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Identification, Mechanisms and Kinetics of Macrolide Degradation Product Formation under Controlled Environmental Conditions | OMICS International
ISSN: 2380-2391
Journal of Environmental Analytical Chemistry
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Identification, Mechanisms and Kinetics of Macrolide Degradation Product Formation under Controlled Environmental Conditions

Igal Gozlan1,2,3, Ilana Koren1,2 and Dror Avisar1,3*

1The Hydrochemistry Laboratory, Department of Geography and Environmental Studies, Tel Aviv University, Tel Aviv, Israel

2The Porter School of Environmental Studies, Tel Aviv University, Tel Aviv, Israel

3The Water Research Center, Tel Aviv University, Tel Aviv, Israel

*Corresponding Author:
Avisar D
The Water Research Center
Hydrochemistry Laboratory
Tel Aviv University
Tel Aviv 69978, Israel
Tel: 97236405534
E-mail: [email protected]

Received Date: December 29, 2015; Accepted Date: January 08, 2016; Published Date: January 25, 2016

Citation: Gozlan I, Koren I, Avisar D (2016) Identification, Mechanisms and Kinetics of Macrolide Degradation Product Formation under Controlled Environmental Conditions. J Environ Anal Chem 3:171. doi:10.4172/2380-2391.1000171

Copyright: © 2016 Gozlan I, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Erythromycin, azithromycin, clarithromycin and roxithromycin are antibiotics belonging to the widely used macrolide group. Their presence in the environment has been much investigated, despite the rapid degradation of Erythromycin to its spiroketal degradation product. In this study, the formation of macrolide degradation products was investigated in various aqueous solutions, each containing 100 μg/mL of the respective macrolide, under controlled artificial conditions: three phosphate buffer solutions (pH 5, pH 7 and pH 8.5), and a buffer solution at pH 7 with the addition of humic acids. Two solutions from natural sources were also examined: secondary effluent and tap water. The obtained degradation products were identified by their HRMS and NMR spectra (for Erythromycin-spiroketal, obtained from pure compounds isolated by preparative HPLC) as: N-oxide, N-desmethyl and N-didesmethyl forms of all examined macrolides. These degradation products were obtained only under irradiation by sunlight, while the Erythromycin-H2O degradation products were also obtained in the shade. The secondary effluent was the most significant medium for achieving macrolide degradation products. According the degradation product’s t1/2 values obtained in the secondary effluent, the azithromycin was most rapidly degraded (23 hours). Furthermore, results suggested that the degradation process was activated by sunlight irradiation energy, and that the degradation mechanism started with the transfer of an electron from the amine group to O2 to produce the radical ions RMe2N·+ and O2 ·- as intermediates and production of the N-oxide and N-desmethyl macrolide degradation products. The kinetics of macrolide degradation was calculated as a first-order reaction.


Macrolide; Degradation product; Secondary effluent; Humic acid; Photodegradation; LC-HRMS


Degradation products (DPs) obtained from antibiotic residues are a recognized but mostly under-studied group of contaminants [1,2]. They may find their way into the aquatic environment where they are widely dispersed and persist for much longer than previously thought [3,4]. Moreover, drug DPs may be the result of natural biodegradation and/or chemical degradation (including advanced oxidation processes) during wastewater treatment. These DPs are suspected of being more resistant to degradation, and potentially more toxic, than their parent compounds [5-8]. For instance, the macrolide DPs N-desmethyl and N-didesmethyl macrolides have been reported to be biologically active [9,10].

The macrolides are an important group of antibacterial compounds that are commonly used for the treatment of upper and lower respiratory-tract infections. Erythromycin (ERY) is the first and most widely prescribed, orally administered member of this group. Due to its severe side effects, pharmaceutical manufacturers modified this compound to produce three other macrolide drugs:clarithromycin (CLA), roxithromycin (ROX) and azithromycin (AZI), which are widely used in livestock and human medicine. This group of molecules consists of a 14-membered (ERY, CLA and ROX) or 15-membered (AZI) lactone ring, with 10 asymmetric centers and 2 groups of sugar residues: L-cladinose and D-desosamine (Figure 1). ROX and AZI are distinguished from ERY by modifications in ERY’s 14 lactone membered ring that prevent production of the undesirable DP “ERYspiroketal”, which is obtained after internal ketalization processes [11]. Additionally, CLA is distinguished from ERY by replacing the R2 position from OH to O-CH3 (Figure 1).


