Identification of photocatalytic degradation products of bezafibrate in TiO2 aqueous suspensions by liquid and gas chromatography

Available online at www.sciencedirect.com

Journal of Chromatography A, 1183 (2008) 38–48

Identification of photocatalytic degradation products of bezafibrate in TiO2 aqueous suspensions by liquid and gas chromatography D.A. Lambropoulou a , M.D. Hernando b , I.K. Konstantinou c , E.M. Thurman b , I. Ferrer b , T.A. Albanis a , A.R. Fern´andez-Alba b,∗ a Department of Chemistry, University of Ioannina, Ioannina 45110, Greece Department of Analytical Chemistry, University of Almeria, 04120 Almeria, Spain c Department of Environmental and Natural Resources Management, University of Ioannina, Agrinio 30100, Greece b

Received 9 October 2007; received in revised form 4 December 2007; accepted 10 December 2007 Available online 23 December 2007

Abstract In the present study the photocatalytic degradation of bezafibrate (BZF), a lipid regulator agent, has been investigated using TiO2 suspensions and simulated solar light. The study focus on the identification of degradation products (DPs) using powerful analytical techniques such as liquid chromatography time of flight mass spectrometry (LC–TOF–MS), gas chromatography mass spectrometry (GC–MS), and high-performance liquid chromatography with diode-array detection (HPLC–DAD). Each technique provided complementary information that enabled the identification of 21 DPs. Accurate mass measurements obtained by LC–TOF–MS provided the elucidation of 17 DPs. Mass errors lower than 2 mDa, allowed the assignment of empirical formula for the mayor DPs to be determined confidently. Three DPs were identified by GC–MS through the structural information provided by full scan mass spectra obtained by electron impact (EI) ionization and two more by HPLC–DAD by comparing the retention times (tR ) and the UV spectra of the unknown DPs with those of commercial standards. Based on this by-product identification a possible multi-step degradation scheme was proposed. The pathways include single or multiple hydroxylation of BZF with subsequent phenoxy ring opening and the cleavage of the amide and ether bonds. © 2008 Elsevier B.V. All rights reserved. Keywords: Photocatalysis; Titanium dioxide; Bezafibrate; LC–TOF–MS; Degradation products; Pharmaceutical residues

1. Introduction Degradation products (DPs) of pharmaceuticals in the environment or formed during wastewater treatments have attracted an increasing interest in the last years, since they also may be potentially toxic [1,2]. Bezafibrate (BZF, p-[4-[chlorobenzoylamino-ethyl]-phenoxy]-b-methylpropionic acid) is a lipid regulator agent extensively used as prescription drug, with an estimated annual consumption in developed countries of several hundreds of tonnes [3]. Environmental concern of BZF could be associated with its wide use but also with its persistence. BZF has been included in several monitoring studies carried out in various aquatic systems and has been detected in sewage treatment plant (STP) effluent, surface water and drinking waters

∗

Corresponding author. Tel.: +34 950 01 50 34; fax: +34 950 01 50 84. E-mail address: [email protected] (A.R. Fern´andez-Alba).

0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.12.030

at concentration as high as 4.6, 3.1 and 27 ng/L, respectively [1,4–6]. At present, scarce information is available concerning DPs of BZF in water. Few studies available have been based on biodegradation process [7]. An improved knowledge regarding the degradation of BZF in wastewater treatments and the identification of its metabolites or DPs should provide useful information for determining its fate in the environment. Photocatalytic processes with irradiated semiconductors involving oxido-reductive reactions can be useful either for decontamination [8–17] or as techniques for studying the formation of DPs in environment media mainly by hydrolysis or photolysis. It is important to note that the assessment of the reliability of a degradation treatment for a pollutant relies also on the nature of DPs generated, which can be a great concern as they can be even more toxic than the precursor itself. For that, the application of analytical tools capable of providing the complementary information is suitable to complete the puzzle, leading to the final structural assignment for the wide range

