5H2 O, 0.1 g; Na2 MoO4 ·2H2 O, 0.1 g; H2 SO4 98%, 0.5 mL. For the biodegradation assays 250 mL flasks containing 50 mL of MM were supplemented with sodium acetate (Merck) at a concentration of 100 mg L−1 and with each FQ individually at a concentration of 10 mg L−1 . Each flask was inoculated with 750 ␮L

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of the microbial consortium to obtain an initial optical density of about 0.150 measured at  = 600 nm. An uninoculated assay containing the four compounds was also included to assess abiotic degradation. Cultures were incubated with agitation at room temperature during 2 month under natural day light cycles. Aliquots were collected every 3 days in the first 19 days of the study. After the 19th day, cultures were supplemented with fresh MM to recover the volume of 50 mL and the same amount of FQ was added (10 mg L−1 ). The supplementation was carried out 3 times, on the 19th, 33th and 47th days for the cultures with OFL, NOR, CPF and MOX, individually. The abiotic assay was supplemented only on the 19th day. Growth was assessed by measuring the optical density of the cultures at 600 nm. The biodegradation was followed by monitoring the FQ using the LC–FD validated method and by measuring fluoride release. For this, 3 mL aliquots were collected, centrifuged for 10 min at 14,000 rpm and the supernatant analyzed. All conditions were assessed in duplicate and the samples collected were analyzed for two consecutive times by LC–FD. 3. Results and discussion 3.1. LC–FD method development and validation The optimized chromatographic conditions were achieved in isocratic elution with a mobile phase composed by a mixture of 0.1% of triethylamine in ultra-pure water acidified to pH 2.2 with trifluoroacetic acid and ethanol, 64:36 (v/v). The choice of ethanol was according to the green solvent selection guide [38,39]. The optimized flow rate was set to 0.6 mL min−1 and the column oven temperature at 38 ◦ C. The increase in the temperature of the column oven allowed to diminish the chromatographic run and also to improve the chromatographic separation of the FQ. Selectivity of the method was demonstrated comparing chromatograms from the supernatant of the matrix spiked with the FQ (corresponding to day 0 of the biodegradation assays) and the standards at the same concentration. Chromatograms in Fig. 1 show that the microbial consortium did not interfere with the FQ detection. The linearity was achieved in the range of 0.5–12.0 mg L−1 with correlation coefficients always higher than 0.999 (Table 1). Accuracy and precision were assessed analyzing the results achieved with three different concentrations of the FQ, as described above. The values obtained for accuracy were between 86.4 and 104.7%. In order to estimate the method precision, repeatability (%RSD in

Fig. 1. LC–FD chromatograms obtained with exc = 290 nm and em = 460 nm of OFL, NOR, CPF and MOX at 10.0 mg L−1 in (a) ultra-pure water and (b) MM inoculated with the microbial consortium.

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Table 1 Calibration curves, detection limits and quantification limits. Analyte

Linear regression Range (␮g mL−1 ): [0.5–12.0]

RSD (%)

Correlation coefficient (r2 )

DL (ng mL−1 )

QL (ng mL−1 )

Ofloxacin Norfloxacin Ciprofloxacin Moxifloxacin

y = 282,575.91x − 45,92.06 y = 543,558.76x − 37,180.96 y = 491,996.72x − 28,611.27 y = 125,265.30x − 10,348.26