Figure 1: Structures of macrolides and their degradation products.

Macrolide DPs are frequently found in aquatic environments and their presence has been widely investigated, with a focus on their parent compounds ERY, CLA, ROX and AZI [12-18]. Moreover, several studies have reported detection of the ERY DP ERY-H2O, which is probably the spiroketal product [14,19-22]. In fact, ERY-spiroketal is the only macrolide DP ever detected in the environment. In addition, a non-environmental study designed to obtain macrolide DPs recently revealed a potential effect of ERY on its tissue distribution and bioaccumulation in fish, and its metabolisis via demethylation to its N-desmethyl and N-didesmethyl DPs [23]. Studies analyzing related manufactured drug substances in macrolides found N-desmethylerythromycin E, erythromycin E N-oxide, anhydroerythromycin C, N-desmethylerythromycin B, anhydro-Ndesmethylerythromycin A and pseudoerythromycin E enol ether [24]. Others, examining benzamycin which is a combination of benzoyl peroxide and ERY by liquid chromatography–mass spectrometry (LCMS) showed the underlying oxidation process that produces DPs such as ERY-desmethyl and ERY-N-oxide [25].

In all of the studies in which macrolide DPs have been obtained, these compounds were synthesized. Freiberg [9] synthesized macrolides in which the two 3-dimethyl amino substituents of the desosamine and mycaminose moieties were N-demethylated and N-didemethylated by reaction with a halogen (preferably iodine) in the presence of a base to control pH. Napoletano [26] synthesized ERY, CLA and AZI DPs using UV irradiation, describing the synthesis of N-desmethylated macrolide DPs using methanol with sodium acetate and iodine. Similarly, Jakopovic et al. [27] produced N-oxide, N-desmethylated, and N-didesmethylated CLA and AZI DPs.

To the best of our knowledge, there has been no report on the chemical behavior or identification of macrolides under controlled environmental conditions. Thus, the main objective of this study was to use controlled environmental conditions to obtain selected macrolide DPs, to verify their chemical structure, and to understand their degradation mechanisms.


Standards and reagents

ERY (95.5%), AZI (96–102%), CLY (96-102%) and ROX (>90%, HPLC grade) analytical standards were purchased from Fluka (Israel). Acetonitrile, methanol, ethanol and water (all ULC/ MS grade) were purchased from Bio-Lab (Israel). Humic acid was purchased from Fluka. Ammonium formate (>98%) was purchased from Fisher and ammonia (32%) from Merck. Phosphoric acid (H3PO4, 80-90%) was purchased from Fluka, sodium phosphate (98-100.5%) from Riedal-de Haen and hydrogen peroxide (H2O2, 30 wt. % in water) was purchased from Sigma. Tap water (TW) was collected from the Hydrochemistry Laboratory at Tel Aviv University. Field secondary effluent (SE) was taken from the Shafdan wastewater-treatment plant.

Obtaining DPs under sunlight irradiation and shade

Four solutions containing, respectively, ERY, CLA, AZI and ROX (each at a concentration of 100 μg/mL) were prepared. Three different phosphate buffer solutions were examined under controlled artificial conditions pH 5, pH 7 and pH 8.5 and a fourth phosphate buffer solution at pH 7 contained 5 mg/L humic acid. The three different pH’s were selected to examine the behavior of macrolides under natural environmental conditions, which typically present a pH range of 5 to 8.5. In addition, two solutions from natural sources were examined SE and TW to simulate environmental conditions. The initial, nonenvironmental macrolide concentration of 100 μg/mL was chosen because it was high enough to enable monitoring DPs at the various obtained concentrations, but low enough to avoid intermolecular reactions. The sample solutions were prepared in sealed Pyrex glass bottles under natural sunlight (winter at 18°C, latitude: 32°, altitude: sea level), and in the shade (as a control). They were kept for 14 days (336 h), with sampling at 0, 2, 6, 32, 120 and 336 h. These times were chosen after preliminary tests to determine the optimal period for the degradation process. Each experiment was run in triplicate and relative standard deviation (RSTD) was calculated.

N-oxide, N-desmethyl and N-didesmethyl ERY, CLA, AZI and ROX DPs were obtained only under solar irradiation. These DPs were also obtained for ERY-H2O (Table 1).