D.A. Lambropoulou et al. / J. Chromatogr. A 1183 (2008) 38–48

of compounds expected in such studies. Gas chromatography mass spectrometry (GC–MS) and liquid chromatography mass spectrometry (LC–MS) techniques are powerful analytical techniques, because of their selectivity and sensitivity, which allow the identification of DPs and tentative degradation pathways can be proposed [18–23]. The work presented inhere describes, the use of LC–TOF–MS, GC–EI-MS and HPLC–DAD systems for identifying the structure of BZF DPs during TiO2 -mediated photocatalysis under solar irradiation. In particular, information obtained from all strategies has been interpreted in terms of molecular structures, and a tentative scheme for the BZF degradation has been proposed.

39

2. Experimental

irradiated separately under the same experimental conditions in order to provide an adequate concentration factor for our applications. Finally, photolysis experiment in the absence of TiO2 was performed under the same irradiation conditions. Irradiation was performed using a Suntest CPS+ apparatus from Heraeus (Hanau, Germany) equipped with a xenon arc lamp (1500 W) and special glass filters restricting the transmission of wavelengths below 290 nm. An average irradiation intensity of 750 W/m2 was maintained throughout the experiments and was measured by internal radiometer. The corresponding light dose for 10 min of irradiation was 450 kJ/m2 . Chamber and black panel temperatures were regulated by pressurized aircooling circuit and monitored using thermocouples supplied by the manufacturer. The temperature of samples did not exceed 25 ◦ C using tap-water cooling circuit for the UV reactor.

2.1. Reagents and materials

2.3. Sample preparation

Analytical grade BZF (99.5% purity) was purchased from Promochem (Germany). 4-Chlorobenzoic acid (4-CBA) (97%, purity) and 4-hydroxybenzoic acid (99%, purity) were purchased from Flucka and sodium sulfate (proanalysis) and HK2 PO4 were from Merck (Darmstadt, Germany). Pesticidegrade methanol and acetonitrile were purchased from Labscan (Dublin, Ireland) and Merk (Darmstadt, Germany), respectively. Titanium dioxide, TiO2 (Degussa P25), a mixture of 80% anatase and 20% rutile with an average particle size of 30 nm, nonporous with a reactive surface area of 50 m2 /g was used for the degradation experiments. Doubly distilled water, filtered through 0.45 mm highly anisotropic membranes (HA cellulose acetate, Millipore) was used. Oasis HLB cartridges (6 mL, 200 mg) were purchased from Waters. Stock standard solutions of 1000 mg/L of each compound were prepared in methanol. Working standards solutions were prepared by diluting the stock solutions with methanol. The stock and working standards were stored at 4 ◦ C. BZF has a molecular weight of 361.83 with very low water solubility (0.355 mg/L at 25 ◦ C) [24]. Aqueous solutions were prepared by spiking the water with an appropriate amount of the stock solution in methanol, to have methanol content <0.05%.

The irradiated solution (50 mL) was extracted by means of solid-phase extraction (SPE). Oasis HLB (hydrophilic– lipophilic balance) cartridges (6 mL, 200 mg) were placed in a filtration apparatus (Supelco, Bellefonte, PA, USA) and washed with 5 mL of methanol and 3 mL of deionized water, without vacuum. Then, the irradiated solution was extracted at a flowrate of 10 mL/min. The compounds trapped in the cartridges were collected by using 2 × 5 mL of methanol as eluting solvent. The eluents were evaporated under a gentle stream of nitrogen to 500 ␮L of methanol. A 250 ␮L aliquot of the SPE extract was completely dried down and taken up in 100 ␮L of ethyl acetate for GC–MS analysis (injection volume 2 ␮L). The second aliquot of 250 ␮L of SPE extract was concentrated by solvent evaporation with a gentle stream of nitrogen and recomposed to a final volume of 200 ␮L in water/methanol (50:50, v/v) prior to analysis into LC–TOF–MS and HPLC–DAD instruments.