<2.6 <2.7 <2.7 <3.2

0.99998 0.99984 0.99990 0.99990

3.0 3.0 3.0 5.0

5.0 5.0 5.0 20.0

the intra-day assay) and intermediate precision (%RSD in the interday assays) were considered, and the results obtained showed that the method is precise, as the %RSD calculated for both parameters were lower than 2.51 and 2.74, respectively (Table 2). DL were 3.0 ␮g L−1 for OFL, NOR and CPF and 5.0 ␮g L−1 for MOX and QL were 5 ␮g L−1 for OFL, NOR and CPF and 20 ␮g L−1 for MOX (Table 1). The QL is appropriate to quantify the FQ in the concentration normally found in effluents of WWTP [28]. A direct approach for the FQ quantification is presented, without previous extraction or pre-concentration stages. Samples were injected into the chromatographic system with only a previous centrifugation step. The LC–FD method described can hereafter adapted for the analysis of extracts previously subjected to a pre-concentration procedure. 3.2. Degradation assays After assembly of the degradation assays, samples were collected every three days until the FQ degradation was almost complete. Once it was achieved at the 19th day, a new supplementation with MM and FQ was performed. The following supplementation events were then planed for every two weeks. Biotic and abiotic degradation of the target FQ were followed by the developed and validated LC–FD method and by fluoride release during sixty days. LC–FD chromatograms indicated the primary degradation of FQ and formation of metabolites and/or transformation products whereas fluoride release indicated the defluorination degree of the compounds. Fig. 2a–d shows the degradation pattern observed during the 60 days of assays. Removal percentages presented in this section should be interpreted as an integrated degradation, considering both biological and abiotic phenomena, such as photolysis. OFL exhibited a slightly higher extent of degradation regarding the individual biotic assays. OFL was almost totally consumed (98.3%) until the 19th day, although stoichiometric fluoride release

accounted only for 21.7%, which indicates that the initial compound was only partially mineralized (Fig. 2a). Over the successive feedings, the extent of biodegradation was slightly reduced. During the first 33 days of the assay OFL biodegradation was above 90% at the end of each supplementation, with remaining concentrations of the antibiotic of 1.7 and 6.5% on days 19 and 33, respectively. The highest value for fluoride release was observed at day 47, suggesting a mineralization extent of 50.5%, whereas the chromatographic data showed a degradation extent of 73.2% at the same day (Table 3). These results indicate that mineralization does not occur at the same rate or velocity as the parent compound is eliminated. The LC–FD clearly revealed the appearance of metabolites/transformation products after the successive OFL supplementation (Fig. 3a). After the last supplementation and until the last day of the assay the extent of degradation of OFL was the lowest obtained along the study regarding this FQ, with only 58.2% degradation expressing a decrease of ca. 40% of the degradation extent obtained within the first feeding period (Fig. 2a, Table 3). High biodegradation extent was also observed for NOR, with 96.1% of the compound degraded at the 19th day, although stoichiometric fluoride release accounted only for 23.8% of degradation. After the first supplementation, 89.8% of the compound was degraded until the 33rd day. Over the successive feedings, the extent of biodegradation was slightly reduced, as previously mentioned for OFL (Fig. 2b). After the two subsequent supplementations, 62.2 and 55.4% was degraded at the 47th and the 60th days, respectively (Table 3). The higher fluoride release from NOR, with stoichiometric fluoride release accounting for up to 60.5% of the degraded compound at the 60th day, suggests higher mineralization than the compound OFL. The formation of metabolites and/or transformation products was also observed in the LC–FD analysis (Fig. 3b). CPF biodegradation assay showed 94.7% of the compound degraded at the 19th day, and 95.6% degraded at the 33rd day.

Table 2 Recovery, accuracy, intra- and inter-day precision of OFL, NOR, CPF and MOX. Nominal concentration (␮g mL−1 )

1st day

Accuracy (%)

2nd day

3rd day

Recovery (%)

RSD (%)

RSD (%)

Accuracy (%)

RSD (%)

Accuracy (%)

RSD (%)

98.9 95.6 88.3

0.44 1.54 2.13

100.2 96.4 89.0

0.53 1.71 2.13

101.5 96.2 88.5

1.16 1.83 2.21

105.1 104.8 92.1

7.36

100.4 93.3 88.4

0.28 2.28 0.95

104.2 95.5 90.2

0.29 2.47 1.09

104.7 93.9 88.0

1.64 2.74 1.17

106.4 108.4 92.1

8.72

Ciprofloxacin 1 5 11

96.5 92.0 87.8

1.43 1.69 0.69

101.3 93.8 89.7

1.57 1.88 0.87

100.8 92.1 87.3

1.39 2.21 0.92

103.5 104.1 90.9

7.47

Moxifloxacin 1 5 11

95.1 89.7 86.4

1.96 1.44 0.80

98.1 90.7 88.5

1.46 1.76 0.85

97.8 90.4 86.7

2.05 1.95 1.06

98.4 102.4 90.1

6.47

Ofloxacin 1 5 11 Norfloxacin 1 5 11

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Fig. 2. Degradation pattern during 60 days of: (a) OFL; (b) NOR; (c) CPF and (d) MOX based on LC–FD FQ quantification and fluoride release 1 LC–FD CPF data considered until the 54th day due to sample interferences that precluded the quantification of the compound at the 60th day.