Name Azithromycin Clarithromycin Roxythromycin
DIDES N-Oxide DES Parent N-Oxide DIDES DES Parent DIDES DES N-Oxide Parent
[MH]+ 721.49 765.51 735.50 749.52 764.48 720.45 734.47 748.48 809.5 823.52 853.53 837.53
ConditionsRT (min) 8.93 9.23 9.26 9.95 10.50 10.65 11.02 11.83 11.02 11.33 12.07 12.31
Phosphate Buffer
pH 5
n/d n/d n/d 100% n/d n/d n/d 100% n/d 0.4% n/d 99%
Phosphate Buffer
pH 7
n/d n/d n/d 100% 0.4% n/d 0.8% 99% n/d 0.5% n/d 99%
Phosphate Buffer
pH 8.5
0.8% 0.8% 8.4% 90% 0.4% n/d 0.7% 98% n/d 4.2% 0.6% 94%
Humic acids
(5mg/L) pH 7
0.8% 1.1% 10.3% 88% 0.3% n/d 1.7% 98% n/d 3.7% 0.6% 96%
(measured pH 8.2)
2.3% n/d 11.3% 86% 0.2 n/d 0.8% 99% n/d 4.0% n/d 96%
(measured pH 7.7)
23% 1.4% 76% n/d 4.6% 0.5% 79% 16% 3.2% 86% 3.7% 8.5%

Table 1: Azithromycin, clarithromycin and roxithromycin degradation products (area%) under various conditions after 14 days under sunlight irradiation. TW, tap water; SE, secondary effluent.

Analytical measurements

LC-MS analysis of macrolides and their DPs (after exposure to sunlight irradiation) was performed by high-performance liquid chromatography (HPLC, Agilent 1100) coupled to MS (Q-Tof, Waters, model Premier) via an ESI interface in positive mode, using a C18 ACE column (250 × 2.1 mm, 5 μm particle size). The column temperature was set to 28°C, the flow rate to 0.5 mL/min, and the injection volume was 10 μL. The HPLC mobile phase consisted of water with ammonium formate (0.05 M) adjusted to pH 8 with ammonia (A) and acetonitrile (B). The elution gradient was initiated with 20% B, increased to 80% over 14 min, and then held at 80% for 5 min. 1H and 13C nuclear magnetic resonance (NMR) analyses were carried out for ERY and ERY-spiroketal using a Bruker 500 MHz model ADVANCE II. The samples were dissolved in CDCl3.

Sample preparation for N-oxide DPs

The four N-oxide DPs for ERY, CLA, AZI and ROX, respectively, were prepared and used as markers. In this procedure, 1 g of macrolide (ERY, CLA, AZI or ROX) was dissolved in 8 mL of methanol and 2 mL of H2O2 (30% in water), then heated to 60°C for 4 h. The four products were diluted to the appropriate concentration and then injected into the LC-MS. According to this analysis, the four N-oxide DPs were obtained at high conversion (more than 90% according to area percent).

Sample preparation of ERY-H2O for NMR analysis

ERY-H2O [peak at retention time (RT)=10.7 min] was isolated from the ERY working solution after its degradation (at pH 5), using semi-preparative HPLC (Agilent 1100) with a Vydac C18 column (250 mm length, 10 mm I.D. and 10 μm particle size). The mobilephase composition and the elution-gradient program were the same as for the HPLC analytical method (section 2.3), except that the flow rate was set to 5 mL/min. The obtained fractions were lyophilized; they were chromatographically similar before and after the lyophilization procedure. The isolated ERY-H2O was identified by MS and NMR spectra and used as a marker for the ERY-spiroketal compound (Tables 3-5).