2.2. Photocatalytic degradation experiments Irradiation experiments of BZF were performed on stirred aqueous solutions using a 100 mL pyrex glass UV reactor containing 50 mL of BZF aqueous solution (1 mg/L) and 100 mg/L of TiO2 at natural pH. Before irradiation, separate adsorption experiments were conducted under dark conditions. The suspensions were continuously stirred under darkness for 4 h, and aliquots (1 mL) were collected and filtered (0.2 mm Anatop 25 plus, Whatman) at different time intervals, and the concentration of BZF remaining in the filtrates was analyzed and compared to the total concentration initially added. Concentration levels were monitored by using the HPLC–DAD system. Before irradiation the solutions were kept in the dark for 60 min under stirring to reach adsorption equilibrium on to semiconductor surface. For the identification of photoproducts, 50 mL of BZF solution was

2.4. LC–TOF–MS analysis LC–TOF–MS system was firstly applied for the identification of DPs of BZF. The separation of BZF and the DPs was carried out using an HPLC system (Agilent Series 1100, Agilent Technologies, Palo Alto, CA, USA) equipped with a C8 analytical column, 150 mm × 4.6 mm, 5 ␮m particle size (Zorbax Eclipse XDB-C8). Column temperature was maintained at 25 ◦ C. The injected sample volume was 50 ␮L. Mobile phases A and B were acetonitrile and water with 0.1% formic acid, respectively. The chromatographic method held the initial mobile phase composition (10% A) constant for 5 min, followed by a linear gradient to 100% A in 25 min. The flow-rate was 0.6 mL/min. The HPLC system is connected to TOF MSD (Agilent Technologies) with an electrospray interface, using the operational parameters optimized in a previous study [12]. 2.5. HPLC analysis The HPLC system consisted of a Shimadzu (Kyoto, Japan) Model LC-10ADVp pump associated with a 7725i Rheodyne

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six-port valve and a Shimadzu Model SPD-10AVp UV–vis diode-array detector connected to a Shimadzu Model Class VP 5 integration software. The analytes were separated by means of a Discovery C18 (Supelco), (250 mm length × 4.6 mm i.d., 5 ␮m particle size) analytical column from Supelco that was fitted with a guard column of the same material. A gradient elution was performed by a binary gradient composed of solvent A (aqueous solution of HK2 PO4 , 5 mM) and solvent B (acetonitrile) according to the following program: 10–20% B in 15 min (1 mL/min); 20–50% B in 5 min (1 mL/min); 50–70% B in 10 min (1 mL/min); 70–100% B in 10 min (1.0 mL/min); 100% B for 5 min (1 mL/min) to return to the initial conditions. Column temperature was set at 30 ◦ C. The detector was set at 227 nm for BZF, 254 nm for 4-hydroxybenzoic acid and 237 nm for 4-CBA. The peak identification of the target species was based both on the tR and the spectra matching to standards. 2.6. GC–EI-MS analysis A GC–MS, QP 5000 Shimadzu instrument equipped with a DB-5-MS capillary column (J&W Scientific) of 30 m length and 0.25 ␮m i.d., coated with 5% phenyl 95% methylpolysiloxane was used for identifying DPs under the following chromatographic conditions: injector temperature 250 ◦ C, oven temperature program from 55 (held for 2 min) to 210 ◦ C at a rate of 5 ◦ C/min (held for 20 min) and to 270 ◦ C at a rate of 10 ◦ C/min. Helium was used as the carrier gas and the interface was kept at 290 ◦ C. The MS system was operated in electron impact (EI) mode with an ionization potential of 70 eV and the spectra were obtained in full scan mode. 3. Results and discussion 3.1. Kinetics of disappearance by TiO2 -photocatalytic process Fig. 1 shows the kinetics of BZF degradation, while in the insert the logarithm of the ratio of the initial concentration (C0 ) to the concentration at a given time (C) versus time (t), over 250 min, is plotted. BZF was shown to degrade in less than

Fig. 1. Degradation kinetics of bezafibrate (C0 = 1 mg/L) in aqueous TiO2 suspensions (100 mg/L) under simulated solar light. The inset represents the semi-logarithm plot of the curve.