Table 3 Degradation for OFL, NOR, CPF and MOX during successive feedings as measured by LC–FD and corresponding F− release. Time (days) 19

OFL NOR CPF MOX a

33 −

60a

47 −

−

HPLC–FD

F

HPLC–FD

F

HPLC–FD

F

HPLC–FD

F−

98.3% 96.1% 94.7% 80.5%

21.7% 23.8% 22.6% 9.2%

93.5% 89.8% 95.6% 77.7%

32.5% 123.0% 119.3% 30.8%

73.2% 62.2% 58.5% 61.5%

50.5% 60.2% 72.9% 44.5%

58.2% 55.4% 31.8% 46.3%

43.5% 60.5% 62.0% 44.5%

CPF values refer to the 54th day due to the presence of interferences which precluded quantification of the compound in the 60th day.

Fig. 3. LC–FD chromatograms of OFL (a), NOR (b), CPF (c) and MOX (d) biodegradation samples collected at days 0, 19, 33 and 47.

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After the two subsequent supplementations (33th and 47th days) CPF presented 58.5% and 31.8% of degradation on the 47th and the 54th days, respectively (Table 3). At the 54th day the stoichiometric fluoride release accounted for 62.0% of CPF degradation, similar to that obtained for NOR at the 60th day (Fig. 2c). The presence of secondary peaks observed in the chromatograms and the non-completed fluoride release also indicated the presence of metabolites and/or transformation products (Fig. 3c). The biodegradation extent of MOX was lower than that observed for the other three FQ (Fig. 2d). The highest values of MOX degradation were verified at the 19th and 33rd days, with a remaining concentration of MOX of 19.5% and 22.3%, respectively (Table 3). After the 33rd day the extent of the biodegradation was slightly reduced. The degradation values for the 47th and 60th days were 61.5 and 46.3%, respectively. The highest values for fluoride release were verified at the 47th and the 60th days, representing a degradation extent of 44.5%. Fluoride release was lower in the MOX assay, suggesting lower mineralization of this compound. MOX is the compound with the more complex structure considering the four FQ studied, since it presents the bulkiest C-7 substituent. MOX has a diazabicyclo ring at C-7 instead of a piperazinyl moiety [40]. This additional complexity may represent a slower defluorination step. A MOX photocatalytic degradation pathway including several fluorinated transformation products has been reported [41]. The appearance of secondary peaks in the chromatograms was also observed for MOX degradation samples (Fig. 3d). The uninoculated assay with the mixture of the FQ was performed to assess abiotic degradation in the presence of natural light. In a previous assay in dark conditions FQ compounds did not present any degree of degradation, fact that probably points to the absence of thermo-degradation and hydrolysis [42]. The FQ resistance to hydrolysis has been previously described by other authors [26,27]. Direct and indirect photolysis is the predominant route of abiotic degradation for hydrolysis resistant pharmaceuticals [43]. At the 19th day of the abiotic assay in the presence of natural light, the target FQ had decreased to 43.8, 66.2, 65.8 and 73.2% regarding OFL, NOR, CPF and MOX, respectively, although the fluoride released indicated 23.3% of total degradation. These results indicate that in the biotic assays both biological degradation and photolysis could be occurring. At the 19th day, the abiotic assay was supplemented with fresh MM and the FQ. The analysis of the subsequent collected samples revealed that the pattern of abiotic degradation was similar to that observed after the first FQ addition. Considering that fact, no additional supplementation with the FQ was performed. Even so, collection of aliquots for analysis continued in the remaining sampling times established. By the end of the assay, at the 60th day, 38.7% of the fluoride was released, while a reduction ranging from 29.7 to 44.8% in FQ concentration was observed by LC–FD, with NOR presenting the highest degradation extent. Moreover, in this study NOR presented the highest degradation extent in the abiotic condition. Babic´ et al. [27] in a recent study reported that solar irradiation contributes significantly to NOR degradation, which also presents a different abiotic degradation pathway from CPF. FQ can suffer degradation by non-biological mechanisms, as previously reported [44–46], and are described to be degraded by ultraviolet radiation [47], by radical-mediated photolysis [46,48] and by photocatalytic processes [49,50]. Photodegradation is influenced by the light intensity, pH, phosphate levels and the presence of co-solutes and organic compounds [51–53]. FQ containing tertiary aliphatic piperazinyl nitrogens (such as OFL) can also generate active intermediates after photochemical transformations [54]. The bacterial consortium was able to degrade the FQ antibiotics in aerobic conditions into high extent in the individual assays, when compared with efficiencies obtained in WWTP and OECD

