Analytical Methods
Roxithromycin degradation by acidic hydrolysis and photocatalysis
Journal: Analytical Methods
Manuscript ID: AY ART 03 2014 000708.R2
Article Type: Paper
Date Submitted by the Author: 28 May 2014
Complete List of Authors: Kwiecień, Anna; Jagiellonian University, Collegium Medicum, Department of Inorganic and Analytical Chemistry Krzek, Jan; Jagiellonian University, Inorganic and Analytical Chemistry Żmudzki, Paweł; Jagiellonian University, Collegium Medicum, Department of Medicinal Chemistry Matoga, Urszula; Jagiellonian Univeristy, Collegium Medicum, Department of Inorganic and Analytical Chemistry Długosz, Maciej; Jagiellonian University, Faculty of Chemistry Szczubiałka, Krzysztof; Jagiellonian University, Faculty of Chemistry Nowakowska, M; Jagiellonian University, Faculty of Chemistry
Page 1 of 23 Analytical Methods
1 2 3 Roxithromycin degradation by acidic hydrolysis and 4 5 6 photocatalysis 7 8 9 10 11 12 13 Anna Kwiecie ń*1 , Jan Krzek 1, Paweł Żmudzki 2, Urszula Matoga 1, Maciej Długosz 3, 14 3 3 15 Krzysztof Szczubiałka , Maria Nowakowska 16 17 18 1 Department of Inorganic and Analytical Chemistry, Jagiellonian University Medical 19 20 College, Faculty of Pharmacy, Medyczna 9, 30-688 Kraków, Poland 21 2 Department of Medicinal Chemistry, Jagiellonian University Medical College, Faculty of 22 23 Pharmacy, Medyczna 9, 30-688 Kraków, Poland 24 3 25 Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 *Corresponding author: 42 43 Dr Anna Kwiecie ń 44 Department of Inorganic and Analytical Chemistry 45 Jagiellonian University, Medical College, Faculty of Pharmacy 46 9 Medyczna Street 47 30-688 Kraków, Poland 48 phone: +48 12 620 54 80 49 fax +48 12 620 54 05 50 e-mail: [email protected] 51 52 53 54 55 56 57 58 59 60 1 Analytical Methods Page 2 of 23
1 2 3 Abstract 4 5 6 The purpose of this work was to study two methods of degradation of roxithromycin; acidic 7 8 hydrolysis and photocatalytic degradation under illumination with near-UV light in the 9 10 11 presence of the TiO 2 photosensitizer. For acidic degradation studies a TLC-densitometric 12 13 method have been developed using TLC aluminium plates precoated with silica gel F 254 and 14 15 the mobile phase composed of methanol-acetone-ammonia 25% (1:14:0.1, v/v/v) which gives 16 17 compact spots for roxithromycin and its degradation products. Photocatalytic degradation 18 19 studies in the presence of TiO were monitored by HPLC . The obtained results have shown 20 2 21 22 that both methods of degradation of roxithromycin are quite efficient. Kinetics of first order 23 24 photocatalytic degradation reaction follows the Langmuir-Hinshelwood theory for 25 26 photocatalytic microheterogeneous systems. The chemical structures of the degradation 27 28 products obtained during acidic degradation and photocatalytic degradation were proposed 29 30 31 based on UPLC-MS/MS technique. The general mechanisms of both processes were 32 33 proposed. 34 35 36 37 38 39 Key words: roxithromycin, degradation studies, kinetic evaluation, TiO 2 photosensitizer, 40 41 quantitative determination, photocatalysis 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 Page 3 of 23 Analytical Methods
1 2 3 Introduction 4 5 Pharmaceuticals are a class of substances which raise increasing environmental 6 7 concern due to their strong bioactivity [1], on one hand, and their growing concentration and 8 9 10 widespread distribution in the environment, on the other [2]. This is, at least in part, due to the 11 12 fact that the pharmaceuticals present in the hospital wastewater are almost always introduced 13 14 untreated to the municipal sewage system and ultimately to the municipal wastewater 15 16 treatment plants (WWTPs). The most potent and harmful pharmaceuticals in the hospital 17 18 wastewater include antibiotics, hormones, enzymes, MRI and X-ray contrast agents, 19 20 21 anesthetics, psychopharmaceuticals, disinfectants and cytostatics [3-5]. Among these 22 23 substances, antibiotics are of special interest because they cause antibiotic resistance of 24 25 (pathogenic) bacteria that is one of the most important challenges of the modern medicine 26 27 worldwide [6]. 