Site dC(ppm) dH  (ppm) dC(ppm) dH (ppm) dC(ppm) differences
1 175.91 - 181.31 - 5.4
2 44.96 2.88 47.54 3.42 2.6
3 80.02 3.94 77.63 4.38 -2.4
4 39.51 1.97 44.32 2.11 4.8
5 83.68 3.56 87.15 3.48 3.5
6 74.96 - 82.59 - 7.6
7 38.55 1.90, 1.70 42.91 2.42, 1.52 4.4
8 45.14 2.49 42.88 2.33 -2.3
9 221.90 - 117.57 - -104.3
10 37.96 3.09 50.97 3.10 13.0
11 68.93 3.50 87.86 3.53 18.9
12 74.73 - 83.86 - 9.1
13 77.32 5.05 82.86 5.19 5.5
14 21.16 1.93, 1.50 25.4 2.02, 1.54 4.2
15 10.69 0.85 11.56 0.87 0.9
16 15.94 1.19 14.52 1.12 -1.4
17 9.18 1.12 18.45 1.23 9.3
18 26.90 1.47 28.56 1.45 1.7
19 18.32 1.17 12.68 1.09 -5.6
20 12.04 1.15 15.17 1.33 3.1
21 16.23 1.14 25.37 1.32 9.1
1’ 103.23 4.39 103.64 4.31 0.4
2’ 70.99 3.24 70.04 3.28 -0.9
3’ 65.56 2.71 66.30 2.86 0.7
4’ 28.90 1.73, 1.26 31.9 1.70, 1.25 3.0
5’ 68.93 3.46 69.53 3.56 0.6
6’ 21.40 1.23 21.35 1.28 0.0
3’-N(CH3)2 40.33 2.32 40.23 2.57 -0.1
1’’ 96.37 4.90 96.75 5.23 0.4
2’’ 35.01 2.31, 1.58 35.99       2.30,1.56 1.0
3’’ 72.68 - 74.57 - 1.9
4’’ 78.01 3.02 79.73 3.03 1.7
5’’ 65.68 4.01 66.96 4.01 1.3
6’’ 18.63 1.29 18.45 1.16 -0.2
7’’ 21.52 1.25 21.84 1.23 0.3
3’’-OCH3 49.52 3.32 50.01 3.29 0.5

Table 3:1H and 13C NMR data of erythromycin (ERY) and ERY-spiroketal. In red: the most significant obtained shift between ERY and ERY-sprioketal.

Name Fragments ERY AZI CLA ROX
Parent [MH]+ 734.4713 749.5183 748.4838 837.5335
[MH]+-H2O 716.4688      
[MH]+-2H2O 698.4579      
[MH]+-Clad 576.3818 591.4232 590.3889 679.4383
[MH]+-Clad-H2O 558.3715      
[MH]+-Clad-2H2O 540.3604      
[MH]+-Clad-3H2O 522.3492      
[MH]+-Clad-OCH2O(CH2)2OCH3       573.3760
Clad 158.1183   158.1221  
[(M/2)+H]+z=2   375.2558   419.264
N-Oxide [MH]+ 750.4677 765.5155 764.4803 853.5284
[MH]+-OCH2O(CH2)2OCH3       748.4897
[MH]+-H2O 732.4518      
[MH]+-Clad 592.3673      
[MH]+-Clad-H2O 574.3579      
[(M/2)+H]+ z=2   383.2537    
Desmethyl [MH]+ 720.4538 735.5018 734.4701 823.5194
[MH]+-H2O 702.442      
[MH]+-2H2O 684.4321      
[MH]+-OCH2O(CH2)2OCH3       718.3818
718-CH3       703.3809
[MH]+-Clad 562.3575 577.4067 576.3784 665.4236
[MH]+-Clad-H2O 544.3472      
[MH]+-Clad-2H2O 526.3369      
[MH]+-Clad-3H2O 508.3257      
[MH]+-Clad-OCH2O(CH2)2OCH3       559.3606
Clad-CH3 144.101 144.1016    
[(M/2)+H]+ z=2   368.2518   412.2583
Didesmethyl [MH]+ 706.4390 721.4846 720.4559 809.5029
[MH]+-Clad 548.342 563.3903 562.3591 651.4084
[MH]+-Clad-2H2O 512.3196      
[MH]+-Clad-3H2O 494.3099      
M -H2O (1)
[MH]+ 716.4612      
[MH]+-H2O 698.4497      
[MH]+-CH3 684.4348      
[MH]+-Clad 558.3657      
[MH]+-Clad-H2O 540.3535      
[MH]+-Clad-2H2O 522.3433      
[MH]+-Clad-3H2O 500.3229      
M -H2O (2) [MH]+ 716.4612   730.4780  
[MH]+-Clad 558.3666      
Clad 158.1165      
N-Oxide -H2O [MH] + 732.4569   746.4740  
Desmethyl -H2O [MH] + 702.4420      

Table 4: Main MS fragments of macrolides erythromycin (ERY), azithromycin (AZI), clarithromycin (CLA) and roxithromycin (ROX) and their degradation products.

