Ozonation of Ciprofloxacin in Water - American Chemical Society

May 23, 2008 - Degradation products formed during ozonation of an aqueous solution of the antibiotic ciprofloxacin in a bubble column are analyzed by ...
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Environ. Sci. Technol. 2008, 42, 4889–4895

Ozonation of Ciprofloxacin in Water: HRMS Identification of Reaction Products and Pathways BAVO DEWITTE, JO DEWULF,* KRISTOF DEMEESTERE, VINCENT VAN DE VYVERE, PATRICK DE WISPELAERE, AND HERMAN VAN LANGENHOVE Research Group EnVOC, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium

Received January 8, 2008. Revised manuscript received April 1, 2008. Accepted April 8, 2008.

Degradation products formed during ozonation of an aqueous solution of the antibiotic ciprofloxacin in a bubble column are analyzed by HPLC-UV high-resolution mass spectrometry. Based on the identification of the reaction products, reaction pathways are proposed starting with (1) degradation at the piperazinyl substituent, (2) degradation at the quinolone moiety with formation of isatin analogues, and (3) degradation at the quinolone moiety with formation of anthranilic acid analogues. Unlike H2O2 addition (10 µM), pH (3, 7, and 10) strongly affects degradation product formation during ozonation. Degradation at the quinolone core is favored at pH 7. Addition of t-butanol, a hydroxyl radical scavenger, ruled out formation of isatin and anthranilic acid analogues. Because the carboxylic group and the keto group at the quinolone moiety are essential for antibacterial activity, degradation at pH 7 seems to be promising for reduction of bacterial resistance against quinolones in contaminated water.

Experimental Section

Introduction The occurrence, fate, and behavior of pharmaceutical compounds in the environment have become an emerging issue. Points of concern involve the interference of hormone disrupting agents with natural hormones and the high toxicity of some groups of pharmaceuticals, e.g., neoplastics. There is a growing awareness toward antibiotics as well, since their discharge in the environment can induce bacterial resistance (1), including broad spectrum antibiotics like quinolones (2). Within Europe, the most widely prescribed quinolone antibiotic was ciprofloxacin (3). Ciprofloxacin has been detected at concentrations up to 124.5 µg · L-1 in raw sewage hospital water (4), up to 5.6 µg · L-1 in wastewater treatment plant (WWTP) effluents originating from household wastewaters (5) and even up to 31 mg · L-1 in effluents of WWTP, treating wastewaters of pharmaceutical manufacturers (6). Similar to many other pharmaceutical compounds, ciprofloxacin is poorly biodegradable (7) making physical-chemical technologies indispensable for its degradation prior to discharge in the environment. Advanced oxidation processes (AOPs), characterized by the generation of hydroxyl radicals at ambient conditions, gain large interest for degradation of pharmaceuticals (8). * Corresponding author phone: ++32 9 264 59 49; fax ++32 9 264 62 43; e-mail:[email protected]. 10.1021/es8000689 CCC: $40.75