28 29 30 Macrolides (MLs) such as erythromycin, clarithromycin, roxithromycin, and 31 32 azithromycin, are the second most important class of antibiotics used in human therapy after 33 34 the β-lactams [7]. Roxithromycin (Roxy), ((E)-erythromycin-9-[O-[(2-methoxyethoxy)- 35 36 methyl]oxime]) is a second generation semi-synthetic macrolide antibiotic derivative of 37 38 39 erythromycin A (Figure 1). It exists in an E-configuration [8]. This molecule is composed of a 40 41 14-membered lactone ring, a desosamine sugar unit and a cladinose sugar unit. A significant 42 43 difference between the structures of both macrolides lies at position 9 of the lactone ring, 44 45 where Roxy has an etheroxime chain instead of a carbonyl group at the same position in 46 47 erythromycin A. 48 49 50 51 52 53 54 55 56 57 58 59 60 3 Analytical Methods Page 4 of 23
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Figure 1. Chemical structure of roxithromycin 20 21 22 Roxy is the least active of the 14-membered macrolides having activity against gram- 23 24 positive and gram-negative cocci, gram-positive bacilli and some gram-negative bacilli [9,10]. 25 26 27 Roxy has been frequently detected worldwide in the effluents of WWTPs [11,12], 28 29 surface waters [13], and even a drinking water [14]. Due to the widespread use of Roxy it is 30 31 important to find out its metabolites and the most efficient methods of its degradation before it 32 33 reaches the environment. Oxidative dealkylation of the alkylether side chain, hydrolytic 34 35 36 cleavage of the sugar cladinose, and O-demethylation at the oxime side chain are the major 37 38 metabolic pathways of Roxy in humans [8]. Metabolic isomerization of the oxime is 39 40 stereoselective leading to the formation of Z-oxime isomers [15,16]. 41 42 The acidic hydrolysis of Roxy in simulated gastric fluid (SGF) has been already 43 44 reported [17]. Moreover, the degradation of clarithromycin and Roxy adsorbed on to iron(III) 45 46 47 and manganese(IV) oxides was studied [7]. It was found that the degradation of these MLs 48 49 proceeds both by the hydrolysis of the cladinose sugar and the lactone ring, with the N- 50 51 dealkylation of the amino sugar being the minor pathway. Sorption and transformation of 52 53 MLs on clays have been also described [18]. Biodegradation studies of Roxy in activated 54 55 56 sludge process have shown that hydrolysis of Roxy is negligible and the antibiotic removal 57 58 proceeds mainly through adsorption [19]. 59 60 4 Page 5 of 23 Analytical Methods
1 2 3 The aim of this work was to study the degradation of Roxy by acidic hydrolysis and 4 5 sensitized photodegradation as possible methods of its treatment before it is transferred into 6 7 the environment. We have performed a detailed analysis of the degradation pathway under 8 9 10 these conditions. We also propose the alternative degradation method, a photocatalytic 11 12 degradation of Roxy using TiO 2 dispersion. To the best of our knowledge no papers 13 14 presenting UV-light induced photocatalytic degradation of Roxy sensitized by suspended 15 16 TiO 2 are available in the literature, although systems containing TiO 2 coated on carriers such 17 18 as cenospheres have been reported [20]. 19 20 21 22 23 24 Experimental 25 26 27 28 Apparatus 29 30 31 The degraded Roxy samples were analyzed using UPLC-MS/MS system - ACQUITY 32 33 UPLC and TQD mass spectrometer (Waters Corporation, Milford, MA, USA) and a HPLC 34 35 system composed of a Waters 515 HPLC pump, a 2996 Photodiode Array Detector, and a 717 36 37 Plus Autosampler (Waters Corporation, Milford, MA, USA). TLC analyses were performed 38 39 40 using a TLC Scanner 3 densitometer with Cats 4 software (Camag, Muttenz, Switzerland). 41 42 Irradiations were performed using a Rayonet RPR-100 photoreactor (Southern New England 43 44 Ultraviolet Company, Brandford, CT, USA). Samples were dried using a drying oven with 45 46 natural convection – Binder ED 115/E2 (Binder GmbH, Tuttlingen, Germany) and a muffle 47 48 furnace FCF 5S HM (Czylok, Jastrz ębie Zdrój, Poland). 49 50 51 52 53 54 55 56 57 58 59 60 5 Analytical Methods Page 6 of 23