biodegradation tests reported elsewhere [55]. Other published studies reported low biodegradability of these antibiotics under the close bottle test with wastewater bacteria [56,57]. In the study performed by Kümmerer and his co-workers, OFL and CPF exhibited less than 20% of biodegradability after 28 days [56]. The referred study claimed that there was no biodegradation or adsorption of the FQ to the bacterial sludge, suggesting that none of the antibiotics can be classified as “readily biodegradable” [56]. Other biodegradation studies using activated sludge from WWTP indicated CPF as being recalcitrant to biodegradation and transformation in aqueous systems under the OECD 301 test conditions, after incubation for 29 days [55]. According to the same study, no mineralization was observed in the aqueous system over 29 days of incubation and the chromatographic analysis showed the presence of metabolites, further identified by LC–MS/MS. Biodegradation studies of FQ using fungus have also reported the formation of metabolites during the process [58–62]. This study contributes to understanding the microbial community behavior at high concentrations of antibacterial fluorinated compounds and its ability to induce FQ-degrading enzymes. Induction of bacterial degradation relies on the concentration of the target compound in the medium and may be limited if the substrate concentration is below the threshold level necessary to activate the enzymes responsible for its biotransformation [63]. Another study has used mg L−1 levels of a FQ antibiotic to enrich FQ-degrading bacterial strains, increasing CPF biotransformation as well as providing the possibility to detect its transformation products [64]. The FQ concentration used in this study is above different EC50 values described in the literature for several microorganisms [55,56,65,66]. The use of a high FQ concentration is relevant to aerobic biological treatment of antibiotic containing wastewaters, as referred elsewhere [67]. Reduction of the degradation extent after the successive feedings may be due to the high FQ concentration and to possible toxic effects in the bacterial community. Several studies have stated FQ induced toxicity in microorganisms [65,68–70]. FQ bacterial killing effects against Escherichia coli were described as concentration-dependent [71]. Even when exposed to such high levels of antibacterial compounds, the microbial consortium was still able to carry out biodegradation at considerable extents, fact that contributes to the relevance of this work [44–54]. All the biodegradation assays performed in the present study clearly expressed the formation of metabolites and/or transformation products. The comparison between the data collected from the chromatographic analysis and from the fluoride release stresses the importance to distinguish between the concepts mineralization/degradation/transformation. The non-stoichiometric amount of fluoride released indicates incomplete mineralization. 3.3. Identification of transformation products by LC–MS/MS The major degradation/transformation products from OFL, NOR, CPF and MOX detected by LC–FD at different days of incubation were analyzed by LC–MS/MS. The proposed chemical structures of the detected metabolites are given in Tables 4–7 for OFL, NOR, CPF and MOX, respectively. Most of the proposed structures were based on data published elsewhere [44,50,55,59,62,72–76]. QqTOF data obtained with molecular formulas C16 H19 FN3 O4 and C16 H20 N3 O4 , allowed the proposition of new structures for metabolites OFL-C (Table 4) and NOR-C (Table 5), respectively. Besides the metabolites/transformation products characterization, QqTOF confirmed the identity of the compounds previously detected by TQD analysis. OFL transformation products identified in degradation samples are presented in Table 4. OFL-A, m/z 348.00, and its representative fragments were identified by TQD and confirmed by the QqTOF analyzer as C17 H19 FN3 O4 , m/z 348.1348 (QqTOF obtained MS2 products

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Table 4 Proposed chemical structures, formulas, molecular ions and TQD obtained MS2 products of ofloxacin and its transformation products. Products

Experimental (m/z)