Published on Web 05/23/2008

Due to the chemical complexity of the target compounds, AOP-based degradation of pharmaceuticals proceeds through quite complicated multistep pathways and complete mineralization is reported to be not cost-effective so far (9). Most studies focus on degradation kinetics of the target compound. However, identification of the intermediates formed during degradation of pharmaceuticals is necessary to fully optimize AOPs (9). Vogna et al. (10), for example, found that carbamazepine degradation by UV/H2O2 led to acridine intermediates, which are more toxic than their parent compound because of their mutagenic and carcinogenic activity. Considering in particular the AOP mediated degradation of quinolones, recent work on the removal kinetics of enrofloxacin (11–14), flumequine (14), norfloxacin (14), and ciprofloxacin (13–16) and toxicity evaluation of ofloxacin (17) can be found. Little attention has been paid, however, to the identification of reaction products. Only Paul et al. (14) confined six degradation products when studying ciprofloxacin degradation by visible-light mediated TiO2. Previously, we reported the parameters influencing ciprofloxacin degradation yield by ozonation such as ozone inlet concentration, ciprofloxacin concentration, pH, and H2O2 concentration (16). However, reaction pathways were not studied, and only one intermediate (desethylene ciprofloxacin) was identified. MS fragmentation of both ciprofloxacin and desethylene ciprofloxacin was reported. Up to 52-64% of ciprofloxacin was found to degrade through desethylene ciprofloxacin at pH 10 compared to 20-25% at pH 3 and 7 which could be explained by the acid-base centers of ciprofloxacin. In the present paper, the elaboration of the degradation mechanism is the main subject. By use of innovative high resolution mass spectrometry (HRMS), up to 17 reaction products have been proposed. Next, reaction pathways are proposed and the effect of pH, H2O2 and t-butanol on degradation product formation is investigated for the first time which can lead to reaction optimization and enhanced reduction of antibacterial activity.

 2008 American Chemical Society

Chemicals and Stock Solutions and Experimental Setup. Detailed information on chemicals and stock solutions and experimental setup can be found in the Supporting Information. Analytical Procedures. Gas phase ozone analysis and ciprofloxacin and desethylene ciprofloxacin analysis were done as recently described (16). For the analysis of degradation products, 25 mL liquid samples were taken by a liquid syringe (Hamilton, U.S.) with a 30 cm long needle through a septum on top of the reactor. Immediately after sampling, samples were flushed for 3 min with nitrogen at 15 mL · min-1 in order to remove residual ozone. All degradation products, except desethylene ciprofloxacin, were preconcentrated by a factor of 125 with solid phase extraction (SPE) on Oasis MAX cartidges (30 µm; 150 mg; 6 mL, Waters, U.S.). The SPE procedure was modified from the Atlantis columns applications notebook (18). First, the SPE cartridge was conditioned with 3 mL methanol, 1 mL 5 N NaOH solution and 1 mL of deionized water. Next, samples were mixed with 5 mL 25% NH4OH solution and loaded onto the cartridge. Then the cartridge was washed with 1 mL 5% NH4OH solution and 1 mL methanol. After that, the cartridge was allowed to run dry for 5 min before elution of retained analytes with 5 mL methanol containing 1% formic acid. Samples were evaporated to dryness in a water bath (50 °C) under a gentle stream of nitrogen (86 kPa) in a Turbovap LV (Caliper LifeScience, VOL. 42, NO. 13, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Ciprofloxacin Degradation Productsa tRb (min)

[M+H]+ measured

nominal mass (Da)

molecular formula

errorc (ppm)

10.90 11.65 15.58 16.98 17.79 18.69 19.69 21.84 22.65 23.80 37.01 37.14 40.14 40.49 40.95 41.33 41.95

310.120 336.135 346.119 282.125 308.140 264.114 290.131 292.110 306.125 332.141 310.120 362.113 350.114 334.120 263.076 348.135 337.119

309 335 345 281 307 263 289 291 305 331 309 361 349 333 262 347 336

C14 H16 O4 N3 F C16 H18 O4 N3 F C17 H16 O4 N3 F C13 H16 O3 N3 F C15 H18 O3 N3 F C13 H14 O2 N3 F C15 H16 O2 N3 F C14 H14 O3 N3 F C15 H16 O3 N3 F C17 H18 O3 N3 F C14 H16 O4 N3 F C17 H16 O5 N3 F C16 H16 O5 N3 F C16 H16 O4 N3 F C13 H11 O3 N2 F C17 H18 O4 N3 F C16 H17 O5 N2 F

