Antibiotics have found to show an increasing stability and consistency within environmental samples. Partially metabolized and partially removed in WWTP processes, with an increasing antibiotic resistance trend and the potential ecotoxicological effects, it has become crucial to monitor them within the environment.
In the study the aim was to develop a proficient analytical method for the assessment of low antibacterial concentration of 3 classes of antibiotics; Fluoroquinolones (Ciprofloxacin, Enrofloxacin, Marbofloxacin, Ofloxacin, Norfloxacin), Sulfonamides (Sulfadimethoxine, Sulfamethoxazole) and Amphenicols (Florfenicol) in Tartu city river water body Emajogi.
Emajõgi is a river in Estonia which flows from Lake Võrtsjärv through Tartu County into Lake Peipsi, crossing the city of Tartu for 10 km and a length of approximately 100 km. The first-time analysis of river water samples in Tartu city with the developed offline solid phase extraction (SPE), LC-MS/MS method has shown to be able to detect and quantify antibiotics at low concentration levels within river water samples with good recovery and repeatability. The work done provided a reliable data that could be used to potentially monitor the selected antibacterial compounds in other river water environments and potentially lakes.
Initial results indicated good recovery of the selected analytes through solid phase extractions from river water samples at >5 °C. Precleaning and filtration of the samples before SPE extraction is crucial to minimize all potential matrix effects, a tedious, but otherwise necessary and efficient step. After the initial results, an ecotoxicological assessment was made considering the provided impactful concentrations. All antibiotics were found to be below the threshold limit.
The novelty of the method exists in its first-time applicability and first-time assessment of river water samples from Emajõgi. It could be potentially used for routine monitoring of these analytes within the river water throughout the year and to assess different concentration ranges and influences within the water and through it, the relative ecosystem.
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Antimikroobsete ainete määramine jõevees kasutades tahke faasi ekstraktsiooni ja LC-MS/MS meetodit
Waseem Ahmad Iftikhar Kokkuvõte
Antimikroobsete ainete jääke leitakse järjest sagedamine keskkonnaproovides. Kuigi need ained osaliselt metaboliseeruvad ja osaliselt eraldatakse reoveepuhastites, siis ikkagi suureneb antibiootikumiresistentsuse ja ökotoksikoloogiliste mõjude risk. See toob kaasa vajaduse määrata nende ainete sisaldusi keskkonnaproovides.
Käesoleva töö eesmärgiks oli analüüsimetoodika arendamine kolme eri rühma kuuluvate antibiootikumide jääkide määramiseks jõevees; fluorokinoloonid (tsiprofloksatsiin, enrofloksatsiin, marbofloksatsiin, ofloksatsiin, norfloksatsiin), sulfoonamiidid (sulfadimetoksiin, sulfametoksasool) ja amfenikoolid (florfenikool).
Emajõgi on Eesti üks tuntumaid jõgesid, mis voolab Võrtsjärvest Peipsi järve ja läbib seejuures Tartu linna. Käesolevas töös arendatud metoodikat kasutati antibiootikumijääkide määramiseks Emajõe veeproovides, mis olid võetud Tartu reoveepuhastusjaama lähedalt ning sellest alla- ja ülesvoolu. Kasutatud tahke faasi ekstraktsiooni (SPE) ja LC-MS meetodit kasutav analüüsimetoodika võimaldas tuvastada antibiootikumijääkide madalaid sisaldusi hea saagise ja korratavusega. Arendatud metoodikat on võimalik kasutada antibiootikumide jääkide monitoorimiseks jõgedes ja tõenäoliselt ka järvede vees.
Valideerimise tulemusena selgus, et kasutatud SPE metoodikal on uuritud analüütide suhtes rahuldav saagis. Seejuures leiti, et maatriksiefektide alandamiseks on proovide SPE-eelne filtreerimine väga oluline. Selle mõju ekstraheerimise saagisele vajab siiski täiendavat uurimist.
Analüüsil leitud antibiootikumide kontsentratsioonidele anti ka esialgne ökotoksikoloogiline hinnang. Kõigi uuritud antibiootikumide sisaldused jäid allapoole kehtestatud piirnormi.