TQD MS2 products

Proposed formula [M+H]+

OFL

361.19

261.19 318.25 344.23 362.10 316.19

C18 H20 FN3 O4

OFL-A

348.00; (348.1348 QqTOF)

304.25 330.16 261.15 282.24

C17 H19 FN3 O4 +

[44] [76] [77]

OFL-B

392.00

374.17

C18 H21 FN3 O4 +

[44]

OFL-C

336.10; (336.1354 QqTOF)

298.20 279.16 261.23 316.17 235.15

C16 H19 FN3 O4

Structure proposal

Fig. 4. QqTOF MS/MS spectrum on OFL-C and proposed structures for the transformation product and its major fragments.

Refs.

94

A.S. Maia et al. / J. Chromatogr. A 1333 (2014) 87–98

Table 5 Proposed chemical structures, formulas, molecular ions and TQD obtained MS2 products of norfloxacin and its transformation products. Products

Experimental (m/z)

TQD MS2 products

Proposed formula [M+H]+

NOR

319.92

302.21 233.19 276.18 219.06 205.14

C16 H18 FN3 O3

NOR-A

335.90

318.18 86.16

C16 H19 FN3 O4

[62]

NOR-B

293.93; (294.1252 QqTOF)

256.13 276.28 227.79

C14 H17 FN3 O3

[62]

NOR-C

317.93; (318.1449 QqTOF)

300.18 187.13 216.37

C16 H20 N3 O4

304.1451, 261.1030, 284.1391 and 330.1253). OFL-A was described as a demethylated specie originated by the initial photoinduced transformations, which are confined to the piperazine moiety and to the methyl groups, while leaving intact the fluoroquinolone core [44]. The same metabolite was also identified after sonophotocatalytic treatment of OFL in secondary treated effluent [76] and in a proposed metabolic pathway based on photocatalytic and biological transformations [77]. OFL-B, m/z 392.00, resembles to a bihydroxylated-monooxidated compound, according to the above mentioned published study [44]. OFL-A and OFL-B were already described as products of photoinduced transformations [44]. OFLC, m/z 336.10, and its representative fragments were identified by TQD and confirmed by the QqTOF analyzer as C16 H19 FN3 O4 , m/z 336.1354 (QqTOF obtained MS2 products 279.0770, 298.1185, 261.0664, 318.1245 and 235.0873). The molecular formula and the fragments data indicate the presence of a new metabolite for OFL. The data allowed the suggestion of a structure for this unknown transformation product (Fig. 4). NOR transformation products identified in degradation samples are presented in Table 5. NOR-A, m/z 335.90, involves the formation of a carboxylic acid in the amino group after the opening of the piperazinyl ring while NOR-B, m/z 293.93, represents a net loss of C2 H2 at the piperazinyl substituent [62]. NOR-A and NOR-B were first described in a degradation study performed with a white-rot fungus [62]. NOR-B and its representative fragments were identified by TQD and confirmed by the QqTOF analyzer as C14 H17 FN3 O3 , m/z 294.1252 (QqTOF obtained MS2 products 276.1142, 233.1083, 256.1083). NOR-C, m/z 317.93, and its representative fragments

Structure proposal

Refs.

were identified by TQD and confirmed by the QqTOF analyzer as C16 H20 N3 O4 , m/z 318.1449 (QqTOF obtained MS2 products 300.1339, 274.1545, 231.1124, 205.0973, 188.0708 and 217.0964). The molecular formula and the fragments data indicate the presence of a new metabolite for NOR. The data allowed the suggestion of a structure for this unknown transformation product (Fig. 5). NOR-C represents the opening of the piperazine ring and the amide formation. These transformations have been described and confirmed by NMR spectroscopy in a NOR metabolite formed by the fungus P. guepini [72]. NOR-C MS2 product 300.1339, C16 H18 N3 O3 , represents the H2 O loss from the amide via nucleophilic attack by the primary amine, as described elsewhere [78]. CPF was the fluoroquinolone with further metabolites recognized from literature (Table 6). CPF-A, m/z 262.90, was previously described as originated during OECD tests 301B and 307 [55] and as a metabolic product of different fungus species and basidiomycetes indigenous from agricultural sites [62,72,73], representing the loss of the piperazine ring. CPF-B, m/z 305.93, has been stated as a product generated by the loss of C2 H2 at the piperazinyl substituent [62]. CPF-B species were also referred to as desethylene ciprofloxacin [62] and have been well reported not merely in biological organisms like brown-rot fungus [58] and white-rot fungus [62] but also after photodegradation processes [44,50,74,79] and ozonation treatment [80]. CPF-B and its representative fragments were identified by TQD and confirmed by the QqTOF analyzer as C15 H17 FN3 O3 , m/z 306.1249 (QqTOF obtained MS2 products 288.1140, 245.1089, 268.1087). CPF-A and CPF-B species are recognized as mutual metabolites in mammals with little antibacterial action [81,82].