200 min following pseudo-first order kinetics and the apparent first order rate constant k was 2.81 × 10−2 min−1 (Fig. 1). The results from both adsorption experiments and the dark control demonstrate low BZF adsorption to TiO2 surfaces and no detectable degradation of BZF was observed within 4 h in the dark at room temperature indicating that hydrolysis of BZF can be neglected. Similarly, blank experiments under simulated solar light without catalysts, using the same initial concentration as in photocatalytic experiments, confirmed the absence of photolysis. 3.2. Identification of photoproducts by LC–TOF–MS The LC–TOF–MS total ion chromatogram (TIC) (Fig. 2) shows the formation of various BZF DPs that could be identified by accurate mass determination to deduce molecular formulae. Up to 17 compounds could be detected as possible DPs. DPs were unequivocally identified by using: (a) an identification program of TOF instrument with an accuracy threshold of 5 ppm (standard for unknown identification—error obtained were less than 2 ppm in most cases), (b) the abundance of the chlorine isotope in the molecular ion, (c) a characteristic fragment ion used as diagnostic ion (DI) for the DPs, and (d) the presence of the sodium adduct as an additional peak together with the peak of the protonated molecule. In Fig. 2, the TIC shows the molecule of BZF at m/z 362 as base peak (tR 21.9 min). The accurate mass of BZF was 362.1154 with a 37 Cl isotope signal of 364.1128 with a relative intensity of about one-third of the main peak. The protonated form of BZF is also accompanied by a sodium adduct giving a positive charged ion at m/z 384.097. The fragment ion with accurate mass of 138.9965 and with a signal at m/z 140.99 was used as the DI for examining the accurate mass spectra of BZF DPs. From both, the relative intensity of these signals and the difference between the two masses, it can be deduced that the chlorine atom is present in the molecular ion of this fragment. The m/z 138.9965 of this DI gave a unique elemental composition [C7 H3 ClO] in the calculator tool and the structure (with an error of <2.0 ppm in most cases) is presented in Fig. 2. In this way, the presence and number of chlorine atoms in the suspected DPs can be easily attained taking into account both the relative intensity of the 37 Cl/35 Cl signals and the accurate mass differences between the two masses. Targeting in the m/z of DI, numerous chlorine-containing suspected species were found in the TIC, at retention times between 15 and 23 min. The tR for most DPs are shorter than that of BZF, probably due to an increased polarity and/or shorter structure of the molecules. Exact mass measurements, elemental compositions and retention times of BZF and identified DPs are presented in Table 1. Various hydroxylated DPs were detected by LC–MS at the first stages of the treatment as a consequence of the • OH radical attack on the aromatic moiety. There are two possibilities for these structural features, either hydroxylation on the 4-chlorobenzoyl ring or hydroxylation of the phenoxy moiety. Examination of the product ion mass spectra of the target compounds revealed that • OH radical attack at the phenoxy

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Fig. 2. Total ion chromatogram (TIC) by LC-TOF-MS obtained from a SPE extract of bezafibrate solution after 60 min of irradiation with simulated solar light in the presence of TiO2 suspension (100 mg/L) showing the protonated molecular ion of BZF at m/z 362 and chlorinated by-products. Peak assignment is based on the molecules illustrated on Table 1. Identification criteria for chlorine by-products, (i) searching of diagnostic ion – accurate mass: 138.9965 (remaining fragment ion from BZF) and (ii) chlorine isotopic profile.

