LC-MS residue analysis of antibiotics, what - Wageningen UR

352 Pages·2013·8.65 MB·English

LC-MS residue analysis of antibiotics
What selectivity is adequate?

Bjorn J.A. Berendsen
Thesis committee
Promotor
Prof. dr. M.W.F. Nielen
Professor of Analytical Chemistry, with special emphasis for the detection of
chemical food contaminants
Wageningen University
Co-promotor
Dr. A.A.M. Stolker
Business Unit manager, veterinary drugs
RIKILT, Wageningen UR
Other Members
Prof. dr. A.H. Velders, Wageningen University
Prof. dr. D.G. Kennedy, Agri-Food and Biosciences Institute, Belfast, UK
Prof. dr. W.M.A. Niessen, VU University, Amsterdam
Dr. H. van den Bijgaart, Qlip N.V., Zutphen

This research was conducted under the auspices of the Graduate School VLAG
(Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health
Sciences).
LC-MS residue analysis of antibiotics
What selectivity is adequate?

Bjorn J.A. Berendsen

Thesis
submitted in fulfillment of the requirements for the degree of doctor
at Wageningen University
by the authority of the Rector magnificus
Prof. Dr. M.J. Kropff,
In the presence of the
Thesis Committee appointed by the Academic Board
to be defended in public
on Friday 14 June 2013
at 11 a.m. in the Aula.
Bjorn J.A. Berendsen
LC-MS residue analysis of antibiotics, what selectivity is adequate?

352 pages
PhD thesis, Wageningen University, Wageningen, NL (2013)
With references, with summaries in Dutch and English

ISBN 978-94-6173-569-0
Abstract
In residue analysis of antibiotics quantitative and qualitative aspects are involved
in declaring a sample non-compliant. The quantitative aspect regards the
determination of the amount of the compound present in the sample. Validation
procedures are available to determine the uncertainty of this result, which is
taken into account in the decision making process. The qualitative aspect regards
the confirmation of the identity of the compound present. In this, selectivity is
the main parameter which is defined as the ability of a method to discriminate
the analyte being measured from other substances. A trend observed in residue
analysis is towards more generic methods for the detection of a broad range of
compounds in a single run. As a result, by definition, selectivity is compromised.
Procedures to determine the uncertainty of the qualitative aspect are lacking
and, as a result, whether or not a method is adequately selective is a matter of
experts’ judgment.
In this thesis a method is presented for grading selectivity of methods using liquid
chromatography coupled to tandem mass spectrometry. Based on the outcome it
can be stated if selectivity is adequate and thus if a confirmatory result stands
strong when challenged in a court case. If selectivity is found inadequate,
additional measures can be taken like the selection of another product ion or the
use of a third product ion to obtain adequate selectivity.
Furthermore, two examples of analyses are presented in which selectivity plays
an important role. First, the analysis of the banned antibiotic chloramphenicol
(CAP). CAP contains two chiral centers and the nitro-group can either be para- or
meta-substituted. Therefore, eight different isomers of CAP occur of which only
RR-p-CAP is antimicrobially active. In the analysis of CAP, extreme selectivity is
needed to distinguish the antimicrobially active compound from its inactive
isomers. A method applying chiral liquid chromatography with tandem mass
spectrometry was developed to discriminate antimicrobially active CAP for its
inactive isomers. Also the research for the possible natural occurrence of this
drug is presented. It is shown that CAP can be produced in unamended soil by
Streptomyces venezuelae in appreciable amounts and that crops can take up CAP
from soils. Therefore, it is concluded that CAP can occur in crops and animal
feed due to its natural production by soil bacteria.
Second, the development of a multi-ß-lactam method is presented. In this
method a derivatization is applied to be able to effectively detect off-label
ceftiofur use. In this selectivity is intentionally compromised and no unequivocal
confirmation can be carried out using this method. The developed method is
applicable to a wide range of ß-lactam antibiotics including penicillins,
cephalosporins and carbapenems and is the best method available today for
effective monitoring of off-label ß-lactam usage in poultry breeding.
Table of contents
Chapter 1

General introduction 9
Chapter 2
Selectivity in the sample preparation for the analysis of drug residues in 55
products of animal origin using liquid chromatography coupled to mass
spectrometry
Chapter 3
The (un)certainty of selectivity in liquid chromatography coupled to 81
tandem mass spectrometry
Chapter 4
The analysis of chloramphenicol 113
4.1. General introduction on chloramphenicol analysis 114
4.2. Evidence of the natural occurrence of the banned antibiotic 119
chloramphenicol in crops and soil
4.3. Discrimination of eight chloramphenicol isomer by liquid 134
chromatography tandem mass spectrometry in order to investigate
the natural occurrence of chloramphenicol
4.4. Quantitative trace analysis of eight chloramphenicol isomers in 152
urine by chiral liquid chromatography coupled to tandem mass
spectrometry
4.5. The occurrence of chloramphenicol in crops through the natural 173
production by bacteria in soil
Chapter 5
The analysis of ß-lactam antibiotics 193
5.1. General introduction on ß-lactam antibiotic analysis 194
5.2. Newly identified degradation products of ceftiofur and cefapirin 202
impact the analytical approach for the quantitative analysis of kidney
5.3. Assessment of liquid chromatography tandem mass spectrometry 230
approaches for the analysis of ceftiofur metabolites in poultry muscle
5.4. Comprehensive analysis of ß-lactam antibiotics including 248
penicillins, cephalosporins and carbapenems in poultry muscle using
liquid chromatography coupled to tandem mass spectrometry
Chapter 6
General conclusions and future perspectives 287

Summary 325
Samenvatting 332
Dankwoord 339
About the author 345

Introduction
In animal breeding the use of antibiotics has become common practice.
Antibiotics are used to treat bacterially infected animals but are also
administered as a preventive measure. From an animal and human health
perspective, responsible use of antibiotics is of importance and therefore
extensive monitoring programs are in place within the European Union. The
methods used for analysis of antibiotics aim for the detection, quantitation and
confirmation of the antibiotic present in products of animal origin. In this
chapter the use of antibiotics and its drawbacks are discussed followed by a
description of the legal framework for antibiotic analysis and the methods
employed. Finally some challenges are discussed that are subject of this thesis.
Antibiotics and their veterinary usage
Antibiotics
Antibiotics are used to treat infections caused by bacteria and other micro-
organisms. Traditionally, the term “antibiotics” is used to describe any substance
produced by a micro-organism that is effective against the growth of another
micro-organism [1]. Nowadays the term “antibiotics” is used interchangeably
with the term “antibacterials”, and includes synthetic substances like
sulfonamides and quinolones as well.
Definition:
“Antibiotic ► noun, a medicine (such as penicillin or its derivatives) that
inhibits the growth of or destroys micro-organisms.” [2]
In 1928, Alexander Flemming discovered the antibiotic action of the fungus of the
genus 'Penicillium' and attributed it to one of its constituents: penicillin [3,4]. In
1932, the first antibiotic substance, developed by Gerhard Domagk, became
commercially available: prontosil. This was the first commercially available
synthetic antibiotic belonging to the sulfonamide group and has a broad activity
against Gram-positive bacteria, but not against enterobacteria (Gram negative)
[5]. In 1939, Howard Florey and Ernst Chain continued the study on penicillin [6]
10