Macrolide Name Measured m/z Calculated m/z Accuracy (mDa) Accuracy (ppm) DBE [MH]+ formula RT (min)
ERY Parent 734.4713 734.4691 2.2 3.0 4.5 C37H68NO13 9.84  
N-Oxide 750.4677 750.4640 3.7 4.9 4.5 C37H68NO14 9.30  
Desmethyl 720.4538 720.4534 0.4 0.6 4.5 C36H66NO13 9.10 11.26
Didesmethyl 706.4390 706.4378 1.2 1.7 4.5 C35H64NO13 8.86  
M -H2O 716.4612 716.4585 2.7 3.8 5.5 C37H66NO12 10.89 12.47
N-Oxide -H2O 732.4569 732.4534 3.5 4.8 5.5 C37H66NO13 10.20  
Desmethyl -H2O 702.4420 702.4429 -0.9 -1.3 5.5 C36H64NO12 9.10 9.91
AZI Parent 749.5183 749.5164 1.9 2.5 3.5 C 38H 73N 2O 12 9.95  
N-Oxide 765.5155 765.5113 4.2 5.5 3.5 C 38H 73N 2O 12 9.23  
Desmethyl 735.5018 735.5007 1.1 1.5 3.5 C 37H 71N 2O 12 9.26  
Didesmethyl 721.4846 721.4851 -0.5 -0.7 3.5 C 36H 69N 2O 12 8.93  
Parent 749.5183 749.5164 1.9 2.5 3.5 C 38H 73N 2O 12 9.95  
CLA Parent 748.4838 748.4847 -0.9 -1.2 4.5 C38H70NO13 11.83  
N-Oxide 764.4803 764.4796 0.7 0.9 4.5 C38H70NO14 11.50  
Desmethyl 734.4701 734.4691 1.0 1.4 4.5 C37H68NO13 11.02  
Didesmethyl 720.4559 720.4534 2.5 3.5 4.5 C36H66NO13 10.65  
ROX Parent 837.5335 837.5324 1.1 1.3 4.5 C41H77N2O15 12.31  
N-Oxide 853.5284 853.5273 1.1 1.3 4.5 C41H77N2O16 12.07  
Desmethyl 823.5194 823.5167 2.7 3.3 4.5 C40H75N2O15 11.33  
Didesmethyl 809.5029 809.5011 1.8 2.2 4.5 C39H73N2O15 11.02  

Table 5: Measured m/z, calculated m/z, accuracy (mDa and ppm), double-bond equivalent (DBE), formulae of protonated ion and retention time (RT) of macrolides erythromycin (ERY), azithromycin (AZI), clarithromycin (CLA) and roxithromycin (ROX) and their degradation products.

Quality Control

All data were obtained in triplicate and the deviations (RSTD) were always less than 20%, and usually less than 10%. The correlations (R2) were higher than 0.98 (except for experimental conditions with a very low degradation process) for the results obtained in the experiments run under sunlight.

Kinetics calculations

Kinetics calculations were based on the following equations:

k1 calculation

ln [C] =-k1t+Ln[C0], first order,

[C0] ERY, CLA, AZI, ROX=0.136, 0.133, 0.134, 0.120 mM, respectively


[C] (mM)=macrolide concentration at a given time

[C0] (mM)=macrolide concentration at starting time

k1 (hr-1)=macrolide degradation-rate constant

t=time (hr)

Results and Discussion

The macrolide DPs were obtained in controlled laboratory experiments under sunlight irradiation.

Macrolide DPs laboratory experiments

A variety of macrolide DPs have been produced under controlled environmental conditions. The main DPs of ERY, CLA, AZI and ROX (obtained only under sunlight irradiation) were: N-oxide, N-desmethyl and N-didesmethyl (Figure 1). All results were obtained in triplicate and calculated as area percentage obtained from the MS detector. Tables 1 and 2 present the results after 336 h. In addition, DP’s identity was confirmed by their MS spectra (Tables 4 and 5).

Name Erythromycin
DIDES DES-H2O(1) (spiroketal) DES N-Oxide Parent DES-H2O(2) N-Oxide -H2O ERY-H2O(1) (spiroketal) ERY-H2O(2)
[MH]+ 706.45 702.44 720.45 750.46 734.47 702.44 732.45 716.46 716.46
RT (min) 8.86 9.10 9.10 9.30 9.84 9.91 10.20 10.89 12.47
Phosphate Buffer
pH 5
n/d n/d n/d n/d 5.5% 0.5% 0.5% 90% 2.0%
Phosphate Buffer
pH 7
n/d n/d n/d 3.8% 7.5% 0.5% 0.5% 83% 3.5%
Phosphate Buffer
pH 8.5
n/d n/d 0.3% 0.2% 48% n/d n/d 17% 26%
Humic acids
(5mg/L) pH 7
n/d n/d n/d 3.0% 4.5% 0.5% 1% 83% 4.0%
(measured pH 8.2)
n/d n/d 0.5% n/d 95% n/d n/d 2.1% n/d
(measured pH 7.7)
1.0% 2.0% 70% 5.5% 19.5% n/d n/d n/d% n/d

Table 2: Erythromycin degradation products (area %) obtained under various conditions after 14 days under sunlight irradiation.

