Testing slaughter animals for residues; what is the state of the art?

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1 Testing slaughter animals for residues; what is the state of the art? Leendert A. van Ginkel, RIVM, The Netherlands Bruno LeBizec, ENVN LABERCA, France

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4 Healthy Food Source: New Scientist 2003

5 Healthy food

6 Safe food

7 Contaminants and Residues Natural Toxines Veterinary Drugs Industrial Contaminants

8 Hormonal Growth Promoters

9 Council Directive 96/23 ANNEX I GROUP A Substances having anabolic effect and unauthorized substances 1 - Stilbens, stilben derivatives, and their salts and esters 2 - Thyrostats 3 - Steroids 4 - Resorcylic acid lactones including zeranol 5 - β-agonists 6 - Compounds included in Annex IV of EC n 2377/90 GROUP B Veterinary drugs and contaminants 96/23

10 One cornerstone: network of laboratories EU-Member States Current and future (NRLs) Community Reference Laboratories European Commission

11 EU MS now and possible extensions Total 39 Countries EU MS Oct 2006 Acceding MS 2007 Candidate MS Potential candidates EFTA countries

12 Growth promoting molecules DEMETHYLATION Anabolic activity is increased ESTERIFICATION Activity is prolonged DESHYDROGENATION Masculinising activity is decreased SUBSTITUTION Anabolic activity is increased ALKYLATION Oral activity is incr FLUOROACYLATION Anabolic activity is increased SUBSTITUTION Anabolic activity is increased REDUCTION Masculinising activity OH is increased O CH 3 O HO OH

13 Athletes and farmers exchange information on best practices Stanozolol A problem in sport A problem in farming

14 Biotransformation of (pro) hormones prohormones excretion activation and metabolism

15 Different approaches for residue testing Using the activity: functional test Using the chemistry: (fysical) chemical test

16 Functional tests Effect assays : e.g. hormonal effects Mouse utereus weight test for oestrogens Rat seminal vesicle test for androgens

17 The Estrogen Responsive - Chemically Activated LUciferase expression Light (ER CALUX ) assay Add substrate (luciferine) ER-CALUX uses a genetically modified T47D human breast adenocarcinoma cell expressing an endogenous estrogen receptor. Ligand Ligand binding Transport protein Chemical receptor Hsp Proteins Enzymes Luciferase Chemical Responsive Element (CRE) Transcription Nucleus Cytosol

18 Hormonal Effect Assay Advantages: * total estrogenic effect * sensitive and rapid * high sample throughput * also unknown compounds Disadvantages: * no absolute amount per substance * endocrine disruptors also measured * biological effect, no identification individual compounds

19 Chemical test Use physical chemical properties of molecules to determine the identity and concentration Several possibilities, but in residue analyses Mass measurements are the most important

20 OH CH 3 O CH 3 H 288 H H H

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22 Technique LC-TOF-MS Ion Formation Fragmen tation Trans port Beam shaping 6

23 From the ion source into the vacuüm chamber

24 Through the heated capillary to the first stage vacuum chamber

25 Fragmentation

26 Focussing the ions and transport to the third stage

27 Acceleration into the fourth stage

28 Quad + Slits (Beam shaping) Slits (Beam shaping) 1

29 Into the Flight Tube (Separation/Detection) ions travel through the flight tube, which is about 1 meter of length. At the opposite end of the flight tube is an ion an ion mirror, which reflects the ions that arrive to the detector Ion Mirror Flight Tube 1

30 Mass fragmentogram : mass spectrum RACTO-04 1 (0.060) Daughters of 302ES+ 2.97e Mass fragmentgram : mass spectrum % fragment fingerprints identify the molecule m/z

31 A new possibility: detection of steroid-(esters) in hair

32 G A S C H R O M A T O G R A P H Y METHYLTESTOSTERONE. Long term detection in hair. NT-d 3 GC-MS/MS, EI, SRM, compliant hair sample blanc poil Sm (SG, 1x1) MRM of 9 Channels EI > e5 Height % MRM of 9 Channels EI > e % MRM of 9 Channels EI > e % MRM of 9 Channels EI > e3 % MRM of 9 Channels EI > e4 % Time MT

33 Case study 3 = METHYLTESTOSTERONE. Long term detection in hair. G A S C H R O M A T O G R A P H Y NT-d 3 GC-MS/MS, EI, SRM, 1 ng.g -1 in hair ajout 1 ppb Sm (SG, 1x1) MRM of 9 Channels EI > e5 Height % Sm (SG, 1x1) MRM of 9 Channels EI > e5 Height % Sm (SG, 1x1) MRM of 9 Channels EI > e4 Height % Sm (SG, 1x1) MRM of 9 Channels EI > e4 Height % Sm (SG, 1x1) MRM of 9 Channels EI > e Height % 10 pg injected MT Time

34 G A S C H R O M A T O G R A P H Y % % esters esters % GC-MS/MS, EI, SRM, kinetic of fixation M e th ylte s to s te ro n e bb M e th ylte s to s te ro n e bb > F 2 :M R M o f F2:MR M of 6 channels,ei 446.3> e+00 m Abondance Relative Signal D y = x R 2 = ppb D2 Concentration (ppb) 34.6 ppb BEFORE IM DAY ppb DAY ppb

35 Somatotropine (growth hormone) analysis PST 190 amino acids / Two disulphide bonds , Mw Estradiol Mw 272,4

36 Somatotropine (growth hormone) analysis Dia-filtration: vivaspin devices Immuno affinity Chromatography

37 Somatotropine (growth hormone) analysis Serum 10 microgr/ml LC-MS/MS separation on mass difference between rpst and PST

38 Other new possibilities Discriminating natural from synthetic steroids (GC-C-MS) Measuring conjugated steroids with LC- MSMS

39 Testing samples for their biological (hormonal) effect can also detect unkown compounds.

40 Modern physical chemical methods can detect and confirm the presence of analytes beyond reasonable doubt

41 The misuse of natural hormones can be detected using GC- C-IRMS

42 Analysing samples of hair greatly improves the possibilities of detecting abuse of hormones

43 Cooperation between scientist and using the NRL- CRL network for residue testing contribute to the safety of our food