ring is favored and that attack at 4-chlorobenzoyl moiety is a minor route. The DI (138.9965 u) corresponding to the loss of C12 H18 NO3 group from BZF is still present in the spectra of all target DPs, as can be noted from the typical isotopic distribution, so excluding that the • OH radical has attacked the 4-chlorobenzoyl moiety, otherwise this peculiar ion would be absent. Therefore, the • OH attack occurred preferably on the phenoxy ring. This is also supported by the effect on substituents of the aromatic ring. • OH radical had strong electrophilic character and tended to attack the carbon atoms with the highest electron density. The chlorine-containing ring is characterized by a lower reactivity with respect to the unchlorinated one due to the electron withdrawing effect of the chlorine itself. In addition, the –CONH group attached to the chlorinated ring is also a deactivating substituent. On the contrary, the electron donating R-oxy substituent (–O–C(CH3 )2 COOH) has an activating effect increasing the electron density of the unchlorinated ring and the subsequent electrophilic attack by • OH. R-oxy substituents have an activating effect on aromatic rings similar to that of hydroxy substituents [13,25]. Similarly, the hydroxylation of triclosan by the electrophilic attack of • OH and O3 have been found to proceed primarily on the phenol group but not on the o-dichloro-phenoxy group [26,27]. The monohydroxylated products that have been identified (see structures 14a and b in Table 1) were characterized by different m/z ratios along with different peaks. The two peaks eluted prior to BZF at 19.7 and 20.9 min and yielding an accurate mass m/z of 378.1100, gave the best-fit formula C19 H20 ClNO5 (neu-

tral molecule). These spectra correspond to the addition of one radical, consistent with the formation of monohydroxylated derivates. Additionally, the protonated form of the target DPs is also accompanied by one sodium adduct giving a positive charged ion at m/z 400.0925. The two spectra are almost identical and one of them (tR 20.9) is shown in Fig. 3. Since the R-oxy group has similar activating effect on aromatic rings to that of –OH group the hydroxylation would be facilitated by the ortho and para orientation. The para position is not available thus, the hydroxylation should take place predominately in the ortho position in respect to the R-oxy group and the formation of meta-hydroxylated derivative would be expected as a minor route. However, a steric effect of the bulky R-oxy group cannot be excluded leading to an increase in the formation of meta-derivative. Considerations concerning the polarity of the molecules could help also in discriminating between them. In the case of the ortho isomer, an intramolecular hydrogen bond between the hydrogen of the hydroxyl group borne by the ring and the oxygen of the ether group could be expected to take place as observed for 2-methoxyphenol [28,29]. In that case the polarity of the compound should be decreased, increasing its tR . The order of elution of photoproducts was a positive indication that 14a (tR = 19.7 min) was the meta-derivative and 14b (tR = 20.9 min) the ortho-derivative. Based on the above indications, 14a and b products appear to correspond to the possible isomers arising from • OH monosubstitution on the benzene ring. Continuous • OH attack could lead either to the formation of more hydroxylated derivatives (compound 12, m/z 410) and/or • OH

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Table 1 Exact mass measurements and elemental compositions of bezafibrate, its degradation products and their fragment ions using LC–TOF–MS analysis Observed mass ([M+H]+ )

tR (min)

Elemental compositions

Calculated mass

Error (ppm)

1

224.1281

11.5

C12 H18 NO3 +

224.1282

0.4

2

138.0912

12.3

C8 H12 NO+

138.0913

1.0

3

240.0419

15.4

C11 H11 ClNO3 +

240.0421

1.24

4

300.0628

15.7

C13 H15 ClNO5 +

300.0633

1.7

5

156.0208

16.1

C7 H7 ClNO+

156.0210

1.7

6

298.0474

16.3

C13 H13 ClNO5 +

298.0476

0.9

7

228.0417

16.6

C10 H11 ClNO3 +

228.0421

2.2

8

292.0730

17.3

C15 H15 ClNO3 +

292.7349

1.7

9

282.0522

17.6

C13 H13 ClNO4 +

282.0527

1.9

10

308.0682

18.1

C15 H15 ClNO4 +

308.0684

0.7

11

266.0576

18.4

C13 H13 ClNO3 +

266.0578

0.9

12

410.0999

18.9

C19 H21 ClNO7 +

410.1001

0.5

13

384.0843

19.5

C17 H19 ClNO7 +

384.0845

0.407

DPs

Chemical structure

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43

Table 1 (Continued ) Observed mass ([M+H]+ )

tR (min)

Elemental compositions

Calculated mass

Error (ppm)