Three different phosphate buffer solutions (pH 5, 7 and 8.5), containing 100 μg/mL of each macrolide drug (prepared separately), were examined under controlled artificial conditions. In addition, the pH 7 macrolide-containing solution was also examined with the addition of humic acid. Solutions from natural sources SE and TW were also examined (Tables 1 and 2).

According to our observations, the photodegradationmechanism responsible for these DPs has never been presented. Chen et al. [28] investigated the autoxidation of tertiary amines using trimethylamine as a model, at a temperature of 100°C and pressure of 153 atm of O2 to obtain the N-desmethyl and N-oxide products (Figure 2a). That study presented a mechanism that starts with electron transfer from the amine group to O2 to obtain the radical ions Me3N+ and O2 - (a second-order reaction). In the present study, sunlight irradiation at room temperature was used to obtain the N-oxide and N-desmethyl products (Figure 2b) in different solutions (Tables 1 and 2), and the O2 dissolved naturally in those solutions (first-order reaction, Figure 3 and Table 6). Production of the radical ions RMe2N·+ and O2 ·- was activated by the sunlight irradiation energy (Figure 2b). The same mechanism can be suggested for the formation of the N-didesmethyl DP from the N-desmethyl DP (Figure 2c). Chen et al. [28] claimed that in the autoxidation process of N-oxide and N-desmethyl DP formation, one is not a side product of the other, meaning that N-desmethyl is not obtained from N-oxide. To examine this claim, an additional experiment was conducted. Artificially prepared CLA-N-oxide (section 2.3.1) was exposed to sunlight irradiation for 14 days. No N-desmethyl product was detected (Figure 2d). In support of this, Hill et al. [29] described CLA oxidation using meso-tetraarylmetalloporphyrins and NaOCl to obtain N-oxide and N-desmethyl products, and demonstrated that the latter was not obtained from CLA-N-oxide.

Solution k1 (hr-1) t1/2 (hours) k1 (hr-1) t1/2 (hours) k1 (hr-1) t1/2  (hours) k1 (hr-1) t1/2  (hours)
SE 4.88E-03 142 3.04E-02 23 5.56E-03 125 7.60E-03 91
TW 6.60E-05 10500 3.2 0E-04 2310 2.76E-05 25109 9.00E-05 7700
pH 7 (HA) * * 3 .9 0E-04 1733 6.01E-05 11531 5.30E-05 13076
pH 5 * * 0 - 3.62E-06 191436 8.80E-05 7875
pH 7 * * 0 - 2.74E-05 25292 2.00E-05 34650
pH 8.5 * * 3.20E-04 2310 5.02E-05 13805 1.58E-04 4386

Table 6: Kinetics rate parameters of macrolides erythromycin (ERY), azithromycin (AZI), clarithromycin (CLA) and roxithromycin (ROX) in various solutions under sunlight irradiation. SE, secondary effluent; TW, tap water; HA, humic acid.


Figure 2: Mechanism underlying autoxidation of tertiary amines, demonstrated by trimethylamine (a), and by exposing tertiary amines at room temperature to sunlight irradiation (b, c and d).



Figure 3: Degradation rates of macrolides spiked in various solutions and exposed to solar irradiation. (a, b, c and d) Erythromycin (ERY), azithromycin (AZI), clarithromycin (CLA) and roxithromycin (ROX) respectively, in secondary effluent. (a1, b1, c1 and d1) ERY, AZI, CLA and ROX, respectively, in phosphate buffer solutions at pH 5, 7 and 8.5 in the pH 7 solution with added humic acid (HA) and in tap water (TW).

According to the above suggested mechanism, the macrolide DPs N-oxide, N-desmethyl and N-didesmethyl cannot, for the most part, be produced at low pH (5 and 7), due to unavailability of the amine’s two non-bonding electrons as a result of its protonation according to its high pKa level (~9) (Table 1) [15]. In contrast, in a solution with high pH (8.5), the DPs were obtained, but at relatively low levels. The N-oxide DP was obtained at 0.2%, 0.8%, 0.4% and 0.6% for ERY, AZI, CLA and ROX respectively; the N-desmethyl DP was obtained at 0.3%, 8.4%, 0.7% and 4.2% for ERY, AZI, CLA and ROX respectively (Tables 1 and 2).

