Florida International University FIU Digital Commons FIU Electronic Theses and Dissertations University Graduate School 7-3-2013 Assessment of the Occurrence and Potential Risks of Antibiotics and their Metabolites in South Florida Waters Using Liquid Chromatography Tandem Mass Spectrometry Venkata Reddy Panditi [email protected] DOI: 10.25148/etd.FI13080706 Follow this and additional works at: https://digitalcommons.fiu.edu/etd Part of the Analytical Chemistry Commons, Environmental Chemistry Commons, Environmental Indicators and Impact Assessment Commons, and the Environmental Monitoring Commons Recommended Citation Panditi, Venkata Reddy, "Assessment of the Occurrence and Potential Risks of Antibiotics and their Metabolites in South Florida Waters Using Liquid Chromatography Tandem Mass Spectrometry" (2013). FIU Electronic Theses and Dissertations. 916. https://digitalcommons.fiu.edu/etd/916 This work is brought to you for free and open access by the University Graduate School at FIU Digital Commons. It has been accepted for inclusion in FIU Electronic Theses and Dissertations by an authorized administrator of FIU Digital Commons. For more information, please contact [email protected]. FLORIDA INTERNATIONAL UNIVERSITY Miami, Florida ASSESSMENT OF THE OCCURRENCE AND POTENTIAL RISKS OF ANTIBIOTICS AND THEIR METABOLITES IN SOUTH FLORIDA WATERS USING LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in CHEMISTRY by Venkata Reddy Panditi 2013 To: Dean Kenneth G. Furton College of Arts and Sciences This dissertation, written by Venkata Reddy Panditi, and entitled Assessment of the occurrence and potential risks of antibiotics and their metabolites in South Florida waters using liquid chromatography tandem mass spectrometry, having been approved in respect to style and intellectual content, is referred to you for judgment. We have read this dissertation and recommend that it be approved. _______________________________________ Rudolf Jaffe _______________________________________ Yong Cai _______________________________________ Berrin Tansel _______________________________________ Jeffrey Joens _______________________________________ Piero R. Gardinali, Major Professor Date of Defense: July 3rd, 2013 The dissertation of Venkata Reddy Panditi is approved. _______________________________________ Dean Kenneth G. Furton College of Arts and Sciences _______________________________________ Dean Lakshmi N. Reddi University Graduate School Florida International University, 2013 ii © Copyright 2013 by Venkata Reddy Panditi All rights reserved. iii DEDICATION This thesis is dedicated to the memory of my beloved mother, Samrajyam. She inspired me through her pain and suffering. I would also like to dedicate this thesis to my wife, Sudha Rani and loving daughter, Hasini, for their understanding, support and unconditional love. iv ACKNOWLEDGEMENTS I would like to express the deepest appreciation to my major professor, Piero Gardinali for being such an inspiring mentor, who has the attitude and the substance of genius. Every conversation with him made me to question my propositions and think critically to find the answer that led me to become a better scientist. Without his guidance and persistent support this dissertation would not been possible. I would like to thank my committee members Dr. Rudolf Jaffe, Dr. Yong Cai, Dr. Jeffrey Joenes and Dr. Berring Tansel for sharing their ideas and constructive comments that helped as check points during the course of my research study. I would also like to thank Professor Dr.Kati Migliaccio and her research crew from University of Florida, Tropical Research and Education Center (TREC), Homestead, for helping me in ground water sample collection. My special thanks to one of my best friends Natalia. She was always helpful and guided me during difficult times. I also would like to thank my loving brother, Sridhara Reddy for his moral support. I feel he is always with me. Finally, I wish to thank all my co-workers, fellow students and friends for leaving me such a memorable time in college. v ABSTRACT OF THE DISSERTATION ASSESSMENT OF THE OCCURRENCE AND POTENTIAL RISKS OF ANTIBIOTICS AND THEIR METABOLITES IN SOUTH FLORIDA WATERS USING LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY by Venkata Reddy Panditi Florida International University, 2013 Miami, Florida Professor Piero R. Gardinali, Major Professor An automated on-line SPE-LC-MS/MS method was developed for the quantitation of multiple classes of antibiotics in environmental waters. High sensitivity in the low ng/L range was accomplished by using large volume injections with 10-mL of sample. Positive confirmation of analytes was achieved using two selected reaction monitoring (SRM) transitions per antibiotic and quantitation was performed using an internal standard approach. Samples were extracted using online solid phase extraction, then using column switching technique; extracted samples were immediately passed through liquid chromatography and analyzed by tandem mass spectrometry. The total run time per each sample was 20 min. The statistically calculated method detection limits for various environmental samples were between 1.2 and 63 ng/L. Furthermore, the method was validated in terms of precision, accuracy and linearity. The developed analytical methodology was used to measure the occurrence of antibiotics in reclaimed waters (n=56), surface waters (n=53), ground waters (n=8) and drinking vi waters (n=54) collected from different parts of South Florida. In reclaimed waters, the most frequently detected antibiotics were nalidixic acid, erythromycin, clarithromycin, azithromycin trimethoprim, sulfamethoxazole and ofloxacin (19.3-604.9 ng/L). Detection of antibiotics in reclaimed waters indicates that they can’t be completely removed by conventional wastewater treatment process. Furthermore, the average mass loads of antibiotics released into the local environment through reclaimed water were estimated as 0.248 Kg/day. Among the surface waters samples, Miami River (reaching up to 580 ng/L) and Black Creek canal (up to 124 ng/L) showed highest concentrations of antibiotics. No traces of antibiotics were found in ground waters. On the other hand, erythromycin (monitored as anhydro erythromycin) was detected in 82% of the drinking water samples (n.d-66 ng/L). The developed approach is suitable for both research and monitoring applications. Major metabolites of antibiotics in reclaimed wates were identified and quantified using high resolution benchtop Q-Exactive orbitrap mass spectrometer. A phase I metabolite of erythromycin was tentatively identified in full scan based on accurate mass measurement. Using extracted ion chromatogram (XIC), high resolution data-dependent MS/MS spectra and metabolic profiling software the metabolite was identified as desmethyl anhydro erythromycin with molecular formula C36H63NO12 and m/z 702.4423. The molar concentration of the metabolite to erythromycin was in the order of 13 %. To my knowledge, this is the first known report on this metabolite in reclaimed water. Another compound acetyl-sulfamethoxazole, a phase II metabolite of sulfamethoxazole was also identified in reclaimed water and mole fraction of the metabolite represent 36 %, of the vii cumulative sulfamethoxazole concentration. The results were illustrating the importance to include metabolites also in the routine analysis to obtain a mass balance for better understanding of the occurrence, fate and distribution of antibiotics in the environment. Finally, all the antibiotics detected in reclaimed and surface waters were investigated to assess the potential risk to the aquatic organisms. The surface water antibiotic concentrations that represented the real time exposure conditions revealed that the macrolide antibiotics, erythromycin, clarithromycin and tylosin along with quinolone antibiotic, ciprofloxacin were suspected to induce high toxicity to aquatic biota. Preliminary results showing that, among the antibiotic groups tested, macrolides posed the highest ecological threat, and therefore, they may need to be further evaluated with, long-term exposure studies considering bioaccumulation factors and more number of species selected. Overall, the occurrence of antibiotics in aquatic environment is posing an ecological health concern. viii TABLE OF CONTENTS CHAPTER PAGE CHAPTER 1 ....................................................................................................................... 1 INTRODUCTION .............................................................................................................. 1 1.1. Overview .................................................................................................................. 2 1.2. What are Antibiotics? .............................................................................................. 4 1.3. Sources of Antibiotics in the environment ............................................................... 6 1.3.1. Natural sources .............................................................................................. 6 1.3.2. Antibiotic production .................................................................................... 6 1.3.3. Usage............................................................................................................. 7 1.3.4. Occurrence .................................................................................................... 9