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Anabolics: the situation in Belgium G Maghuin-Rogister, P Gaspar, M Vandenbroeck, P Schmitz, G Degand, G Pelzer, J Degraeve

To cite this version:

G Maghuin-Rogister, P Gaspar, M Vandenbroeck, P Schmitz, G Degand, et al.. Anabolics: the situation in Belgium. Annales de Recherches Vétérinaires, INRA Editions, 1991, 22 (3), pp.305-309. ￿hal-00902032￿

HAL Id: hal-00902032 https://hal.archives-ouvertes.fr/hal-00902032 Submitted on 1 Jan 1991

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Anabolics: the situation in Belgium

G Maghuin-Rogister P Gaspar M Vandenbroeck P Schmitz G Degand G Pelzer J Degraeve

1 Laboratoire d Analyse des Denrées Alimentaires d’Origine Animale, Faculté de Médecine Vétérinaire, Université de Liège, Bât 8 42 Sart-Tilman, B-4000 Liège; 2 Laboratoire de Spectrométrie de Masse, Chimie Médicale, CHU, Université de Liège, Bât B 23 Sart-Tilman, B-4000 Liège, Belgium

(27-28 March 1990: International Meeting on Anabolics, Toulouse, France)

Summary ― The illegal use of anabolics in 1989-1990 and its control in Belgium are reviewed. After a short presentation of the Belgian legislation and EEC directives, a recent hormone scandal published by a consumer magazine is related and the composition of 17 hormone cocktails found on the black market is given. The strategy of control involves screening of the main artificial anabolics in by radioimmunoassay (RIA) and confirmation by thin-layer chromatography (TLC). Samples of fat and injection sites are also examined by TLC. In the future, RIA will probably be replaced by en- zyme immunoassay and TLC by mass spectrometry. anabolics I anabolizing agents / hormone residues / immunoassay / control strategy

Résumé ― Hormones anabolisantes : la situation en Belgique. L’utilisation illégale d’hormones anabolisantes en 1989-1990 et son contrôle en Belgique sont passés en revue. Après une brève présentation de la législation belge et des directives CEE, on relate un scandale récent dénoncé par un organisme de défense des consommateurs ainsi que les résultats d’une enquête portant sur l’analyse de 17 fioles d’anabolisants vendues au marché noir. La stratégie du contrôle comporte un criblage des principales hormones anabolisantes artificielles par dosage radio-immunologique (RIA) et une confirmation par chromatographie sur couche mince (CCM). Des échantillons de graisse et des sites d’injection sont aussi soumis à une analyse par CCM. Dans le futur, les RIA seront proba- blement remplacés par des dosages immuno-enzymatiques et la CCM par la spectrométrie de masse. hormone anabolisante / résidu d’hormone / dosage immunochimique / stratégie de contrôle

* Correspondence and reprints INTRODUCTION ban on anabolics for meat production blocked application of the part of this law The consequences of the application of concerning tolerance in favor of natural the Directive (88/146/EEC) prohibiting the sexual hormones and other sub- use of anabolics and taking into account stances having a hormonal action that the limitations of control methods are the have been recognized as safe for the meat following: i), the development of a black consumer. market of hormone cocktails including po- tentially dangerous substances such as synthetic , , progest- EEC ban agens, corticosteroids and (3-adrenergic . ii), As a consequence of this Directives (88/146/EEC) replacing (85/649/ black market, a risk exists for the meat EEC) (1 ), (86/469/EEC) (2) and the Com- consumer to absorb amounts of residues, mission decision (87/410/EEC, 89//EEC) at sites, of hormonal and toxico- injection (3) concern: i), the prohibition of the use in An increased fi- logical significance. iii), livestock farming of certain substances nancial effort towards control due to the having a hormonal action and of substan- use of increasingly sophisticated analytical ces having a thyrostatic action; ii), the con- methods. Limitations of ac- iv), therapeutic trol of hormone and thyrostatic residues in tion in medicine. veterinary v), Disloyal living animals and fresh meat; iii), the between meat competition European pro- methods to be used for residues ducers and livestock farmers of third-world detecting of substances having a hormonal action countries. In fact, control of nude carcass- and of substances having a thyrostatic ac- es to EEC countries is still imported impos- tion. sible in the case of natural hormones and rather difficult for artificial anabolizing agents. RECENT EVENT Our presentation will be limited to the il- legal use of anabolics in Belgium and its control. &dquo;Test-Achats&dquo; (a consumer magazine)