Arendatud metoodika oli uudne laborile, kus töö läbi viidi – varem ei ole seal uuritud ravimite jääkide sisaldust jõevees. Samuti lisandus uuritavate analüütide hulka kolm uut antibiootikumi.
Metoodikat saab edasi arendada lisades veelgi analüüte ja kontrollides selle toimivust ka teiste veekogude vete analüüsil. Arendatud metoodika võimaldab keskkonna seisundit paremini hinnata.
29
Acknowledgements
First, I would like to thank my parents for their ever-growing love, support and for teaching me values that have always proven to be useful. To my brothers Ijaz, Fraz, Rashid, Qamar, Umar and my sisters Khadijah and Maryam for always being there for me when I needed them and for never letting me settle for less.
I would like to very humbly and sincerely thank my supervisor, Professor Koit Herodes who has taught me the immense value of understanding through patience. Even when my knowledge would fail me, he would uphold me to do better and to try and think of a solution. He will always answer my questions no matter how basic or how trivial they may seem and always hear my concerns before providing a sound solution. You have been the best mentor I could have asked to learn from and I’m forever thankful that I had the chance to learn from the best.
I would like to thank Professor Ivo Leito for his immense wisdom and guidance through all matters of my student life and for helping me solve all the uncertainties in my student life. We always had you by our side to guide us through all the deviations, and for that I am grateful.
My other sources of knowledge and support have been immense, and I would like to thank them as well. I would like to thank Christina for sharing her knowledge with me and for always making sure I had the help I need, even on weekends. I would like to thank Toiv, Hanno, Anu for molding us into practical analytical chemists with a lot of patience and care put into our learning. Ernesto and Nhung for all your advice and help and for your candid impression of my work and what could be done better.
I would like to thank my dear friends, Helmi and Zen for their honest inputs. Finally, I would like to thank my lab mate Joshua for being a valuable friend and colleague. I would have never learnt as much as I did about LC-MS if it weren’t for our common passion for it and for our constant discussions back and forth. I appreciate and cherish all my time at the University of Tartu and firmly believe that my professors, colleagues and friends have imparted me with the gift of knowledge, humility and steadfastness in accepting and overcoming my limits. Thank you.
This work was supported by AMRITA project, which is financed by Estonian Research Council from ERF resources of RITA programme for Ministries of Rural Affairs, Environment and Social Affairs (contract no 7.2-2/19/5).
30
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Annexes
Annex 1: List of Figures and Tables.
Annex 2: Structure of Antibiotics.
Annex 3: Antibiotic resistance data from ECDC (European commission of disease prevention and control)
Annex 4: Sample collection points.
Annex 5: MRM Chromatograms of antibiotics in samples
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Annex 1: List of Figures and Tables.
Figures:
Figure 1. Flow of the LC-MS Project. ... 12
Figure 2. Gradient elution method showing MRM of 8 different antibiotics. ... 17
Figure 3. Blank Chromatogram to check for possible carryover effects. ... 22
Figure 4. Process Efficiency ... 25
Tables: Table 1. Antibiotic Classification based on their mechanism of action ... 7
Table 2. LC-MS/MS Conditions for the analysis of antibiotics by MRM using Waters XBridge RP column... 16
Table 3. LC Gradient Elution for the analysis of antibiotics. ... 17
Table 4.Calibration Ranges, Regression coefficients and LOD and LOQ values. ... 19
Table 5. Antibiotics at Upstream, Midpoint and Downstream of the WWTP. (ND: Not detected, NA: Not applicable) ... 20
Table 6. Percentage Accuracy of spiked samples. ... 21
Table 7. Concentrations of Analytes before and after washing. ... 21
Table 8. Gradient to flush the column ... 22
Table 9. Matrix Effect Evaluation at different concentration levels. ... 23
Table 10. Recovery of antibiotics ... 24
Table 11. Ratio of Measured values and PNEC values to assess ecotoxicological and resistance promotion impact. ... 26
37
Annex 2: Structures of Antibiotic Compounds.
