Thermal Degradation of Antibiotic Residues: Amphenicols as a Case Study By Lei Tian ...

143 Pages·2016·5 MB·English

by  Lei Tian, Miss

Thermal Degradation of Antibiotic Residues: Amphenicols as a Case
Study

By

Lei Tian

Department of Food Science and Agricultural Chemistry
Macdonald Campus, McGill University
Montreal, Canada

April, 2016

A thesis submitted to McGill University in partial fulfillment of the
requirements of the degree of Master of Science

©Lei Tian, 2016
ABSTRACT
Veterinary drug residues, though carefully regulated, remain commonly detected in food of
animal origin (meat, seafood, dairy products etc.). Worryingly, new pharmaceutically active
contaminants are continuously being detected in food. Recently, human pharmaceuticals and
other antimicrobial residues occurring as environmental contaminants in aquatic systems were
observed to accumulate in various seafood. At the same time, most foods of animal origin are
cooked before consumption. In terms of food safety, it is therefore necessary to understand the
fate of drug residues in the food supply chain, notably during thermal processing.
Amphenicols (e.g. florfenicol, chloramphenicol) are one class of antibiotics commonly reported
in fish/seafood and aquatic system worldwide. In this study, the thermal degradation kinetics of
amphenicols were explored using high performance liquid chromatography triple quadrupole
tandem mass spectrometry (HPLC-QqQ-MS/MS). Results indicated that the chloramphenicol
and florfenicol followed the first-order degradation kinetic, and the degradation rate constant k
increased with temperature increase. Then, the identity of thermal degradation products was
investigated through two strategies. First, pyrolysis-gas chromatography-mass spectrometry
(Py-GC-MS) was applied to investigate the volatile degradation products of three antibiotics and
three additional pharmaceuticals. Chloramphenicol, lincomycin, gemfibrozil and
diphenhydramine were shown to degrade into a range of previously unidentified volatile
compounds. No volatile degradation products were identified for florfenicol. Overall, Py-GC-MS
was a fast and effective method to explore the thermal degradation of pharmaceuticals. Finally,
II
HPLC coupled with high resolution tandem mass spectrometry (in our case
HPLC-QTOF-MS/MS) coupled with bioinformatics was applied to identify the thermal
degradation products of chloramphenicol in water and to confirm the degradation products in
cooked mussels. This approach allowed to find previously unidentified degradation by-products.
This work further highlights the need to take into account the impact food processing for the

food safety risk assessment of amphenicol antibiotics.
III
RÉSUMÉ
Des résidus de médicaments vétérinaires, bien qu’ils soient strictement régulés, sont
régulièrement détectés dans les aliments d’origine animale (viande, poisson et fruits de mer,
produits laitiers…). De manière inquiétante, de nouveaux résidus de médicaments sont
continuellement détectés dans les aliments. Récemment, on a observé que divers médicaments et
produits antimicrobiens, contaminant les milieux aquatiques, pouvaient s’accumuler dans les
poissons et fruits de mer. En termes de sécurité sanitaire des aliments, il est nécessaire de
comprendre le devenir des résidus de médicaments dans la chaine de production agroalimentaire,
notamment au cours des traitements thermiques.
Les phénicols (chloramphénicol, florfénicol) constituent une famille d'antibiotiques détectés
internationalement dans le poisson, fruits de mer et systèmes aquatiques. Dans la présente étude,
la cinétique de dégradation thermique des phénicols a été étudiée en utilisant la chromatographie
liquide haute performance couplée à la spectrométrie de masse triple quadrupôle
(HPLC-QqQ-MS/MS). Les résultats ont montré que les cinétiques de dégradation du
chloramphénicol et du florfénicol suivent un modèle de premier ordre, et la constante de
dégradation thermique, k, croît avec la température. Par la suite, deux stratégies ont été mises en
œuvre pour l’identification des produits de dégradation thermique. Tout d’abord, la
pyrolyse-chromatographie en phase gazeuse-spectrométrie de masse (Py-GC-MS) a été utilisée
pour l’étude des produits de dégradation volatils de trois antibiotiques et trois autres composés
pharmaceutiques. Ainsi, cette approche a montré que le chloramphénicol, la lincomycine, le
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gemfibrozil et la diphénhydramine se dégradent en une série de composes volatils. Aucun
compose volatil n’a été détecté au cours de la dégradation du florfénicol. Py-GC-MS s’est
montre être une technique efficace et rapide pour l’étude de la dégradation thermique des résidus
de médicaments. Enfin, la HPLC couplée à la spectrométrie de masse haute résolution
(HPLC-QTOF-MS/MS dans notre cas), combinée à la bioinformatique a été utilisée pour
identifier les produits de dégradation thermique du chloramphénicol dans l’eau, et confirmer leur
présence au cours de la cuisson d’une autre matrice, de la moule. Cette approche a permis à
nouveau de détecter des nouveaux produits de dégradation. Ce travail de recherche met en
exergue la nécessité de prendre en compte l’impact de la transformation alimentaire pour