1 2 3 Dynamic light scattering 4 5 Dynamic light scattering (DLS) of TiO 2 dispersed in water was used to estimate the 6 7 size of the average TiO 2 particle. The measurement was carried out using Malvern Zetasizer 8 9 10 Nano-ZS. Anatase refractive index of 2.55 was used in the calculations. 11 12 13 14 UV-Vis reflectance spectroscopy 15 16 Reflectance spectrum of the TiO 2 powder was acquired using an Ocean Optics 17 18 USB2G5726 spectrophotometer equipped with an AvaLight-DHS lamp and an integration 19 20 21 sphere. The bandgap energy was estimated using Tauc plot. 22 23 24 25 Powder X-Ray diffraction (PXRD) 26 27 The PXRD analyses were carried out to assess the crystal structure of the obtained 28 29 TiO 2. The measurement was carried out using X’PERT PRO diffractometer. Scherrer 30 31 equation was used to approximate the crystallite size: 32 33 34 35 36 37 38 where: 39 40 L – average crystallite size, λ – the wavelength of X-ray radiation, K – shape constant equal to 41 42 43 0.9 for spherical particles, β1/2 – the line broadening at half the maximum intensity, θ - Bragg 44 45 angle 46 47 48 49 Materials and reagents 50 51 52 Roxithromycin - (3R,4S,5S,6R,7R,9R,11S,12R,13S,14R) - 6 - {[(2S,3R,4S,6R) - 4- 53 54 (dimethylamino) - 3 - hydroxy - 6 - methyloxan – 2 - yl]oxy} - 14 - ethyl - 7,12,13 - 55 56 trihydroxy - 4 - {[(2R,4R,5S,6S) - 5 - hydroxy - 4 - methoxy - 4,6 - dimethyloxan - 2 -yl]oxy} 57 58 59 60 6 Page 7 of 23 Analytical Methods
1 2 3 - 3,5,7,9,11,13 - hexamethyl - 10 - (2,4,7 - trioxa - 1 - azaoctan - 1 - ylidene) - 1 - 4 5 oxacyclotetradecan-2-one was purchased from LGC Standards, EPR 1500000, Teddington, 6 7 London, England). The following commercial Roxy preparations were used: Rulid 150 mg 8 9 10 (Sanofi-Aventis, Paris, France), Xitrocin 150 mg (Polfa Pabianice, Pabianice, Poland), 11 12 Rolicyn 150 mg (Polfa Tarchomin, Warsaw, Poland), Renicin 150 mg (Sandoz, Kundl, 13 14 Austria). Ethanol (POCh), methanol (Merck), acetone (Chempur), ammonia 25% (POCh), 15 16 sulfuric acid 95% (POCh), hydrochloric acid (POCh), water (Merck), acetonitrile (Merck), 17 18 formic acid (Merck), titanium (IV) isopropoxide 97% (Sigma-Aldrich), KH PO (POCh) were 19 2 4 20 21 of analytical or HPLC purity and used as received. 22 23 24 25 Standard solutions 26 27 Standard solutions were prepared by dissolving an appropriate amount of the standard 28 29 30 substance of Roxy in ethanol to give a concentration of 0.025% (w/v) for linearity and 31 32 precision studies and 0.0025% (w/v) for the determination of LOD and LOQ. Standard 33 34 solution of Roxy at a concentration of 0.6 % (w/v) was prepared for the degradation studies in 35 36 acidic solutions. 37 38
39 40 41 TLC-densitometric conditions 42 43 Standard solution of Roxy was applied in 8 mm bands, 10 mm apart, and 10 mm from 44 45 the lower edge to TLC silica gel 60 F 254 plates (10 ×15 cm) using Camag sample applicator 46 47 48 Linomat 4 equipped with a 100 L syringe. Chromatograms were developed over a distance 49 50 of 145 mm using methanol-acetone-ammonia 25% (1:14:0.1, v/v/v) in a chromatographic 51 52 chamber saturated for 10 min with mobile phase vapor at room temperature. The mobile 53 54 phase was chosen experimentally after checking 20 different mobile phases. After 55 56 57 development, the TLC plates were dried at room temperature and spots detected with freshly 58 59 60 7 Analytical Methods Page 8 of 23
1 2 3 prepared concentrated sulfuric acid-ethanol (1:4, v/v) mixture. After drying the plates were 4 5 sprayed with the reagent, then heated in an oven at 100 °C for 5 min. After spot detection, the 6 7 plates were scanned in the visible range at 483 nm, which was chosen on the basis of an 8 9 10 absorption spectrum recorded directly from the chromatogram. The analytical wavelength was 11 12 the same for Roxy and its degradation products. Densitometric scanning was carried out using 13 14 a Camag TLC Scanner 3 equipped with a tungsten lamp in the linear absorbance-reflectance 15 16 mode controlled by Cats 4 software resident in the system. The slit dimensions were 8 ×0.6 17 18 19 mm, the scanning speed was 20 mm/s, and data resolution was 100 m/step. Retardation 20 21 factors (R F values) were used to identify Roxy and its possible degradation products. For 22 23 quantitative determination of Roxy in the degradation studies, the scan areas of appropriate 24 25 26 peaks were recorded and the relative percent concentrations computed by employing the 27 28 internal normalization method, according to the formula: i [%]=(x i/Σx) 100, where i [%] is 29 30 constituent concentration, x i is peak area for the determined constituent, and Σx is sum of 31 32 33 peak scan areas in the chromatogram. 