14a

378.1098

19.7

C19 H21 ClNO5 +

378.1106

1.3

15

368.0896

19.9

C17 H19 ClNO6 +

368.0895

1.3

16

276.0781

20.3

C15 H15 ClNO2 +

276.0785

1.7

14b

378.1100

20.9

C19 H21 ClNO5 +

378.1106

0.2

BZF

362.1151

21.9

C19 H20 ClNO4 +

362.1154

0.7

17

318.0893

22.8

C17 H17 ClNO5 +

318.0891

0.5

DPs

Chemical structure

ring opening derivatives (compounds 13 and 15, m/z 384 and 368, respectively). The compound with m/z 368 seems to correspond to a degradation by-product already identified during its treatment with ozone [30], namely, a derivative which comes from the cleavage of the unchlorinated aromatic ring after direct • OH attack. Alternatively, the cleavage of the aryloxy-carbon bond and the loss of the methyl propionic acid group could take place: (a) initially on the BZF molecule to form compound 16 (m/z 276) and can be also followed by ring hydroxylation (compounds 8 and 10, m/z 292 and 308, respectively) and ring opening (compound 4, m/z 300); and, (b) on the ring-hydroxylated and ring-opened derivatives to form compounds 6, 9 and 11 (m/z 298, 282 and 266, respectively). All the fragments in the aboveidentified compounds show an isotopic distribution typical of the presence of chlorine atom. Compound 16 (tR = 20.3 min, m/z 276) that matched a formula of C15 H14 ClNO2 with an accuracy of 1.7496 ppm was one of the most abundant fragment ions in the TIC (Fig. 2). The protonated form of the target DP is also accompanied by sodium adduct giving a positively charged ion at m/z 298. It should be noted that this compound was also detected as a photoproduct of BZF [31] while an analogue product was identified in the photocatalytic degradation of another fibrate pharmaceutical, genfibrozil [32]. The products at 15.7 and 16.3 min (compounds 4 and 6, respectively) showed highly similar spectra and a difference of 2 u in the m/z molecular ions indicating that these compounds were oxo- and hydroxy-derivatives of a base structure. Further steps of oxidation and decarboxylation leads to the for-

mation of compound 3 (m/z 240) and compound 7 (m/z 228), which was the most abundant fragment ion in the product ion mass spectrum. The final product formed after the successive oxidation-decarboxylation cycles (compound 5) was identified as the protonated 4-chlorobenzamide (tR = 16.1 min). As can be seen in Table 1, the accurate mass of this compound (156.0208) gave a unique elemental composition of C7 H7 ClNO+ (error 1.7 ppm). It should be noted that the 4-chlorobenzamide was also identified by using GC–MS. The mass spectra of the major intermediates are depicted in Fig. 3. Of particular interest was also the peak eluting at 22.8 min. Based on exact mass measurements, the elemental composition of this ion was C17 H17 ClNO3 + , indicating that BZF molecule had lost a CH3 CH2 group. Based on the product ion spectra in combination with the presence of DI the unknown compound is proposed to be a cyclic product (DP 17), the structure of which is depicted in Table 1. Along with the chloro-photoproducts, the presence of non-chlorinated products could also be detected during the photocatalysis of BZF. At tR of 11.5 min, the full scan spectrum for the m/z 224 fragment could be assigned to a protonated DP (compound 1) formed by the cleavage of the amide bond followed also by the release of the 4-CBA. The absence of chlorine atom can be also observed by the absence of the DI and the isotope M + 2 ion contribution. Similarly the peak corresponding to compound 2 (tR 12.3, m/z 138) could be assigned to a product formed by the elimination of the methylpropionic acid group from compound 1 following the same pathway as mentioned previously for the formation of compound 16 from BZF.

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Fig. 3. Product ion spectra of the major intermediates and the DP 14b with Rt 20.9 min.