-0.77 -1.85 -0.49 1.64 2.29 -1.41 2.30 2.00 -2.57 -2.34 0.80 -3.83 2.15 -0.90 -3.75 1.52 0.66

difference with ciprofloxacin

degradation pathwayd

- 3C 2H +O -C +O - 2H +O -4C 2H - 2C - 4C 4H O - 2C 2H O - 3C 4H - 2C 2H ciprofloxacin - 3C 2H +O - 2H + 2O - C 2H + 2O - C 2H +O - 4C 7H N +O -CHN + 2O

Pip, Qui Qui Pip, Qui Qui

n°

11 9 10 8

Pip

6

Pip Pip Pip Pip Pip

3 5 4 7 2

a The numbering follows the reaction pathways (Figure 1). Products for which a structure is proposed are presented in bold. b HPLC retention time based on MS detection. c Difference between measured and theoretical mass. d Suggested degradation pathway (pip ) piperazinyl, qui ) quinolonic moiety).

U.S.) followed by immediate dissolution in 200 µL water (0.1% formic acid). Finally, the samples were placed on a vortex at 3000 rpm for 1 min (Bio Vortex V1, Kisker-Biotech, Germany) and centrifuged at 1000 rpm for 2 min (EBA 20, Hettich, Germany) before HPLC/UV/MS analysis. For chromatographic separation, the HPLC needle was rinsed twice with 200 µL methanol (0.1% formic acid) and twice with 200 µL LC-MS grade water (0.1% formic acid) before injection of 10 µL sample into a Surveyor HPLC system (Thermo Finnigan, Germany). A Luna C18 (2) column (150 × 3.0 mm, 3 µm, Phenomenex, U.S.), thermostatted at 35 °C, was used with a mobile phase containing water (0.1% formic acid) as eluent A and methanol as eluent B. Eluent A/eluent B ratios changed from 90/10 (0 min) over 60/40 (23 min) and 80/20 (33 min) to a ratio of 10/90 (34 min) which was kept constant for a final 11 min. UV-vis analysis, coupled to HPLC, was performed on a Surveyor photodiode array detector (Thermo Finnigan) equipped with light pipe flow cell of 50 mm (10 µL) measuring spectra between 200 and 800 nm. For quantification, peak areas were obtained from wavelengths at the absorbance maxima ( 4.5 nm within 24 h after ozonation experiments. MS spectra (m/z ) 149.5-450.5) were recorded on a high resolution multi dimension MAT 95XP-Trap mass spectrometer (Thermo Finnigan) equipped with a TSQ/SSQ 7000 atmospheric pressure ionization source at a resolution of 1000 for low resolution and 8000 for high resolution MS (HRMS) measurements. Analyses were done in positive ionization mode by electrospray ionization (ESI). Polyethylene glycol (PEG) was used as reference for HRMS. Analytes as well as their fragmentation products, created when applying an additional energy of 100 V to the ESI needle (collision induced dissociation, CID), were analyzed as follows. First, the nominal mass was determined by low resolution MS (LRMS) followed by HRMS for elucidation of the molecular formula. Given the molecular formula of protonated ciprofloxacin (C17H19O3N3F), chemical formula containing 0-17 carbon atoms, 0-40 hydrogen atoms, 0-10 oxygen atoms, 0-3 nitrogen atoms, and 0-1 fluoro atoms were taken into account for identification of degradation and fragmentation products. If the measured m/z of the protonated compounds deviated less than 5 ppm from the theoretical values, the chemical formula was retained. Further indications for suggested structure of degradation products were given by (1) UV and (2) CID-HRMS analysis of 4890

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molecules containing functionalities identical to the suggested structures (3), by comparison with literature data and (4) by comparison with desethylene ciprofloxacin degradation products after desethylene ciprofloxacin ozonation.