34 35 36 37 Determination of Roxy in commercial pharmaceutical preparations 38 39 Three L of the Roxy standard solution and the analyzed solutions at a concentration 40 41 42 of 0.025% (w/v) were applied to the chromatographic plates with a Linomat in the form of a 43 44 band 8 mm in width. Chromatograms were developed under the conditions described in the 45 46 TLC conditions section. The content of antibiotics in drugs was computed by comparing the 47 48 peak areas for standard and tested solutions. The results of determination for preparations 49 50 under investigation are listed in Table S2 (see Supplementary Information). 51 52 53 54 55 Acid-induced hydrolysis of Roxy 56 57 Hydrolysis of Roxy was carried out at pH 1.30, 1.70, and 2.30 at 40, 50 and 60 °C. In 58 59 60 8 Page 9 of 23 Analytical Methods
1 2 3 a 8 mL borosilicate glass vial 2 mL of 0.6% (w/v) Roxy standard solution was mixed with 2 4 5 mL of appropriate acid (0.1, 0.04 and 0.01 mol/L HCl) and heated in an oven to the desired 6 7 temperature. Then 500 L of incubated solutions were taken for the analysis in time intervals 8 9 10 given in Figure 2. After cooling the solutions were mixed with ethanol at a 1:1 ratio. 10 L 11 12 aliquots of resultant solutions were applied to the TLC plates in triplicate. The TLC- 13 14 densitometric method was carried out according to the developed method. For quantitative 15 16 17 determination purposes, the surface areas of appropriate peaks were recorded, and the 18 19 percentage concentrations of Roxy were computed by using internal normalization method. 20 21 Presented results are the mean value of three measurements. 22 23 24 25 Kinetic testing 26 27 28 To characterize the degradation processes of Roxy, based on the relationship 29 30 log(c)=f(t), where c is concentration and t stands for time, the order of degradation reaction 31 ++ 32 was determined; then the rate constants k and t 0.1 , t 0.5 , E a, and H were calculated [21]. 33 34
35 36 37 Photocatalytic degradation studies of Roxy in the presence of TiO 2 38 39 Synthesis of TiO 2 40 41 2 mL of titanium (IV) isopropoxide 97% was added to 10 mL of ethanol. Then the mixture of 42 43 2 mL of water in 10 mL of ethanol was added in 0.1 mL portions while stirring vigorously. 44 45 46 The mixture was left on magnetic stirrer for 2 h and then 30 mL of water was added and 47 48 mixture was centrifuged. The obtained solid was washed with water and dried in an oven at 49 50 60°C for 12 h. The obtained dry powder was heated in an oven for 2 h at 80°C and then for 2 51 52 h at 500°C. The dry product was triturated in an agate mortar. 53 54 55 56 57 58 59 60 9 Analytical Methods Page 10 of 23
1 2 3 Photocatalytic degradation studies of Roxy 4 5 5 mg of Roxy was dissolved in 50 mL of 0.01 M HCl to obtain 0.1 mg/mL solution of pH = 2 6 7 (HCl was used to increase solubility of Roxy). 25 mg of synthesized TiO 2 was introduced to 8 9 10 20 mL of the solution. The obtained dispersion was placed on a magnetic stirrer and was 11 12 intensively bubbled with oxygen for 30 min. Then the mixture was irradiated for 30 min in the 13 14 Rayonet reactor equipped with 6 lamps (8 W) at λ=350 nm. 4 mL samples were taken for 15 16 analysis and the mixture was constantly bubbled with oxygen during irradiation. In order to 17 18 19 separate TiO 2 from Roxy solution the samples were centrifuged for 10 min at 18000 rpm. 20 21 Concentration of Roxy was monitored by HPLC method, using as the eluent methanol:0.03M 22 23 KH 2PO 4 (50:50) mixture at a flow rate of 0.7 mL/min and a Biobasic-8 (250mm ×4 mm, 24 25 particle size 5 m) Thermo Scientific column. To find out if the adsorption processes of Roxy 26 27 28 on the surface of the photosensitizer occur the same experiment was conducted in the dark. To 29 30 find out if the process of direct photolysis of Roxy takes place the same experiment was 31 32 conducted in the absence of the photosensitizer. 33 34