The addition of humic acid to the solution was expected to enhance production of the photodegradation products due to its ability to act as a sensitizer [30]. A comparison was made between solutions at pH 7 with and without humic acid to demonstrate the latter’s effect. The humic acid certainly enhanced AZI degradation, based on the level of AZI N-desmethyl: 10.3% with humic acid and 0% without. As for the other macrolides, no significant effect of humic acid was observed (Table 1). The N-desmethyl DPs of all examined macrolides were obtained quite rapidly in SE: 70% for ERY, 76% for AZI, 79% for CLA and 86% for ROX after 2 weeks of sunlight irradiation. The N-didesmethyl DPs of all macrolides were also produced in SE, for at a level of 1.0% for ERY, 23% for AZI, 0.5% for CLA and 3.2% for ROX, after 2 weeks under sunlight irradiation. The relatively high AZI-Ndidesmethyl production can be explained by the possible elimination of methyl from the N-methyl amine, which is part of the lactone ring (Figure 1). Nevertheless, no N-tridesmethyl DP was detected for AZI.

The N-oxide DP also appeared mainly in the SE, for ERY (5.5%), AZI (1.4%), CLA (4.6%) and ROX (3.7%). This can be explained by the fact that SE contains sensitizer molecules such as humic acids and others, which encourage the photodegradation process.

In the shade and in all experimental solutions, the macrolides AZI, CLA and ROX showed high stability with minor degradation, whereas ERY only showed high stability in the TW and SE solutions due to their relatively high pH (8.2 and 7.7 respectively). In contrast to the other solutions [phosphate buffer at pH 5 (90%), pH 7 (87%), pH 7 with humic acids (89%) and pH 8.5 (18%)], ERY degraded rapidly in the first 2 h, producing ERY-spiroketal. The second H2O elimination product [ERY-H2O(2)] degraded less to 2.0% (pH 5), 3.5% (pH 7), 4.0% (pH 7 with humic acids) and 26% (pH 8.5) (Table 2). According to these results, it can be concluded that production of the two ERY-H2O elimination products is pH-dependent: the ERY-spiroketal is obtained mainly at low pH and ERY-H2O (2) mainly at high pH.

Structural elucidation of the DPs

Structural elucidation of the macrolide DPs was carried out using the LC-HRMS and NMR techniques. The NMR analysis was carried out only for the ERY-H2O (spiroketal product), following preparative separation and purification.

NMR analysis of ERY-H2O

Definitive proof for the proposed ERY-H2O structure was obtained by comparing the 1H and 13C NMR spectra of ERY and the isolated ERY-H2O (RT=10.89 min; Table 3). Full assignment was performed for 1H and 13C spectra based on El-Bondkly et al. [31] for ERY and on Alam et al. [32] for ERY-spiroketal.

The most significant indication of the formation of ERY-spiroketal was disappearance of the ERY ketone carbon C(9) appearing at δ 221.90 ppm in the 13C spectrum and the appearance of a new peak, related to C(9), at 117.57 ppm in the ERY-spiroketal spectrum a difference of 104.3 ppm upfield (Table 3).

Further examination of the 13C spectra indicated additional significant differences for the C(9)-adjacent carbon peaks C(10), C(11) and C(12), which were shifted downfield from δ 37.96, 68.93 and 74.73 ppm (for ERY) to 50.97, 87.86 and 83.86 ppm (for ERY-spiroketal), respectively. The two methyl carbon peaks, C (17) and C(21), also showed significant differences in chemical shifts: δ 9.18 and 16.23 ppm in ERY shifted downfield to δ 18.45 and 25.37 ppm in ERY-spiroketal, respectively (Table 3). The differences in chemical shifts of 1H NMR spectral peaks of ERY and the spiroketal product were not as indicative as in the 13C NMR spectra (Table 3).

LC-MS analysis

LC–MS analysis was carried out for macrolides and their DPs using HRMS to examine their structures. The main DPs of the four examined macrolides were identified according to their molecular masses, mass fragmentation, empirical formulas and relative retention times in the chromatographic column (Tables 4 and 5). The MS spectra of the detected parent macrolides consisted of their molecular masses [MH]+, which were 734.4713 (ERY), 749.5183 (AZI), 748.4838 (CLA) and 837.5335 (ROX). Their spectra were also characterized by elimination of the cladinose residue to obtain the main fragment [MH]+ without cladinose as presented by Chitneni et al. [24], Haghedooren et al. [25], Leonard et al. [33] and Barrett et al. [34].

