PITFALLS, METHODS and CRITERIA in the ANALYSIS of RESIDUES and CONTAMINANTS in BIOLOGICAL MATERIAL
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UNIVERSITEIT GENT PITFALLS, METHODS and CRITERIA in the ANALYSIS of RESIDUES and CONTAMINANTS in BIOLOGICAL MATERIAL H. DE BRABANDER Thesis submitted in fulfilment of the requirements for the degree of Doctor (Ph.D) in Veterinary Sciences Promotor: prof.dr. A. De Leenheer Dean: prof.dr. A. de Kruif _____ Faculteit Diergeneeskunde VETERINAIRE VOLKSGEZONDHEID & VOEDSELVEILIGHEID Laboratorium voor Chemische Analyse Salisburylaan,133 B-9820 Merelbeke Copyright The author gives the authorisation to consult and to copy parts of this work for personal use only. Any other use is limited by the Laws of Copyright. Permission to reproduce any material contained in this work should be obtained from the author. ISBN 90-5864-043-4 CONTENTS CHAPTER I: INTRODUCTION and AIM.......................................................................................1 CHAPTER II: PITFALLS in RESIDUE ANALYSIS ........................................................................7 2.1. PITFALLS in MASS SPECTROMETRY .......................................................................7 2.2. PITFALLS through IMPURITIES IN CHEMICALS...................................................21 2.3. PITFALLS due to ENDOGENIC SUBSTANCES .......................................................33 CHAPTER III: METHODS for RESIDUE ANALYSIS..................................................................49 3.1.THIN LAYER CHROMATOGRAPHY .........................................................................51 3.2. USING GC-MS ...........................................................................................................89 THYREOSTATS ...............................................................................................................101 3.3. DETERMINATION OF THYREOSTATIC DRUGS by TLC/GC-MS .......................103 3.4. DETERMINATION OF THYREOSTATS by GC-MS................................................119 3.5. HPTLC of ANABOLICS in MEAT and KIDNEY Fat ...............................................131 3.6. ANABOLICS IN KIDNEY FAT : FROM TLC to GC-MS.........................................145 3.7. ENHANCING SPECIFICITY BY HPLC CLEAN-UP...............................................155 3.8. GC-MS of ANABOLICS IN injection sites................................................................169 3.9. COMPARISON OF GC-MS and GC-MS2 ................................................................179 CHAPTER IV: CRITERIA IN RESIDUE ANALYSIS ..................................................................199 4.1. QUALIFICATION or QUANTIFICATION?.............................................................199 4.2. IDENTIFICATION POINTS.....................................................................................209 CHAPTER V: GENERAL DISCUSSION ....................................................................................219 SUMMARY ..................................................................................................................................223 SAMENVATTING........................................................................................................................226 DANKWOORD............................................................................................................................231 i ABBREVIATIONS ADD 1,4-androstadiene-3,17-dione a.m.u. atomic mass unit analyte substance to be detected And Androsterone CCα Decision limit (2002/657/EC) CCβ Detection capability (2002/657/EC) EGAs Estrogens, gestagens and androgens EI Electron ionisation mode FAVV Federaal Agentschap voor de Veiligheid van de Voedselketen g gram GC Gas chromatography GC-MS Gas chromatography - mass spectrometry GC-MSn Gas chromatography - tandem mass spectrometry HPTLC High performance liquid chromatography I.D. Internal diameter IP Identification point (2002/657/EC) LC-MS Liquid chromatography - mass spectrometry LC-MSn Liquid chromatography - multiple mass spectrometry LOD Limit of detection LR-MS Low-resolution mass spectrometry m meter M Mole mg milligram (10-3 g) min minute ml milliliter (10-3 l) mm millimeter (10-3 m) µm micrometer (10-6 m) µl microliter (10-6 l) MRL Maximum Residue Limit MRPL Minimum Required Performance Limit (2002/657/EC) MS Mass Spectrometry MSTFA N-Methyl-N-(trimethylsilyl)-trifluoroacetamide iii m/z Mass-to-charge ratio ng nanogram (10-9 gram) OD Optical density pg picogram (10-12 gram) PL Permissible level (2002/657/EC) ppb parts per billion (µg/kg) ppm parts per million (mg/kg) ppt parts per trillion (ng/kg) SPE Solid phase extraction SIM Selected ion monitoring S/N Signal-to-noise ratio TLC Thin Layer Chromatography TMS Trimethylsilyl iv INTRODUCTION CHAPTER I: INTRODUCTION AND AIM A question to be asked