Results and Discussion Ozonation of Ciprofloxacin: Reaction Pathways. Degradation Products. Ozonation of 15 mg · L-1 ciprofloxacin (Supporting Information Figure S1) in deionized water at pH 7 by a 120 mL · min-1 O3/air stream ([O3] ) 2500 ppmv) results in a half-life time of 16 min. 95% of the initial ciprofloxacin concentration is degraded after 60-75 min. A representative chromatogram after 30 min of ozonation is given in Supporting Information Figure S2. As can be seen a large number of degradation products could be detected. The molecular formula of 17 degradation products could be determined by HRMS (Table 1). Compounds for which a structure is suggested in next paragraphs are indicated in bold. In Supporting Information Table S1, it is summarized on the basis of which criteria structure assignment for each compound was performed. Ciprofloxacin degradation shows to occur at least at two functionalities of the molecule (1): at the piperazinyl substituent (2), at the quinolone moiety. Degradation at the Piperazinyl Substituent. In Figure 1, compounds 2-7 represent degradation products formed during oxidation of ciprofloxacin at the piperazinyl substituent. One main degradation product formed through a net loss of C2H2 at the piperazinyl substituent is desethylene ciprofloxacin (compound 6). Desethylene ciprofloxacin was univocal identified earlier based on nominal mass and retention time (16). Structures for compounds 2-5 and 7 are proposed based on the molecular formula (Table 1) and UV spectrum. Similar to ciprofloxacin, the UV spectrum of these compounds revealed a large absorbance peak between 272 and 280 nm with a shoulder between 300 and 350 nm, indicating that reaction did not take place at the quinolone core. Interpretation was also partially based on literature. Karl et al. (19) suggested degradation compounds analogous to compounds 2-4 and 7 during enrofloxacin oxidation by Gloeophyllum striatum for which degradation is thought to be based on hydroxyl radicals, generated by reduction of hydrogen peroxide by ferrous iron. The formation of desethylene ciprofloxacin may be explained by a multistep pathway starting with the introduction of a hydroxyl group into the piperazinyl substituent

FIGURE 1. Reaction pathways for ciprofloxacin ozonation. Intermediate A and B were not detected but suggested in agreement with Karl et al. (19). (compound 2; 347 Da) possibly followed by oxidation to the keto-derivative. An additional sequence of incorporation of an oxygen atom results in the formation of the hydroxylketo-derivative (compound 3; 361 Da). Subsequent loss of CO results in opening of the piperazinyl ring (compound 4; 333 Da). A second loss of a CO molecule leads to the formation of desethylene ciprofloxacin. Oxidation at the aldehyde functionality with formation of a carboxylic acid (compound 5; 349 Da) is an alternative degradation pathway for

compound 4, leading to desethylene ciprofloxacin after CO2 extrusion. Third, introduction of two hydroxyl groups into one ethylidene bridge can also lead to desethylene ciprofloxacin and glyoxal formation. Ozonation of desethylene ciprofloxacin at conditions identical to those applied for ciprofloxacin ozonation reveals three degradation products with molecular formula as well as retention time identical to ciprofloxacin degradation products: compound 7 (262 Da, C13H11O3N2F), compound VOL. 42, NO. 13, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. (a) CID-MS fragmentation products of compound [8 + H]+, (b) UV-vis spectrum of compound 8, and (c) molecular structure and UV-vis spectrum of isatin. 10 (263 Da, C13H14O2N3F), and compound 11 (281 Da, C13H16O3N3F). This indicates formation of these compounds out of ciprofloxacin through the intermediate desethylene ciprofloxacin. Compared to desethylene ciprofloxacin, compound 7 reveals further oxidation at the piperazinyl substituent (Figure 1) with loss of a nitrogen atom while compounds 10 and 11 are isatin and anthranilic acid analogues, respectively (see below). No organic amine, ammonia, nitrite, or nitrate was measured, so it can not be said how the nitrogen is present at the end of the reaction. Second, some desethylene ciprofloxacin degradation products revealed a net loss/gain, identical to the net loss/ gain of some ciprofloxacin ozonation products: net differences of -2C 2H O (263 Da) and -2C (281 Da) are observed during desethylene ciprofloxacin ozonation. Analogous losses are observed for compounds 9 and 10, respectively. This may indicate identical oxidation mechanisms for both desethylene ciprofloxacin and ciprofloxacin. Degradation at the Quinolone Moiety: Isatin Analogues. Molecular structures, CID-MS fragmentation products as well as UV-vis spectra of compounds 8 (289 Da) and 10 (263 Da) are given in Figure 2 and Supporting Information Figure S3, respectively. Compared to ciprofloxacin (Supporting Information Figure S1), the UV spectra reveal a shoulder with maximum absorbance at, respectively, 455 (Figure 2) and 463 nm (Supporting Information Figure S3). This shift toward higher wavelengths indicates absorbance of blue light, which might explain the yellow color appearing after 10 min of ciprofloxacin ozonation and disappearing again between 60 and 75 min of ozonation. This might be understood by the presence of a large polycentric molecular orbital (PCMO) or specific chromophoric groups like diketones. CID-MS fragmentation of compounds [8 + H]+ and [10 + H]+ leads to 4892