It is worth mentioning that three additional peaks in the TIC at tR 16.8, 20.7 and 21.6 min were considered as unknown DPs U1, U2a and U2b, respectively, because their concentration increased and decreased as a function of the reaction time. For these compounds it is difficult to find a match with a formula, which could be chemically coherent with BZF and only the atom composition was obtained. Comparing the fragments of this unknown species with the parent molecule, it can be noted that all these molecules contain the DI. A summary of the LC–TOF–MS data for the above compounds is presented

in Table 2. This task could be solved by a structure elucidation using an LC–MSn and accurate mass measurements using QTOF. Both sets of data can be combined and used to construct the chemical structure of the possible DPs [33]. 3.3. Identification of photoproducts by GC–EI-MS The extract of the BZF solution after 60 min of irradiation was also analyzed by using GC–MS. All the identified DPs were eluted before BZF (tR 26.6 min). Most of the by-products

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Table 2 Exact mass measurements and elemental compositions of possible degradation products of bezafibrate Observed mass ([M + H]+ ) U1 U2a U2b

296.0680 376.0943 376.0945

tR (min)

Elemental compositions +

16.8 20.7 21.6

C14 H16 ClNO4 C19 H19 ClNO5 + C19 H19 ClNO5 +

Calculated mass

Error (ppm)

296.0684 376.0946 376.0946

1.393 0.869 0.338

Table 3 Degradation products of bezafibrate using GC–EI-MS analysis Compound

Chemical structure

MW

tR (min)

Formula

m/z

Fitting with the spectra library (%)

P1

164.2

16.6

C10 H12 O2

165, 164, 123, 124

89

P2

155.58

21.7

C7 H6 ClNO

155, 139, 111, 75

92

P3

140.57

25.7

C7 H5 ClO

139, 111, 75

70

observed in the LC–TOF–MS appear to be missing in the EI-MS chromatogram; this is likely related to low ionization efficiency, high polar character and/or thermal instability and low volatility. Three photoproducts (P1, P2 and P3), were unequivocally identified by using NIST library with a fit value >70% in all cases, one of which (4-chloro-benzamide), as mentioned before, was also identified by LC–TOF–MS. A summary of the GC–MS data is reported in Table 3. Fig. 4 shows the EI mass spectra of the products P1, P2 and P3, which was identified by the NIST library as 4-isopropoxybenzaldehyde, 4-chloro-benzamide and 4-chloro-benzaldehyde, with an 89%, 92% and 70% fit, respectively. 3.4. HPLC–DAD identification products

Fig. 4. GC–EI-MS mass spectra of the products P1, P2 and P3.

Using the HPLC–DAD system, two more photoproducts, 4hydroxy-benzoic acid (H1) and 4-chloro-benzoic acid (H2) were detected as the peaks eluting at 11.15 and 23.2 min (Table 4). A comparison between the retention times and the UV spectra of the commercial standards and the unknown DPs under the same chromatographic conditions was performed for their identification. 4-Chloro-benzoic acid, generated via the hydrolytic cleavage of the amide bond of BZF, has been also identified as major hydrolysis product by microbial degradation [7]. 4Hydroxy-benzoic acid is generated from 4-chloro-benzoic acid by the chlorine substitution with hydroxyl group or by reductive dehalogenation and the subsequent electrophilic addition of the • OH radical.

46 D.A. Lambropoulou et al. / J. Chromatogr. A 1183 (2008) 38–48 Fig. 5. Tentative degradation pathways for BZF photocatalytic degradation by TiO2 and simulated solar light. Dotted arrows represent pathways among the DPs that were not detected but are proposed based on previous photocatalytic studies.

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Table 4 Degradation products of bezafibrate using HPLC-DAD analysis Compound

Chemical structure

MW

tR (min)