35 36 37 Studies of the degradation products using UPLC-MS/MS 38 ® ® 39 The UPLC-MS/MS system consisted of a Waters ACQUITY UPLC (Waters 40 41 Corporation, Milford, MA, USA) coupled to a Waters TQD mass spectrometer (electrospray 42 43 ionization mode ESI-tandem quadrupole). Chromatographic separations were carried out 44 45 46 using the Acquity UPLC BEH (bridged ethyl hybrid) C18 column; 2.1 × 100 mm, and 1.7 µm 47 48 particle size. The column was maintained at 40°C, and eluted under gradient conditions from 49 50 95% to 0% of eluent A over 10 min, at a flow rate of 0.3 mL min -1. Eluent A: formic 51 52 acid/water (0.1%, v/v); eluent B: formic acid/acetonitrile (0.1%, v/v). Chromatograms were 53 54 recorded using Waters e λ PDA detector. Spectra were analyzed in 200 – 700 nm range with 55 56 57 1.2 nm resolution and sampling rate of 20 points/s. MS detection settings of Waters TQD 58 59 60 10 Page 11 of 23 Analytical Methods
1 2 3 mass spectrometer were as follows: source temperature 150 °C, desolvation temperature 4 5 350°C, desolvation gas flow rate 600 L h -1, cone gas flow 100 L h -1, capillary potential 3.00 6 7 kV and cone potential 20 V. Nitrogen was used as both nebulizing and drying gas. The data 8 9 10 were obtained in a scan mode ranging from 50 to 1000 m/z at 0.5 s intervals. Collision 11 12 activated dissociation (CAD) analyses were carried out with the energy of 40 eV, and all the 13 14 fragmentations were observed in the source. Consequently, the ion spectra were obtained by 15 16 scanning from 50 to 1000 m/z range. Data acquisition software was MassLynx V 4.1 17 18 (Waters). 19 20 21 22 23 Results and discussion 24 25 The aim of this paper was to study the degradation of Roxy in acidic solutions and its 26 27 photocatalytic degradation process in UV light in the presence of the TiO photosensitizer. 28 2 29 30 For both methods the degradation products of Roxy were identified. 31 32 33 34 Acidic hydrolysis of roxithromycin 35 36 Acid-induced hydrolysis of Roxy was carried out by the developed chromatographic- 37 38 densitometric method. Using TLC F silica gel plates as a stationary phase and methanol- 39 254 40 41 acetone-ammonia 25% (1:14:0.1, v/v/v) as the mobile phase well-developed and resolved 42 43 peaks of Roxy and its degradation products were obtained. The spots were visualized with 44 45 sulphuric acid in ethanol and scanned densitometrically at 483 nm. The method was validated 46 47 according to ICH requirements (see Supplementary information, Method validation, Table 48 49 50 S1). 51 52 Acidic hydrolysis of Roxy was performed at three different temperatures, i.e., at 40, 53 54 50, and 60°C, and for three different pH of 2.30, 1.70, and 1.30 (Figure 2 ). The relationship 55 56 between Roxy concentration and time follows the kinetics of a first order reaction, consistent 57 58 59 60 11 Analytical Methods Page 12 of 23
1 2 3 with the results obtained for hydrolysis carried out in SGF [17]. To obtain full characteristics 4 5 of the degradation process, kinetic parameters such as k, t 0.1 , and t 0.5 values describing Roxy 6 7 acidic degradation rate under different conditions were calculated together with the 8 9 ++ 10 thermodynamic parameters such as E a and H (Table 1 ). 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 Figure 2. The kinetic data for the acidic hydrolysis of Roxy for pH of a) 2.30, b) 1.70, 52 53 and c) 1.30 at ( ) 40, ( ) 50, and ( ) 60°C. 54 55 56 57 58 59 60 12 Page 13 of 23 Analytical Methods