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a net loss of CO and C2O2 (Figure 2a, Supporting Information Figure S3a), being supportive for the presence of a diketone. Other fragmentation losses occurred at the piperazinyl substituent, similar to previously reported losses at the piperazinyl substituent during ciprofloxacin and desethylene ciprofloxacin CID fragmentation (16). The proposed structures for compounds 8 and 10 are tentatively identified based on isatin analysis. Isatin CID fragmentation products show loss of CO and C2O2, whereas the isatin UV spectrum also reveals a shoulder between 400 and 450 nm (Figure 2c). Shifts in the UV spectrum compared to compound 8 and 10 can be explained by the smaller PCMO present. Wetzstein et al. (2) also detected compound 8 during degradation of ciprofloxacin by Gloephyllum striatum. Degradation at the Quinolone Moiety: Anthranilic Acid Analogues. UV-vis-spectra and CID-products are recorded for the degradation products of 307 (compound 9, Supporting Information Figure S4) and 281 Da (compound 11, Supporting Information Figure S5), analogous to previous paragraph. Based on the observed absorbance spectra and the loss of H2O, H2O and CO, and H2O and C2O2 during CID fragmentation, chemical structures for compounds 9 and 11 are proposed (Supporting Information Figures S4 and S5). The suggested structures are tentatively identified by analysis of the analogous N-formylanthranilic acid (Supporting Information Figure S4c). The UV-vis spectrum also shows three maxima (215, 249, and 297 nm) slightly shifted compared to the ozonation products. Combined Reaction Pathway. Additional ozonation experiments with similar ciprofloxacin and ozone concentrations are performed at pH 3 and 10. Next to that, peroxonebased degradation of ciprofloxacin with an initial H2O2 concentration of 10 µmol · L-1 is carried out. At each condition, the suggested structures (Table 1) were detected. Good