Formula

H1

138.12

11.15

C7 H6 O3

H2

156.56

23.2

C7 H5 ClO2

3.5. Mechanism of BZF photocatalytic degradation The great number of identified DPs of BZF shows the complexity of the photocatalysis process and suggests the existence of various reductive and oxidative degradation routes resulting in multi-step and interconnected pathways. On the basis of the identification of intermediates and previous studies on the oxidation of clofibric acid [34,35] and chlorobenzoic acid [36] a tentative photocatalytic degradation pathway of BZF was proposed in Fig. 5. Three pathways (A, B and C) are proposed based upon the organic intermediates identified by LC–TOF–MS, GC–EI-MS and HPLC–DAD systems (Fig. 5). The pathways include single or multiple hydroxylation of BZF via • OH radical electrophilic attack with subsequent ring opening (pathway A) and the cleavage of the ether (pathway B) and amide (pathway C) bonds via positive holes. Pathway B could also take place after primary hydroxylation and ring opening. The three pathways were commonly observed in the photocatalytic degradation of other pharmaceutical and pesticide pollutants having the same functional groups [18,26,32,35]. It should be pointed that in the absence of standards, the assessment of the relative importance of the different pathways is doubtful, but taking into account the similarity of the majority of by-products proposed, a similar response can be assumed in LC–TOF–MS. Considering the number of photoproducts that retained the amide functional group in their structures and their relative responses it can be proposed that pathways A and B are the predominant pathways for the photocatalytic degradation of BZF and that pathway C consist only a minor route. In addition, the monitoring of the DPs at different time intervals reveals that the pathways follow the simplistic model of parallel and consecutive reactions as proposed elsewhere [37]. As can be seen in BZF photocatalytic degradation pathway, the formation of 4-chloro-benzamide, 4-chloro-benzaldehyde and 4-chloro-benzoic acid derivatives, take place at prolonged irradiation times. It is known from previous photocatalytic studies [36,38] that after the formation of such derivatives, 4-chlorophenol and hydroquinone are subsequently produced before complete mineralization. In addition, 4-chlorophenol and hydroquinone was also detected as advanced oxidation DPs of other lipid regulator compounds of the same chemical family as clofibric acid [34,35]. The formation of such intermediates was

not investigated in the present study that has focused more on the initial by-products. 4. Conclusions The photocatalytic degradation of BZF has been studied using TiO2 as catalyst and simulated solar light. The combination of LC–TOF–MS, GC–EI-MS and HPLC–DAD has been shown to be powerful tools in the characterization of BZF intermediates. The number of identified DPs of BZF shows the complexicity of the photocatalysis process and suggests the existence of various degradation routes resulting in multi-step and interconnected pathways. Investigations on the intermediates suggest that the hydroxyl radical attack occur on the phenoxy ring of the BZF before the ring opening, which produced many cycle opening products in the course of the reaction. Alternatively, the cleavage of the ether and amide bonds via positive holes also takes place. Chlorobenzoic acid, chlorobenzaldehyde and chlorobenzamide were found as DPs at prolonged irradiation times. Further investigations using, LC–MSn analysis, among other methods, must be undertaken to attempt to establish the overall degradation pathway. Finally, a thorough evaluation of the toxicity of DPs is needed in order to optimize photocatalytic treatment and assess the relative risks to the environment. Acknowledgements This study was financially supported by the Spanish Ministerio de Educaci´on y Ciencia Projects CTM2004-06265-CO3-01, PPQ2002-04573-C04-03 and CSD2006-00044. Dr M.D. Hernando acknowledges the research contract (contrato de retorno de investigadores) from Consejer´ıa de Educaci´on y Ciencia de la Junta de Andaluc´ıa, Spain. References [1] K. Fent, A.A. Weston, D. Caminada, Aquat. Toxicol. 76 (2006) 122. [2] M. Isidori, A. Nardelli, L. Pascarella, M. Rubino, A. Parrella, Environ. Int. 33 (2007) 635. [3] G.C. Daughton, T.A. Ternes, Environ. Health Perspect. 107 (1999) 907. [4] T.A. Ternes, Water Res. 32 (1998) 3245. [5] E. Zuccato, S. Castiglioni, R. Fanelli, J. Hazard. Mat. 122 (2005) 205. [6] O.A. Jones, J.N. Lester, N. Voulvoulis, Trends Biotechnol. 23 (2005) 163. [7] J.B. Quintana, S. Weiss, T. Reemtsma, Water Res. 39 (2005) 2654.

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