Sulfadimethoxine (SDM) | pKa – 2.11, pKa – 6.17 Sulfamethoxazole (SMX) | pKa - 1.97, pKa -6.67 Ofloxacin (OFL) | pKa – 5.45 pKa – 6.2
Florfenicol (FF) | pKa -3.4, pKa -8.49
38
Annex 3: ECDC Data plot showing the antibiotic resistance increase of some fluoroquinolones
45
Trends in Antibiotic Resistance Towards Fluoroquinolones
Acinetobacter spp.
39
Annex 4: Sample collection points.
WWTP
Upstream
Midpoint
Downstream
40
Annex 5. MRM Chromatograms of antibiotics found in samples.
Ciprofloxacin (CIP)
41 Sulfamethoxazole (SMX)
Sulfadimethoxine (SDM)
S N O
O N
H2
N
O CH3 H
O O N
N N H S
O CH3
O N
H2
CH3
42
Non-exclusive licence to reproduce thesis and make thesis public
I, Waseem Ahmad Iftikhar herewith grant the University of Tartu a free permit (non-exclusive licence) to reproduce, for the purpose of preservation, including for adding to the DSpace digital archives until the expiry of the term of copyright,
DETERMINATION OF ANTIBACTERIALS IN RIVER WATER BY SOLID PHASE EXTRACTION USING LC-ESI-MS/MS
supervised by Associate professor, Dr. Koit Herodes_
2. I grant the University of Tartu a permit to make the work specified in p. 1 available to the public via the web environment of the University of Tartu, including via the DSpace digital archives, under the Creative Commons licence CC BY NC ND 3.0, which allows, by giving appropriate credit to the author, to reproduce, distribute the work and communicate it to the public, and prohibits the creation of derivative works and any commercial use of the work until the expiry of the term of copyright.
3. I am aware of the fact that the author retains the rights specified in p. 1 and 2.
4. I certify that granting the non-exclusive licence does not infringe other persons’ intellectual property rights or rights arising from the personal data protection legislation.
Waseem Ahmad Iftikhar
27/05/2020
43 INFORMATION SHEET
Determination of antibacterials in river water by solid phase extraction using LC -MS/MS
Analytical method was developed for the detection of fluoroquinolones (FQs), sulfonamides and Amphenicol in Emajogi river in Tartu City. The compounds were simultaneously extracted from river water using solid phase extraction (SPE). Identification and quantification was done through Liquid chromatography – tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring (MRM) mode.The recovery of FQs ranged 62% for Enrofloxacin to 76% Marbofloxacin and 68% Florfenicol ,69%
sulfamethoxazole. Limit of Quantification ranged from 0.1 ngg-1 for SA’s ,0.6 - 1.5 ngg-1 for FQ’s, and 0.9ngg-1 for FF. The method was developed and valdated for river water analysis of samples from upstream, midpoint and down stream of the WWTP. The method developed may be used for a more in-depth study on the occurence and fate of these commonly used pharmaceuticals in river water bodies.
Key words: antibiotic residue analysis, LC-MS, method development, solid phase extraction, river water analysis,
CERCS: P300 analytical chemistry. INFOLEHT
Antimikroobsete ainete määramine jõevees kasutades tahke faasi ekstraktsiooni ja LC-MS/MS meetodit
Käesolevad töös töötati välja analüüsimetoodika fluorokinoloonide, sulfoonamiidide ja amfenikoolide rühma kuuluvate antibiootikumide määramiseks jõevees. Analüüdid ekstraheeriti proovist korraga, kasutades tahke faasi ekstraktsiooni (SPE). Identifitseerimine ja kvantitatiivne analüüs teostati vedelikkromatograafia-massispektromeetria (LC-MS/MS) meetodit kasutades valitud ülemineku jälgimise režiimis (MRM). Analüütide saagised jäid vahemikku 62 kuni 78%, mida võib pidada rahuldavaks. Määramispiirid jäid vahemikku 0,1 kuni 1,5 ng g-1. Metoodika arendamisel ja valideerimisel kasutati Emajõe veeproove, mis olid võetud veepuhastusjaama lähistelt ning sellest üles- ja allavoolu. Töö tulemusi saab kasutada antibiootikumijääkide monitoorimiseks jõevees.
Märksõnad: antibiootikumijääkide analüüs, LC-MS, metoodika arendus,dsd tahke faasi ekstraktsioon, jõevee analüüs,
CERCS: P300 analüütiline keemia