FIGURE 3. Time profiles of ciprofloxacin ozonation products for the experiment at pH 7. separation power (Chromatographic Resolution > 1.4) and peak height/noise ratio > 20 are observed for ciprofloxacin degradation products 6, 8, 9, 10, and 11. These compounds proved to be stable for at least one week. Therefore, time profiles for each of these compounds could be plotted. This is illustrated in Figure 3 for the ozonation experiment at pH 7. Compound 9 (anthranilic acid analogue containing the piperazinyl substituent) and compound 8 (isatin analogue containing the piperazinyl substituent) both reach maximum concentrations at 20 min of ozonation. Next to that, the isatin analogue concentration decreases faster between 20 and 30 min of ozonation. This indicates that isatin analogues are not formed through anthranilic acid analogues. The anthranilic acid analogue with loss of ethylene (compound 11) reaches its maximum concentration at about 60 min, being a factor of 2 later than the corresponding isatin analogue (compound 10). Time profiles for the experiments at different pH and/or with H2O2 addition confirmed these findings (data not shown). This indicates that the anthranilic acid analogues, showing loss of C2, are not precursors for the isatin analogues, having loss of C2H2O. Moreover, Karl et al. (19) suggested simultaneous formation of isatin and anthranilic acid analogues from a common intermediate through reactions with hydroxyl radicals. The authors (19) proved that enrofloxacin degradation by Gloeophyllum striatum at the quinolone moiety leads to decarboxylation followed by cleavage of the quinolone moiety between C2 and C3. Finally, decarboxylation at C3 leads to anthranilic acid analogues, whereas deformylation at C2 leads to isatin analogues formation. Formation of the intermediate (Figure 1) can also be explained by direct ozonation at the double bond between C2 and C3 followed by subsequent loss of CO. Next, anomalous ozonolysis at the R,β-unsaturated carbonyl of ciprofloxacin can lead directly to formation of anthranilic acid analogues from ciprofloxacin and desethylene ciprofloxacin (20). Based on the proposed and identified reaction products and the time profiles shown in Figure 3, a combined reaction pathway for ciprofloxacin ozonation is proposed in Figure 1. Ciprofloxacin ozonation leads to degradation at the piperazinyl substituent eventually leading to loss of C4H7N at one hand, and degradation at the quinolone moiety with formation of the anthranilic acid analogue or intermediate A at the other hand. In agreement with Karl et al. (19), intermediate A can lead both to formation of isatin and anthranilic acid analogues. In a similar way, intermediate B is proposed to be a precursor for isatin and anthranilic acid analogues with degradation at the piperazinyl substituent.

