LC-MS Residue Analysis of Antibiotics
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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 Chapter 1 and showed its activity against a broad spectrum of bacteria and proved it to be safe for use in humans. Later many chemically altered semi-synthetic penicillins were developed. The penicillins derive their activity from the 6-aminopenicillinic acid nucleus which is effective against mainly Gram positive bacteria [4,7,8]. Amoxicillin, ampicillin, penicillin G (benzylpenicillin), penicillin V (phenoxymethylpenicillin), cloxacillin, dicloxacillin, oxacillin and nafcillin (figure 1.1) are nowadays registered for veterinary use [9]. Figure 1.1. Molecular structure of the penicillins registered for use in animal practice. 11 The discovery of penicillin led to renewed interest in the search for antibiotic compounds with similar efficacy and safety. In 1956, the first cephalosporin antibiotic, closely related to the penicillins, was isolated from the Acremonium fungus species [10-12]. The six membered dihydrothiazine ring fused with a four membered ß-lactam ring (figure 1.2) is responsible for the biological activity of this group of compounds. Cephalosporins are highly effective antibiotics in the treatment of bacterial infections of the respiratory tract [4,13]. As for the penicillins, many semi-synthetic cephalosporins were developed which are nowadays distinguished in several generations based upon their time of discovery and their range of activity [14]. Cefacetril, cefalonium, cefazolin, cefalexin and cefapirin (all 1st generation), cefoperazone and ceftiofur (3rd generation), and cefquinome (4th generation) are all approved for veterinary use (figure 1.2) [9]. Another ß-lactam group consists