1 2 3 Table 1. Kinetic and thermodynamic parameters calculated for Roxy acidic hydrolysis. 4 5 Temperature (°C) E H++ pH a 6 40 °C 50 °C 60 °C (kJ/mol) (kJ/mol) 7 -1 8 k (h ) 0.02 0.08 0.12 9 2.30 t0.5 (h) 38.45 8.75 5.99 77.64 74.95 10 t0.1 (h) 5.84 1.33 0.91 -1 11 k (h ) 0.17 0.29 0.59 12 1.70 t0.5 (h) 4.06 2.43 1.18 53.92 51.23 13 t0.1 (h) 0.62 0.37 0.18 14 k (h -1) 0.27 0.60 1.29 15 1.30 t (h) 2.59 1.16 0.54 67.76 65.08 16 0.5 17 t0.1 (h) 0.39 0.18 0.08 18 Note: k-reaction rate constant, t -half-life time, t -time in which concentration of Roxy is 19 0.5 0.1 reduced by 10%, E -activation energy, H++ -activation enthalpy. 20 a
21 22 23 Increase in both pH and temperature increases the rate of Roxy hydrolysis. 24 25 TLC analysis of the acidic solutions revealed two degradation products with R F values 26 27 of 0.82 and 0.87 besides the TLC peak of Roxy with R F=0.42 ( Figure 3 ). The obtained 28 29 degradation products were subjected to UPLC-MS/MS analysis in order to propose their 30 31 32 molecular weights and structures. The identification of the degradation products of E-Roxy 33 34 induced by acidic conditions was performed on a basis of UPLC/MS analysis and supported 35 36 with fragmentation patterns obtained from MS/MS experiments. The possible degradation 37 38 products are shown in Table 2 . The acidic degradation was found to affect mainly the L- 39 40 41 cladinose substituent, causing its detachment from macrolide core, and to bring about 42 43 isomerization of the oxime moiety from the native form E to Z. 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 13 Analytical Methods Page 14 of 23
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Figure 3. Densitograms registered for roxithromycin after hydrolysis at pH=1.70 at 47 48 60 °°°C after 0 (a), 30 (b), 60 (c), 90 (d), and 120 (e) min. Peak 1 - roxithromycin, peak 2 49 50 and 3 - degradation products. 51
52 53 54 55 56 57 58 59 60 14 Page 15 of 23 Analytical Methods
1 2 3 Table 2. Proposed structures of the products of acidic hydrolysis of E-roxithromycin 4 5 6 Product Id RT [M+H] + Fragmentation Proposed structure 7 ions 8 9 RP-1 4.16 679.5 115.1, 158.1, 10 176.1, 398.2, 11 416.3, 446.3, 12 573.4, 603.4, 13 661.4 14 15 16 17 18 19 O O 20 RP-2 4.39 679.5 115.1, 158.1, N O 21 176.1, 398.2, 22 416.3, 446.3, OH 23 HO 573.4, 603.4, OH HO NH 24 661.4 25 O O 26 O 27 O OH 28 29 m/z = 679.4 30 31 RP-3 4.86 837.6 115.1, 158.1, 32 176.1, 398.2, 33 416.3, 446.3, 34 573.4, 603.4, 35 661.4, 679.4 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 15 Analytical Methods Page 16 of 23
1 2 3 E- 5.06 837.6 115.1, 158.1, 4 roxithromycin 176.1, 398.2, 5 416.3, 573.4, 6 603.4, 661.4, 7 679.4 8 9 10 11 12 13 14 15 16 17 18 L-cladinose 6.16 177.1 161.1, 147.1, 19 149.1, 129.1, 20 119.1, 103.1, 21 101.1, 85.1, 75.1 22 23 24 25 Structures of the presented stable products of the degradation were proposed based on 26 27 28 Collisionally Activated Dissociation (CAD) experiments. The fragmentation pattern involved 29 30 loss of L-cladinose substituent (-176.1) of E and Z-roxithromycin. In the next step decladinose 31 32 derivatives lost H2O (-18) and subsequently methoxyethene moiety (-58). These products 33 34 underwent further decomposition on two routes: by deformylation (-30) and subsequentially 35 36 loss of D-desosamine substituent, or loss of N,N,2-trimethyl-3,4-dihydro-2H-pyran-4-amine 37 38 39 (-157.1), subsequent deformylation (-30) and loss of H 2O (-18) (see Supplementary 40 41 Information Scheme S1). 42 43 D-Desosamine and its decomposition products were also observed. D-Desosamine 44 45 underwent dehydration (-18) and subsequentially loss of N-methylenemethanamine fragment 46 47 48 (-43) (see Supplementary Information Scheme S2). The main route of fragmentation of L- 49 50 cladinose involved loss of carbon monoxide (-28) and subsequent deformylation (-30) and 51 52 dehydration (-18). Other observed routes involved deformylation (-30) with subsequent 53 54 dehydration (-18) and loss of acetaldehyde moiety (-44), demethylation (-14), or 55 56 57 58 59 60 16 Page 17 of 23 Analytical Methods