first is WHY???? Why should somebody at a blessed age (57 years old) after a doctorate (PhD) in chemistry and a “aggregaat hoger onderwijs in de analytische chemie van eetwaren” (some kind of second PhD in analytical chemistry of food), go for a doctorate again? The reasons are multiple: first of all I want to express my affinity with the Faculty of Veterinary Medicine of Ghent University. Indeed it was the Faculty of Veterinary Medicine who offered me a job after my first doctorate in chemistry. With its ups and downs I was able to have a full career within the Faculty from assistent to full professor. I was able to make many friends from the Faculty staff, from relations of the Faculty and the laboratory with related services. Especially the cooperation with the inspection services (Institute of Veterinary Inspection, the ministry of Agriculture; now together in the FAVV (Federal Agency for the Safety of the Food Chain) was very strong. A lot of veterinary inspectors were closely encountered during the specialised course in veterinary food inspection. Secondly, our rector, Andreas De Leenheer, is well known to be very research-minded. Strong impulses were given to encourage research at our University. However, scientific research is often measured in terms of number of doctorates only. For older post-docs, like me, this may give the impression that the research period is over after the doctorate - except for guiding other doctorates of course. The best way to prove that there is still research after a doctorate is making a doctorate again, this time in veterinary sciences. The current regulations in Flanders allow or did not forbid multiple doctorates. Of course this will not be the doctorate of a young researcher, dedicated to one apparatus or a single subject over a limited period of time (4 – 6 years). It will more be like the long time work of a manager, responsible for the point of view and the final results of a laboratory. During the period covered (1990- 2003) the Laboratory of Chemical Analysis (LCA) of the department of Veterinary Food Inspection has grown from a small HPTLC unit to a laboratory using 5 modern MSn instruments coupled to the neccessary clean-up procedures. Subsequently, the staff has grown from 4 to 16 people, all occupied with education, research and/or service to the community at a University level. The department is accredited under the ISO 17025 with - for LCA - a very flexible scope in analysis of residues and contaminants. A trace is a substance, component, or analyte present in a small concentration in another material called matrix. There is no general agreement on the concentration level of a trace. 1 CHAPTER I With increasing detection power of the analytical techniques this “trace” border continuously shifts to lower concentrations. In most cases the concentration range is expressed in ppm (µg/g) or ppb (ng/g) units or even lower (ppt (pg/g)). A residue may be defined as a trace of a substance, which remains in a matrix after some kind of administration. Some examples are: an animal is injected with methyltestosterone. In meat residues of methyltestosterone may be detected. Blood contains traces of testosterone (no residues of testosterone). The formation of residues may be as well due to the production as to the manufacturing process. In most cases humans are the basic cause of residues and contaminants. Residues may be divided in different groups in different ways. A simplified approach is given below. A. Banned substances such as “hormones” (group A substances) B. Veterinary Medicinal products such as antibiotics (group B substances) C. Contaminants such as PCBs and dioxins (environmental contaminants) Banned substances are mostly indicated by the word "hormones". This is too big a simplification. The growth promotors abused in animal fattening may be divided into four major groups : Thyreostats (e.g. methylthiouracil) may cause a considerable weight gain in a short time. However that weight gain consists mainly of increased filling of the gastro-intestinal tract and increased water retention in the animal. Anabolics or anabolic steroids (e.g. Diethylstilbestrol, nortestosterone) increase weight gain and carcass quality. These substances are also called EGA’s (Estrogens, Gestagens and Androgens). Beta-agonists or repartitioning agents (e.g. clenbuterol) cause also weigth gain in most slaughter animals. Moreover, the amount of muscle tissue (meat) increases and the amount of fatty tissue decreases. Corticosteroids (e.g. dexamethasone) cause an extra weight gain most probably caused by water retention. 2 INTRODUCTION AND AIM Veterinary Medicinal Products (VMP’s) on the other hand are necessary for the treatment of animals in a veterinary practice. A number of these VMP’s are extensively tested for food safety. On the website of EMEA (the European