l’évaluation des risques liés à la présence de résidus de phénicols dans les aliments.

V
CONTRIBUTIONS OF AUTHORS
The authors involved in the thesis and their contributions to the various articles are as follows:

Lei TIAN is the M.Sc. candidate who designed and conducted all the experiments in consultation
with the supervisors. She performed data collection and analysis. Additionally, she prepared
drafts of all the manuscripts for scientific publications.

Dr. Stéphane BAYEN is the thesis supervisor, under whose guidance the research was conducted.
He assisted the candidate in designing and conducting the experiments as well as correcting,
proofreading, reviewing and processing manuscripts for the publications.

The literature review in Chapter 2 is reviewed and organized by Lei TIAN under the supervision
of Dr. Stéphane BAYEN. Salma KHALIL contributed to Chapter 2 and wrote two paragraphs on
tetracyclines in section 3.4. These two paragraphs are part of the manuscript submitted for
publication and are kept in the present thesis to give an overview of the topic for all antibiotics.
VI
LIST OF PUBLICATIONS AND PRESENTATIONS
Part of this thesis has been prepared as manuscripts for publications in refereed scientific
journals:
Lei Tian, Salma Khalil, Stéphane Bayen. Effect of thermal treatments on the degradation of
antibiotic residues in food. Critical Reviews in Food Science and Nutrition. (Status: Accepted)
Lei Tian, Varoujan Yaylayan, Stéphane Bayen. Investigation of the thermal degradation of
veterinary and human pharmaceuticals using pyrolysis-GC-MS. (Status: in preparation)
Lei Tian, Stéphane Bayen. Thermal degradation kinetics of chloramphenicol and its degradation
products investigated by HPLC-QqQ-MS/MS and HPLC-QTOF-MS/MS. (Status: in
preparation)

Part of this thesis has been presented in scientific conferences:
Stéphane Bayen, Lei Tian, Salma Khalil, Varoujan Yaylayan. Unveiling the fate of antibiotic
residues during seafood processing using pyrolysis-gas chromatography-mass spectrometry. IFT
conference, 2015, Chicago, IL, USA.
Stéphane Bayen, Lei Tian. Antibiotic and pharmaceutical residues from field to fork: detection,
thermal degradation and food safety implications. 99th Canadian Chemistry Conference, 2016,
Halifax, NS, CA. (in preparation)
VII
ACKNOWLEDGEMENTS
I would like to thank my supervisor Dr. Stéphane BAYEN for his invaluable guidance and
support. Thanks for his encouragements which help me to solve all the difficulties in research
and help me to be a scientific researcher.

In addition, I would like to thank Dr. YAYLAYAN, Dr. KARBOUNE, Dr. WYKES and Dr.
KERMASHA for sharing the lab materials and equipment.

Also, I would like to thank Dr. Burgos for his comments and suggestions for this thesis.

Finally, my friends and family have supported and encouraged me throughout my masters’ study
to accomplish my goals and I would like to thank them all.