FIGURE 4. Maximum peak area of compound 6, 8, 9, 10 and 11 during ciprofloxacin ozonation in function of pH and H2O2 concentration. The peak area of compound 6 (desethylene ciprofloxacin) was not concentrated by SPE and multiplied with a factor of 25 for reasons of visibility. Effect of pH, H2O2 and t-Butanol on Degradation Product Formation. Maximum peak areas, corresponding to compounds 8, 9, 10, and 11, were determined after concentration with SPE and investigated in function of pH (Figure 4). Compounds 9, 11 (anthranilic acid analogues), and 8 (isatin analogue) reveal maximum concentrations at pH 7, in contrast with previously reported peak area maxima of desethylene ciprofloxacin (16), determined without SPE, which showed maximum concentrations at pH 10 (Compound 7, Figure 4). This indicates the strong influence of pH on degradation product formation. Compound 10, an isatin analogue with degradation at the piperazinyl substituent, does not reveal a clear maximum concentration for any pH. However, reduced formation is observed at pH 3 without H2O2 addition. At neutral pH, degradation at the quinolone moiety is enhanced, probably because of two reasons. First, competitive effects between the piperazinyl substituent and the quinolone moiety for reactive species may occur: when the piperazinyl substituent is less degradable, consumption of the available reactive species for degradation at the quinolone moiety is favored. Second, the concentration ratio between the different active species may be the reason. Ozone and hydroxyl radicals are seen as the most important reactive species during ozonation (21), although other radicals (e.g., O2•-, O3•-, HO2•, HO4•) are also present (16). Direct ozone attack is less probable at the quinolone moiety than at the piperazinyl substituent because the π-electrons and free electron pairs take part in a PCMO. The higher concentration VOL. 42, NO. 13, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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of intermediates formed through degradation at the quinolone moiety may indicate that at pH 7, the concentration of hydroxyl radicals is higher than at pH 3 and 10. This optimum in pH can be explained by two processes. First, the higher the pH, the more ozone decomposes to hydroxyl radicals. Second, radical scavengers (e.g., carbonate ions) decrease the amount of hydroxyl radicals (21). At pH 3, peak areas corresponding to degradation products formed through oxidation at both the piperazinyl substituent and the quinolone moiety are found to be small compared to those at pH 7. The hydroxyl radical concentration is expected to be low at pH 3 (21). Next, reaction between ciprofloxacin and ozone is also expected to be slow at this pH compared to pH 7 and 10 due to protonation of the N-atoms with pKa-values of 8.95, 5.05, and 3.64 (22). Nevertheless, ciprofloxacin degraded significantly faster at acidic pH than at pH 7. As stated in a previous paper (16), this is probably due to the presence of radicals, other than hydroxyl radicals, at acidic pH. Although the chromatogram shows some compounds with higher peak areas at pH 3, they could not be identified so far. In order to rule out the effect of hydroxyl radicals at pH 3, 7, and 10, t-butanol was added as radical scavenger. The reaction constant of t-butanol with hydroxyl radicals amounts 6 × 108 M-1s-1, whereas with ozone, this is less than 0.003 M-1s-1 (23). Independent of the pH, no formation of isatin or anthranilic acid analogues (compounds 8, 9, 10, and 11) is observed in the presence of t-butanol while degradation at the piperazinyl substituent still takes place. This confirms the suggestion that hydroxyl radicals are necessary for degradation of ciprofloxacin at the quinolone moiety while direct ozonation easily occurs at the piperazinyl substituent. Figure 4 also includes peak areas of degradation products 6, 8, 9, 10, and 11 formed when 10 µM H2O2 was added initially to the reactor at pH 3, 7, and 10 (peroxone process). At pH 7, H2O2 addition slightly increases formation of compound 8 and 9, probably because H2O2 addition enhances the concentration of hydroxyl radicals through reaction with ozone (24). Moreover, it was recently proven that addition of 10 µmol H2O2 significantly enhances ciprofloxacin degradation at pH 7 (16). In general, however, it is clear from Figure 4 that H2O2 addition has a limited effect on degradation product formation compared to the effect of pH. The difference between degradation product formation at pH 7 and both acidic and alkaline pH may be important to reduce selective pressure for quinolones. The carboxylic group and the keto group at position 3 and 4, respectively, are considered to be necessary for binding quinolones to the DNA gyrase target (25). Degradation at the carboxylic group (quinolone moiety) with formation of isatin and anthranilic acid analogues reduces the antibacterial potential of the drug and by consequence selective pressure for quinolones (26). In contrast, intermediates which are only formed through degradation at the piperazinyl substituent still have the essential quinolone antibacterial structure (27) but the real antibacterial activity of these intermediates is difficult to predict based on their structure only. Contradictory data can be found in literature. Bielecka-Grzela and Klimowicz (28) mention that desethylene ciprofloxacin is as active toward bacteria as ciprofloxacin. However, Chu and Fernandes (25) report that quinolones with a piperazinyl substituent at C7 have a larger biological activity than quinolones with smaller substituents like -NHCH2CH2NH2. Next, Zeiler et al. (29) found that desethylene ciprofloxacin is more than 100 fold less active than ciprofloxacin against Escherichia coli Further degradation at the piperazinyl substituent leads toward compound 7 which is reported to have e3% antibacterial activity compared to ciprofloxacin (2). Antibacterial activity tests are necessary to further clarify the effect of pH on bacterial resistance. However, the chemical identification 4894

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of the intermediates reported here for the first time indicates that ozonation at pH 7 has higher potential to reduce bacterial resistance than the same process performed at pH 3 and 10. This encourages further optimization of ozonation as treatment technique for wastewater loaded with quinolones.

Acknowledgments We acknowledge financial support from the Flemish Government for the MAT 95XP-Trap in the framework of the Flemish investment support for heavy research equipment.

Supporting Information Available Chemicals and stock solutions, experimental setup, ciprofloxacin structure and UV spectrum, a chromatogram after 30 min of ciprofloxacin ozonation at pH 7 together with CIDMS and UV-vis data of compounds 9, 10, and 11 and a table summarizing the confirmation procedures used for each compound. This material is available free of charge via the Internet at http://pubs.acs.org.

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