LEGISLATION In January 1990, a consumer magazine, &dquo;Test-Achats&dquo;, published a report about the results obtained after analysis of 81 sam- Belgian legislation ples of beef steaks; 16 were considered positive for the presence of hormone resi- Belgian legislation is based on a law (July dues ( acetate, chlor- 15, 1985) about &dquo;the use of substances madinone acetate, nortestosterone, or having a hormonal or an antihormonal ef- 17p-). Out of the 16, 11 samples fect in animals&dquo;. This law planned excep- contained only the artificial progestagens. tions to the ban concerning substances These results contrasted with those of the that could be used for fattening of meat- official controls performed by the Belgian producing animals. Institut d’Expertise Vétérinaire (IEV) on The decision of the EEC ministers in suspect animals that gave < 10% positive December 1985 regarding the complete results in 1987 and 1988. The &dquo;Test-Achats&dquo; information was The Belgian system of control is more largely diffused by the media. Butchers cit- stringent than in other EEC states: i), it is ed in the &dquo;Test-Achats&dquo; paper criticized the applied to all suspect meat-producing ani- method used and brought an action to mals; ii), urine, fat, injection sites and fecal court, mainly arguing on the grounds that samples are taken instead of urine in other they were not responsible for the presence EEC countries; iii), in 1988 and 1989 the of hormone residues in sold meat and that number of analyses was about twice that they had no opportunity to perform a con- imposed by the EEC. tra-expertise. This affair resulted in a dra- matic decrease in beef meat consumption and even difficulty in international trade of OFFICIAL ANALYTICAL METHODS meat with the Netherlands. Radioimmunoassay (RIA) is applied as a screening method to the urine and fecal CONTROLS samples. Samples identified as positive by RIA are confirmed by high performance In Belgium, controls are performed by 3 thin layer chromatography (HPTLC) (Ver- types of organization: i), IEV for the De- beke, 1979). Fat, injection sites, and fecal partment of Public Health; ii), Department samples are also directly examined using of Agriculture; iii), Private organizations. HPTLC.

The lEV Methods under development

The IEV is responsible for controls in the We have developed enzyme immunoas- slaughterhouses. Meat inspectors (veteri- says (Degand et al, 1989) as a screening method in urine for the narians) collect samples of urine, fat, or in- samples presence of residues of the main artificial anabolics jection sites from suspect animals: veal calves, adult cattle, pigs, sheep. and P-agonists: and relat- ed substances, , nortestos- terone, , and The Department of Agriculture clenbuterol. Some of these assays will soon be commercialized by Techland SA, a Belgian company. Urine and meat sam- The Department of Agriculture is responsi- ples can be analyzed by gas chromatogra- ble for the control of living animals. Its in- phy coupled to mass spectrometry (GC- collect urine or fe- spectors (veterinarians) MS). Rapid and sensitive GC-MS methods cal samples. are in progress at our 2 laboratories to bet- ter satisfy the need for practical control. Private organizations RESULTS OF CONTROL The butchers’ trade union, supermarkets, &dquo;hormone-free&dquo; label organizations collect their own samples and organize their own Black market vials control in collaboration with official or non- official laboratories using official or non- In 1989-1990, an analysis of 17 cocktails official analytical methods. was performed. They contained:

- ethinyl-estradiol + dexamethazone isoni- residues in 1987-1988. Lower positive per- cotinate centages were recorded in 1989-1990.

- dexamethazone isonicotinate

- ethinyl-estradiol + dexamethazone isoni- CONCLUSION cotinate + acetate

- dexamethazone With a ban on anabolic hor- - estradiol benzoate + cortisone acetate complete mones, control is very difficult and expen- - 4-vinyl- esters sive: using immunoassay, it takes at least - estradiol benzoate 6 months to set up an assay and another 6 an - estradiol benzoate + testosterone months to perform inter-lab validation; HPTLC is not sensitive enough and some- - nortestosterone decanoate + testoste- times gives false positive results; high per- rone propionate + ethinyl-estradiol formance mass spectrometry (multiple MS) - ethinyl-estradiol + nortestosterone deca- is required in addition to GC-MS for a rapid noate response to the illegal use of new mole- - + ethinyl-estradiol cules.

- estradiol-benzoate + nortestosterone decanoate + ACKNOWLEDGMENT - estradiol benzoate + testosterone cypi- onate We thank the Institut Beige pour - chlormadinone acetate + testosterone I’Encouragement de la Recherche Scientifique cypionate dans I’Industrie et l’Agriculture (IRSIA) for finan-

- estradiol benzoate cial support generously given to the Laboratoire d’Analyse des Denrées Alimentaires d’Origine - estradiol benzoate + testosterone Animale de la Facult6 de M6decine V6t6rinaire - estradiol + methandienone + methyltes- de l’Universit6 de Liege. tosterone + + (isomer) + pregnenedione + chlor-testosterone ace- tate () + (composition REFERENCES established in collaboration with H De Bra- bander, Faculty of Veterinary Medicine, Degand G, Schmitz P, Maghuin-Rogister G of University Ghent, Belgium). (1989) Enzyme immunoassay screening pro- , were also some- cedure for the synthetic anabolic estrogens times found in black market cocktails and androgens: diethylstilbestrol, nortestoste- (for- and trenbolone in mulae shown in of these sub- rone, methyltestosterone fig 1 Most bovine urine. J Chromatogr 489, 235-243 stances were also identified at injection sites collected in Verbeke R (1979) Sensitive multi-residue meth- slaughterhouses. od for detection of anabolics in urine and in Less than 10% of urine samples were tissues of slaughtered animals. J Chromatogr found positive for the presence of anabolic 177, 69-84