A.S. Maia et al. / J. Chromatogr. A 1333 (2014) 87–98

95

Table 6 Proposed chemical structures, formulas, molecular ions and TQD obtained MS2 products of ciprofloxacin and its transformation products. Products

Experimental (m/z)

TQD MS2 products

Proposed formula [M+H]+

CPF

331.91

314.17 245.16 231.14 288.17 204.23

C17 H18 FN3 O3

CPF-A

262.90

245.09 204.10

C13 H12 FN2 O3

[55] [62] [72] [73]

CPF-B

305.93

288.18 245.60 268.14 217.06

C15 H17 FN3 O3

[44] [50] [58] [62] [74] [79] [80]

CPF-C

347.90

330.12

C17 H19 FN3 O4

[44]

CPF-D

315.90

298.11 226.99 212.19

C17 H18 N3 O4

[50]

CPF-E

333.89

316.04 288.34

C16 H17 FN3 O4 +

[44] [79]

Structure proposal

Refs.

96

A.S. Maia et al. / J. Chromatogr. A 1333 (2014) 87–98

Table 6 (Continued) Products

Experimental (m/z)

TQD MS2 products

CPF-F

287.89

270.18 202.15 230.10 242.17

Proposed formula [M+H]+

Structure proposal

Refs.

[74]

CPF-C, m/z 347.90, was labeled as shaped by hydroxylation in the quinolone moiety and referred to as a monohydroxylated specie [44]. CPF-D, m/z 315.90, was previously detected and reported as a CPF defluorinated product complemented by some degree of degradation of the piperazine ring, formed by UVA direct photolysis [50]. CPF-E, m/z 333.89, was recognized in previous works and defined

as one of the most abundant species identified after photoinduced degradation of CPF [44,79]. The formation of CPF-E is probably due to the breakdown of the piperazine moiety with the subsequent opening of the piperazine ring with cleavage of the C2 -N bond [44]. The identified CPF-F compound, m/z 287.89, might resemble to two potential structures, either originated by the loss of the fluorine

Table 7 Proposed chemical structures, formulas, molecular ions and TQD obtained MS2 products of moxifloxacin and its transformation products. Products

Experimental (m/z)

TQD MS2 products

Proposed formula [M+H]+

MOX

401.94

384.18 110.09 261.20 364.23 358.26

C21 H24 FN3 O4

MOX-A

292.90

275.18 206.06 178.11 217.09 234.14

C14 H14 FN2 O4

[75]

MOX-B

415.92

398.55 340.99 355.26 313.12 110.03

C21 H23 FN3 O5

[75]

Structure proposal

Refs.

A.S. Maia et al. / J. Chromatogr. A 1333 (2014) 87–98

97

Fig. 5. QqTOF MS/MS spectrum on NOR-C and proposed structures for the transformation product and its major fragments.

atom in accumulation of the piperazine ring opening or formed by the direct decarboxylation of CPF [74]. The same study that characterized this transformation product also claimed that CPF-F species could be originated by reductive defluorination of CPF-B during photodegradation processes, depending on the existence of HO− and its aptitude to perform as an electron donor. Concerning MOX transformation products MOX-A and MOXB (Table 7) matching to m/z 292.90 and 415.92, respectively, both were described for the first time as the result of (4aS,7aS)-octahydro-6H-pyrrolo[3,4-b]pyridine-6-yl group oxidation present in MOX quinolone moiety [75]. The same study identified MOX-A and MOX-B species as MOX photodegradation products both in solution and in solid phase.