Elimination of H2O from the molecular mass was obtained mainly for ERY (as a significant fragment), due to its sTable product ERY-H2O, compared to other macrolides. AZI has two amine groups (Figure 1), and it was therefore also characterized by the mass m/z 375.2558, which is a result of z=2 (two protonated amines).

The three main DPs, which were obtained only under sunlight irradiation, were N-oxide, N-desmethyl and N-didesmethyl. The spectra of the macrolide N-oxide contained mainly the molecular masses [MH]+, which were 750.4634 (ERY), 765.5107 (AZI), 764.4791(CLA) and 853.5268 (ROX). These DPs’ empirical formulas were confirmed by their HRMS spectra (Table 5). The N-desmethyl and N-didesmethyl DP spectra consisted of their molecular mass as well as their main fragment [MH]+ without cladinose related to the elimination of cladinose (Tables 4 and 5). For ERY, due to its rapid formation to a spiroketal product, two additional photodegradation products were also obtained: ERY-N-oxide-H2O ([MH]+ 732.4529) and ERY-N-desmethyl-H2O ([MH]+ 702.4423) [35].


During the experiments, the macrolides degraded with time mainly under sunlight irradiation, following first-order kinetics (Table 6, Figure 3). No kinetic data could be obtained for the experiments in the shade, due to the relatively high stability of the macrolides under these experimental conditions, except for ERY, which was rapidly degraded in the artificial solutions (different pHs) (section 3.1). Regarding the kinetics data presented in Table 5 and Figure 3, the kinetics values for ERY could be obtained only in natural solutions (SE and TW). Production of the main macrolide DPs only under sunlight irradiation can be explained by the decomposition of the parent molecules due to photoactivation, with degradation that is much faster than in the shade; this is in contrast to ERY in artificial solutions, which rapidly decomposes to its main product ERY-spiroketal [11] (Table 2). In the present study, the kinetics results are demonstrated in various solutions under solar irradiation, producing k1 values ranging from 0.0304 hr-1 for AZI in SE (Figure 3b) to 0 for AZI at pH 5 and 7 (Figure 3b1; Table 6). It was anticipated that humic acids would act as a sensitizer [30] for the induction of photodegradation products (N-oxide, desmethyl and didesmethyl) in comparison to the other solutions. However, no additional effect was observed in CLA or ROX in the presence of humic acids (Figure 3c1 and d1, Table 6), while the k1 of AZI at pH 7 with the addition of humic acids was 0.000390 hr-1, and without humic acids it was 0 (Figure 3b1, Table 6). As discussed in section 3.1, the degradation process is not favored at low pH due to the unavailability of the non- bonding electrons of the amine group. As expected, the degradation rate in SE was relatively high, with k1 values of 0.00488 hr-1 (ERY), 0.0304 hr-1 (AZI), 0.00556 hr-1 (CLA) and 0.00760 hr-1 (ROX) (Figure 3a-d; Table 6).

Summary and conclusions

To the best of our knowledge, this is the first study to examine the formation of a variety of macrolide DPs under controlled environmental conditions, followed by a characterization and kinetics behavior analysis of macrolide degradation under solar irradiation. Three macrolide DPs (N-oxide, N-desmethyl and N-didesmethyl) were produced following exposure of macrolides to solar irradiation. Investigating the N-oxide and N-desmethyl DPs enabled us to understand the reaction mechanism governing DP formation through the intermediate radical ions RMe2N+ and O2 -. The macrolides degraded rapidly under solar irradiation in the investigated aqueous solutions, mainly SE, whereas in the same solutions in the shade, macrolide degradation was negligible, except for ERY in the artificial solutions. The results of this study should direct further research into identified and suggested DPs, not only from macrolides but also from other drugs, which could potentially be found in aquatic environments. The characterization, presented herein, is expected to enable the detection of such DPs in various aquaticenvironments. Moreover, N-desmethyl and N-didesmethyl DPs are still biologically active, potentially increasing their toxicity to humans. Thus, further research is warranted to examine the environmental toxicity and stability of these compounds, which might, through exchange, form other DPs.


We would like to thank Prof. Shmuel Carmeli for his valuable comments and contribution.


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