VIII
CONTENTS
ABSTRACT ................................................................................................................................................ II
RÉSUMÉ ................................................................................................................................................... IV
CONTRIBUTIONS OF AUTHORS ........................................................................................................ VI
LIST OF PUBLICATIONS AND PRESENTATIONS......................................................................... VII
ACKNOWLEDGEMENTS ................................................................................................................. VIII
LIST OF ABBREVIATIONS ................................................................................................................. XII
CHAPTER 1. INTRODUCTION .............................................................................................................. 1
CHAPTER 2. LITERATURE REVIEW .................................................................................................. 7
2.1. Introduction ....................................................................................................................................... 7
2.2. Material and methods ........................................................................................................................ 8
2.2.1 Selected literature ........................................................................................................................ 8
2.2.2 Degradation percentage (DP) and degradation rate constant (k) .............................................. 9
2.2.3 Statistical analysis ..................................................................................................................... 11
2.3. Results and Discussion .................................................................................................................... 11
2.3.1 Experimental assessment of antibiotic degradation .................................................................. 11
2.3.1.1. Experimental design of degradation studies ..................................................................................... 11
2.3.1.2. Quantification of antibiotic degradation ........................................................................................... 12
2.3.2 Thermal kinetics of antibiotics .................................................................................................. 14
2.3.2.1 β-Lactams antibiotics ..................................................................................................................................... 15
2.3.2.2 Tetracyclines ...................................................................................................................................... 22
2.3.2.3 Macrolides ......................................................................................................................................... 23
2.3.2.4 Aminoglycosides ............................................................................................................................... 24
2.3.2.5 Amphenicols ...................................................................................................................................... 24
2.3.2.6 Quinolones ........................................................................................................................................ 25
2.3.2.7 Sulfonamides ..................................................................................................................................... 26
2.3.2.8 Other antibiotics (teicoplanin, polymixin B, vancomycin, oxfendazole and lasalocid) .................... 27
2.3.2.9 Discussion on the influence of the family of antibiotics ................................................................... 27
IX
2.3.3 Degradation kinetics ................................................................................................................. 28
2.3.3.1 Influence of time on the degradation ........................................................................................................... 28
2.3.3.2 Influence of temperature on the degradation ..................................................................................... 29
2.3.3.3 Influence of matrix on the degradation .............................................................................................. 31
2.3.3.4 Influence of cooking methods on the degradation ............................................................................. 33
2.3.4 Identification of degradation products ...................................................................................... 35
2.4. Conclusions ..................................................................................................................................... 36
CHAPTER 3 APPLICATION OF PYROLYSIS-GC-MS IN STUDYING THERMAL
DEGRADATION OF AMPHENICOLS AND OTHER DRUG RESIDUES ....................................... 38
3.1 Introduction ...................................................................................................................................... 38
3.2 Materials & Method .......................................................................................................................... 39
3.2.1 Materials ................................................................................................................................... 39
3.2.2 Pyrolysis GC-MS analysis ......................................................................................................... 39
3.3 Results and discussions .................................................................................................................... 41
3.3.1 Py-GC-MS of Chloramphenicol ................................................................................................ 42
3.3.2 Py-GC-MS of Florfenicol .......................................................................................................... 44
3.3.3 Py-GC-MS of Carbamazepine ................................................................................................... 45
3.3.4 Py-GC-MS of Lincomycin hydrochloride .................................................................................. 46
3.3.5 Py-GC-MS of Diphenhydramine hydrochloride ........................................................................ 48
3.3.6 Py-GC-MS of Gemfibrozil ......................................................................................................... 49
3.4 Conclusions ...................................................................................................................................... 51
CHAPTER 4. THERMAL DEGRADATION KINETICS OF AMPHENICOLS ............................... 53
4.1 Introduction ...................................................................................................................................... 53
4.2 Materials and methods ...................................................................................................................... 54
4.2.1 Materials ................................................................................................................................... 54
4.2.2 Method validation ..................................................................................................................... 54
4.2.3 Experimental procedure ............................................................................................................ 54
4.2.3.1 Kinetic studies at 100° C in water by HPLC-QqQ-MS/MS ..................................................................... 54
4.2.3.2 Kinetic studies at 60 °C and 80 °C in water by HPLC-QqQ-MS/MS ............................................... 56
4.2.3.3 Kinetic studies at 60 °C and 80 °C and 100 °C in water by HPLC-PAD .......................................... 56
X