SFRH/BD/86939/2012, Ana Rita Ribeiro, SFRH/BD/64999/2009 and Catarina L Amorim, SFRH/BD/47109/2008), QREN-POPH, European Social Fund, MCTES and Pest (FCOMP-010124-FEDER-022718; PEst-OE/EQB/LA0016/2011 and CEQUIMED-PEst-OE/SAU/UI4040/2011). This work was also supported by CESPU (09-GCQF-CICS-09) and FCT under the project FluoroPharma PTDC/EBB-EBI/111699/2009. Authors wish to acknowledge ViaAthena and Pedro Batista for the help in the metabolites analysis with TQD and Verena Tellstroem - Bruker - Bremen - for QqTOF analysis. Authors also recognize Virgínia Gonc¸alves for her collaboration.

4. Conclusions

[1] D. Barceló (Ed.), Comprehensive Analytical Chemistry. Wilson & Wilson’s ed., in: M. Petrovic, D. Barcelò (Eds.), Analysis, Fate and Removal of Pharmaceuticals in the Water Cycle, Elsevier, 2007. [2] K. Kümmerer, in: W. Peter (Ed.), Treatise on Water Science, Elsevier, Oxford, 2011, p. 69. [3] C.G. Daughton, Lancet 360 (2002) 1035. [4] D. Barceló, TrAC: Trends Anal. Chem. 22 (2003) 14. [5] T.A. Ternes, Water Res. 32 (1998) 3245. [6] J. Corcoran, M.J. Winter, C.R. Tyler, Crit. Rev. Toxicol. 40 (2010) 287. [7] M. Rabiet, et al., Environ. Sci. Technol. 40 (2006) 5282. [8] S. Kim, D.S. Aga, J. Toxicol. Environ. Health Part B 10 (2007) 559. [9] A. Togola, H. Budzinski, J. Chromatogr. A 1177 (2008) 150. [10] Z. Moldovan, Chemosphere 64 (2006) 1808. [11] R. Loos, et al., Water Res. 44 (2010) 4115. [12] T.V. Madureira, et al., Sci. Total Environ. 408 (2010) 5513. [13] Yiruhan, et al., Environ. Pollut. 158 (2010) 2350. [14] W. Xu, et al., Water Res. 41 (2007) 4526. [15] L. Shi, et al., Water Sci. Technol. 59 (2009) 805. [16] X. Peng, et al., J. Environ. Monit. 13 (2011). [17] D.G.J. Larsson, C. de Pedro, N. Paxeus, J. Hazard. Mater. 148 (2007) 751. [18] D. Li, et al., Water Res. 42 (2008) 307. [19] C.C. Hoerger, et al., Integr. Environ. Assess. Manage. 5 (2009) 331. [20] A.Y.C. Lin, Y.T. Tsai, Sci. Total Environ. 407 (2009) 3793. [21] ECDC, Surveillance of Antimicrobial Consumption in Europe, 2010, Stockholm, 2013. [22] E.M. Agency, Sales of Veterinary Antimicrobial Agents in 19 EU/EEA Countries in 2010, EMA/88728/2012, 2012. [23] X. He, et al., Environ. Geochem. Health 34 (2011) 323–335. [24] T.B. Minh, et al., Mar. Pollut. Bull. 58 (2009) 1052.

The integrated approach using a multi-technique methodology allowed the degradability verification of OFL, CPF, NOR and MOX by a bacterial consortium; the identification of metabolites/transformation products and the proposal of new structures for two of the intermediates. The simple validated LC–FD method proved to be suitable to monitor the biodegradation of the target compounds in the linearity range of 0.5–12.0 mg L−1 . The bacterial consortium was able to degrade each of the four FQ individually, at high extents: 98.3%, 96.1%, 95.6% and 80.5% for OFL, NOR, CPF and MOX respectively, after 19 days of incubation. The fluoride release indicated that the FQ were not completely mineralized in the biodegradation process and several metabolites and/or transformation products were observed by LC–FD. These intermediates were identified using LC–MS/MS and the new structures were proposed based on QqTOF analysis. Acknowledgments This work has been supported by Fundac¸ão para a Ciência e Tecnologia – FCT (PhD grants attributed to Alexandra Sofia Maia,

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