1 2 3 decarbonylation (-28) with subsequent loss of acetaldehyde moiety (-44) and decarbonylation 4 5 (-28) (see Supplementary Information Scheme S3). 6 7 8 9 10 Sensitized by TiO 2 Photodegradation of Roxithromycin 11 12 Although acidic hydrolysis of Roxy is quite efficient, it requires very low pH (<3) and 13 14 elevated temperatures so it may not be always applicable for Roxy hydrolytic degradation [7]. 15 16 Therefore, we propose also an alternative method of Roxy degradation; TiO 2 sensitized 17 18 photocatalytic degradation. The photosensitizer in the form of a powder can be obtained in a 19 20 21 facile way by the hydrolysis of titanium (IV) isopropoxide and can be easily suspended in 22 23 water. 24 25 To find out which isomorphic form is the main constituent of the TiO 2 powder 26 27 obtained the Powder X-Ray Diffractometry (PXRD) measurement was performed. The 28 29 30 presence of Bragg peaks originating only from the anatase crystal structure in the PXRD 31 32 diffractograms confirm that it is the dominant crystal structure of the photocatalyst (see Figure 33 34 S1 in Supplementary Information). There are no peaks originating from rutile or brookite 35 36 indicating that these phases are absent, however, the presence of amorphous TiO 2 can not be 37 38 ruled out. Assuming spherical particle geometry, the average crystallite size calculated from 39 40 41 the line XRD line broadening is around 43 nm, thereby confirming its nanostructural 42 43 morphology. 44 45 The diameter of TiO 2 particles in water colloid found in the dynamic light scattering 46 47 measurements are around 900 nm (Figure S2 in Supplementary Information). It indicates that 48 49 50 TiO 2 crystallites form larger clusters. This effect might be attributed to the temperature 51 52 induced sintering of the grains during calcination step. 53 54 The bandgap energy estimated from the reflectance UV-Vis spectra equals to 3.13 eV 55 56 what further confirms the crystal structure of anatase. The wavelength of the photocatalyst 57 58 59 60 17 Analytical Methods Page 18 of 23
1 2 3 absorption band closely overlaps with the wavelength of the maximum intensity of the 4 5 Rayonet lamps emission. On the other hand, the absorption band of Roxy, which ends at 6 7 about 240 nm, is separated from the emission band of the lamps (see Figure S3 in 8 9 10 Supplementary Information). This implies that the lamp radiation is strongly absorbed by the 11 12 photocatalyst and its absorption by Roxy can be neglected. 13 14 Photocatalytic degradation studies of Roxy revealed that the drug studied undergoes 15 16 photodegradation when irradiated with light at 350 nm (near UV) in the presence of TiO 2. 17 18 Kinetics of the first order reaction follows the suggested theory of Langmuir-Hinshelwood for 19 20 21 photocatalytic microheterogeneous systems ( Figure 4 ) with the reaction rate constant of 22 -1 23 0.00768 min . Since Roxy does not absorb the light used for the irradiations the direct 24 25 photolysis of Roxy did not occur. 26 27 28 29 30 1.0 31 TiO , UV, Roxy 32 2 33 0.8 UV, Roxy 34 35 36 37 0.6
38 ) 0 39 40 0.4 41 42 -ln(C/C 43 44 0.2 45 46 47 0.0 48 49 0 20 40 60 80 100 120 50 51 Time (min) 52 53 Figure 4. Photodegradation of roxithromycin in the absence and in the presence of TiO 2 54 55 photosensitizer. 56 57 58 59 60 18 Page 19 of 23 Analytical Methods
1 2 3 Adsorption process of Roxy on the surface of TiO 2 does not have a significant impact on the 4 5 course of the experiment, as evidenced by minimal changes of the Roxy concentration for the 6 7 experiment carried out in the dark, which also excluded acidic hydrolysis at this temperature. 8 9 10 The possible degradation products are shown in Table 3. 11 12 13 14 Table 3. Proposed products of photocatalytic degradation of E-roxithromycin in the 15 presence of TiO 2 16 17 Product Id RT [M+H] + Fragmentation Proposed structure 18 19 ions 20 RP-UV-1 3.54 693.4 115.1, 158.1, 21 376.2, 500.3, 22 569.3, 587.4, 23 675.4 24 25 26 27 28 29 RP-UV-2 3.70 695.4 115.1, 158.1, 30 571.3, 677.4 31 32 33 34 35 36 37 38 RP-UV-3 3.88 665.4 115.1, 158.1, 39 490.3, 647.4 40 41 42 43 44 45 46 47 RP-UV-4 4.38 649.4 115.1, 158.1, 48 398.2, 416.3, 49 50 446.3 51 52 53 54 55 56 57 58 59 60 19 Analytical Methods Page 20 of 23
