Testing slaughter animals for residues; what is the state of the art? Leendert A. van Ginkel, RIVM, The Netherlands Bruno LeBizec, ENVN LABERCA, France
Healthy Food Source: New Scientist 2003
Healthy food
Safe food
Contaminants and Residues Natural Toxines Veterinary Drugs Industrial Contaminants
Hormonal Growth Promoters
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
One cornerstone: network of laboratories EU-Member States Current and future (NRLs) Community Reference Laboratories European Commission
EU MS now and possible extensions Total 39 Countries EU MS Oct 2006 Acceding MS 2007 Candidate MS Potential candidates EFTA countries
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
Athletes and farmers exchange information on best practices Stanozolol A problem in sport A problem in farming
Biotransformation of (pro) hormones prohormones excretion activation and metabolism
Different approaches for residue testing Using the activity: functional test Using the chemistry: (fysical) chemical test
Functional tests Effect assays : e.g. hormonal effects Mouse utereus weight test for oestrogens Rat seminal vesicle test for androgens
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
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
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
OH CH 3 O CH 3 H 288 H H H
Technique LC-TOF-MS Ion Formation Fragmen tation Trans port Beam shaping 6
From the ion source into the vacuüm chamber
Through the heated capillary to the first stage vacuum chamber
Fragmentation
Focussing the ions and transport to the third stage
Acceleration into the fourth stage
Quad + Slits (Beam shaping) Slits (Beam shaping) 1
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
Mass fragmentogram : mass spectrum RACTO-04 1 (0.060) 100 283.6 Daughters of 302ES+ 2.97e4 283 301 301.8 Mass fragmentgram : mass spectrum % 163.6 fragment fingerprints identify the molecule 163 0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 m/z
A new possibility: detection of steroid-(esters) in hair
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 2 030506011 Sm (SG, 1x1) MRM of 9 Channels EI+ 16.82 100 421.3 > 194.1 391290 3.98e5 Height % 1 030506011 MRM of 9 Channels EI+ 100 446.3 > 301.3 17.62 17.97 5.00e3 16.64 16.70 17.07 17.58 18.33 17.80 18.05 18.23 17.40 18.43 18.69 % 18.78 60 030506011 MRM of 9 Channels EI+ 18.60 100 446.3 > 341.3 17.06 17.18 17.53 6.68e3 17.99 18.03 16.67 16.94 17.32 18.77 17.85 % 17.80 18.16 18.31 50 030506011 MRM of 9 Channels EI+ 100 17.06 17.13 446.3 > 314.3 4.96e3 % 16.89 16.6516.84 17.29 17.38 17.40 17.86 17.81 17.99 18.56 18.43 18.63 61 030506011 MRM of 9 Channels EI+ 100 17.14 446.3 > 356.3 3.53e4 % 17.00 17.19 17.37 18.57 16.68 17.53 17.97 18.22 16.81 18.66 17.80 18.38 18 Time 16.50 17.00 17.50 18.00 18.50 18.73 MT
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 030506002 Sm (SG, 1x1) MRM of 9 Channels EI+ 16.84 100 421.3 > 194.1 583074 5.88e5 Height % 1 030506002 Sm (SG, 1x1) MRM of 9 Channels EI+ 18.36 100 446.3 > 301.3 124279 1.29e5 Height % 3 030506002 Sm (SG, 1x1) MRM of 9 Channels EI+ 18.36 100 446.3 > 341.3 21123 2.49e4 Height % 15 030506002 Sm (SG, 1x1) MRM of 9 Channels EI+ 18.36 100 446.3 > 314.3 7463 1.10e4 Height % 29 030506002 Sm (SG, 1x1) MRM of 9 Channels EI+ 100 446.3 > 356.3 9.06e4 18.36 Height 42781 % 10 pg injected MT 9 16.50 17.00 17.50 18.00 18.50 Time
G A S C H R O M A T O G R A P H Y % 4 0 100 % 0 17.25 esters-281103014 esters-100204021 100 % GC-MS/MS, EI, SRM, kinetic of fixation 1 7.4 3 17.30 17.40 17.50 17.60 17.70 17.80 17.90 18.00 18.10 18.20 18.30 18.40 18.50 18.60 18.70 17.11 M e th ylte s to s te ro n e 1 7.6 0 6 5 0 6 8 bb 19 17.54 17.61 1 7.6 9 17.77 17.84 1 7.4 0 1 7.6 0 1 7.8 0 1 8.0 0 1 8.2 0 1 8.4 0 1 8.6 0 1 8.8 0 1 9.0 0 M e th ylte s to s te ro n e 17.50 1791 bb 17.31 17.68 17.78 446>301 18.19 18.08 1 8.1 6 18.41 1 8.5 1 18.64 F 2 :M R M o f F2:MR M of 6 channels,ei 446.3>301. 2.242e+00 m 17.20 17.40 17.60 17.80 18.00 18.20 18.40 18.60 18.80 19.00 19.20 18.29 18.54 18.63 18.75 Abondance Relative Signal 10.000 9.000 8.000 7.000 6.000 5.000 4.000 3.000 2.000 1.000 18.89 D84 18.98 19.05 19.15 18 y = 0.1798x R 2 = 0.9987 0.000 0 5 10 15 20 25 30 35 40 45 50 1.2 ppb D2 Concentration (ppb) 34.6 ppb BEFORE IM DAY + 2 34.6 ppb DAY + 84 1.2 ppb
Somatotropine (growth hormone) analysis PST 190 amino acids / Two disulphide bonds 53-164,181-189 Mw 21731 Estradiol Mw 272,4
Somatotropine (growth hormone) analysis Dia-filtration: vivaspin devices Immuno affinity Chromatography
Somatotropine (growth hormone) analysis Serum 10 microgr/ml LC-MS/MS separation on mass difference between rpst and PST
Other new possibilities Discriminating natural from synthetic steroids (GC-C-MS) Measuring conjugated steroids with LC- MSMS
Testing samples for their biological (hormonal) effect can also detect unkown compounds.
Modern physical chemical methods can detect and confirm the presence of analytes beyond reasonable doubt
The misuse of natural hormones can be detected using GC- C-IRMS
Analysing samples of hair greatly improves the possibilities of detecting abuse of hormones
Cooperation between scientist and using the NRL- CRL network for residue testing contribute to the safety of our food