1 2 3 RP-UV-5 4.59 711.4 115.1, 158.1, OH OH 4 O 398.2, 470.3, O O 5 488.3, 675.4, N 6 693.4 7 OH 8 HO OH HO NH 9 10 O O 11 O 12 O OH 13 O O 14 RP-UV-6 4.74 853.5 115.1, 158.1, N O 15 398.2, 446.3, 16 573.4, 661.4, 17 OH 679.4 HO HO NH 18 OH 19 O O 20 O 21 O O 22 OH 23 O 24 25 O 26 OH O O 27 RP-UV-7 5.05 869.5 115.1, 158.1, N O 28 398.2, 679.4, 29 851.5 OH 30 HO 31 OH HO NH 32 33 O O O 34 O O 35 OH 36 O 37 O 38 OH 39 OH 40 O O RP-UV-8 5.48 835.5 115.1, 158.1, N O 41 176.1, 398.2, 42 446.3, 573.4, 43 OH 603.4, 661.4, HO 44 OH HO NH 45 679.4 46 O O 47 O O O 48 49 O 50 51 O 52 O 53 54 55 56 57 58 59 60 20 Page 21 of 23 Analytical Methods
1 2 3 The photocatalytic degradation was found to affect mainly the L-cladinose substituent, 4 5 causing its hydroxylation (RP-UV-6 and RP-UV-7) or oxidation of hydroxy substituent (RP- 6 7 UV-8). 8 9 10 In the case of decladinose roxithromycin photocatalytic degradation seems to involve 11 12 oxidation of 2-methoxyethoxymethyl moiety (products RP-UV-4 and RP-UV-5), 13 14 demethylation of 2-methoxyethoxymethyl moiety (product RP-UV-3) or oxidation of the core 15 16 of the antibiotic (RP-UV-1 and RP-UV-2). 17 18 Structures of the presented stable products of photocatalytic degradation were 19 20 21 proposed based on Collisionally Activated Dissociation (CAD) experiments. The 22 23 fragmentation patterns of products RP-UV-3, RP-UV-4 and RP-UV-6 – RP-UV-8 were 24 25 similar to that observed for products of acidic degradation of Roxy. Additionally for RP-UV-7 26 27 fragmentation ion with m/z=851.5 was observed, which was probably the product of 28 29 30 dehydration of 4-methoxy-2,4-dimethyltetrahydro-2H-pyran-2,3,5,6-tetraol moiety (2,5- 31 32 dihydroxy-L-cladinose) to 5,6-dihydroxy-4-methoxy-2,4-dimethyldihydro-2H-pyran-3(4 H) - 33 34 one (2-hydroxy-4-oxo- L-cladinose). Fragmentation pattern for RP-UV-1 and RP-UV-2 is 35 36 similar to previously described, but due to the presence of additional hydroxyl moiety in 37 38 roxithromycin core it involves additional dehydration of fragmentation ions. The 39 40 41 fragmentation path of RP-UV-1 is presented on Scheme S4 (see Supplementary Information). 42 43 In the case of RP-UV-5, possessing additional hydroxy substituents attached to the 2- 44 45 methoxyethoxymethyl moiety, fragmentation involves dehydration of this moiety, besides of 46 47 previously mentioned transitions. The fragmentation path for this product is presented on 48 49 50 Scheme S5 (see Supplementary Information). D-Desosamine and its decomposition products 51 52 were also observed. 53 54 55 56 57 58 59 60 21 Analytical Methods Page 22 of 23
1 2 3 Conclusions 4 5 Detailed studies on the acidic hydrolytic degradation and TiO 2 sensitized 6 7 photodegradation of roxithromycin have shown that both processes are quite efficient. 8 9 10 Although the mechanisms of these processes are quite different, in both of them L-cladinose 11 12 substituent is primarily involved. The structures of the degradation products and the 13 14 mechanisms of the processes studied were proposed. Both types of the processes can be used 15 16 for degradation of Roxy, however, the photocatalytic degradation seems to be more 17 18 promising because it can undergo in mild conditions. 19 20 21 22 23 24 25 References 26 27 28 [1]K. Kümmerer, J. Environ. Manage., 2009, 90 (8), 2354-66. 29 30 31 [2]D.W. Kolpin, E.T. Furlong, M.T. Meyer, E.M. Thurman, S.D. Zaugg, L.B. Barber, H.T. 32 33 Buxton, Environ. Sci. Technol., 2002, 36 (6), 1202-11. 34 35 [3]B. Pauwels, W. Verstraete, J. Water Health , 2006, 4(4), 405-16. 36 37 [4]F. Sacher, F.T. Lange, H. J. Brauch, et al., J. Chromatogr. A, 2001, 938 (1-2), 199-210. 38 39 40 [5]T.A. Ternes, TrAC Trends in Analytical Chemistry , 2001, 20 (8), 419-434. 41 42 [6]D. Debabov, Appl. Biochem. Microbiol., 2013, 49 (8), 665-71. 43 44 [7]J. Feitosa-Felizzola, K. Hanna, S. Chiron, Environmental Pollution, 2009, 157 (4), 1317-22. 45 46 [8] D. Zhong, X. Li, A. Wang, Y. Xu, Sh. Wu, Drug Metab. Dispos. , 2000, 28 , 552-559. 47 48 [9] M. Ostrowski, E. Wilkowska, T. B ączek, Cent. Eur. J. Med ., 2010, 5, 83-90. 49 50 51 [10] W. Yam, H. Wahab, J. Chem. Inf. Model. , 2009, 49 , 1558-1567. 52 53 [11]M. Gros, M. Petrovi ć, D. Barceló, Environ. Toxicology and Chemistry, 2007, 26 (8), 54 55 1553-62. 56 57 58 59 60 22 Page 23 of 23 Analytical Methods
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