APPLICATION OF RADIOIMMUNOASSAY (RIA) FOR THE DETERMINATION OF RESIDUES OF ANABOLIC SEX HORMONES 1

APPLICATION OF RADIOIMMUNOASSAY (RIA) FOR THE DETERMINATION OF RESIDUES OF ANABOLIC SEX HORMONES 1 B. Hoffmann and C. Blietz Institut f~r Veterin~rmedizin des Bundesgesundheitsamtes (Robert yon Ostertag-Institut), D 1OOO Berlin 3 3 Summary Based on the basic principles of radioimmunoassay (RIA), it can be expected that by using this method, residues of anabolic sex hormones in animal tissues can be quantitated in the ng/g to pg/g range with adequate reliability. This was demonstrated for various endogenous and exogenous steroids as well as for stilbeneestrogens. Using the RIA for DES as a regulatory method allowed the successful control of the misuse of this compound in animal production, especially by examination of excreta. Also, for muscular tissue, an interlaboratory validaion study yielded acceptable results. The need for confirmatory methods is stressed. (Key Words: Radioimmunoassay, Residue Analysis, Anabolic Sex Hormones.) I ntroduction Radioimmunoassay (RIA) techniques for the determination of compounds with a low molecular weight, like steroid hormones, have been available for the past decade. Today, millions of assays are performed every year in research and clinical chemistry laboratories. It should be noted that many important and sometimes life saving therapeutic measures are based on data derived from RIA and, recently using a similar assay technique, enzyme-immunoassay (EIA). In general, measurements are performed on body fluids, predominantly blood plasma or serum. Presently, there is no other assay technique that can compete with RIA relative to reliability (i.e., specificity, precision, accuracy, reproducability), sensitivity (quantification in the fmol range) and assay speed. This paper will discuss to what extent these assay criteria are still applicable when Paper presented at the Symposium on "Metabolic Fate of Anabolic Substances in Food Animals" at the meeting of the Amer. Soc. of Anita. Sci., Raleigh, NC, USA, on July 29, 1981. 239 RIA is applied to the determination of residues of anabolic sex hormones in tissues and excreta of food animals. Compounds, Metabolites and Required Assay Sensitivity In table 1 those anabolic compounds and combinations of compounds are listed that have been used most widely or are still in use for fattening of animals in various countries and continents. A distinction has to be made between the endogenous C18-, C19- and C21-steroids (estradiol-17/3, testosterone, progesterone) and the synthetic, nonendogenous steroids trenbolone acetate (TBA), methyltestosterone, metengestrol acetate (MGA) as well as the nonsteroidal exogenous estrogens diethylstilbestrol (DES), hexestrol (HEX), dienestrol (DIEN) and zeranol. Widely varying amounts of endogenous sex steroids occur naturally in food of animal origin (Hoffmann, 1981) in relation to sex, age and reproductive status of the animal. It has been shown (Henricks and Torrence, 1978; Hoffmann, 1978; Reid, 1980) that proper treatment of animals with endogenous sex steroids will not lead to hormone levels in the edible tissues, which at the time of slaughter would exceed the physiological range. Thus the necessity of setting tolerance levels from a toxicological point of view has not arisen. In contrast to this situation, xenobiotic compounds should only be used in animal production, if tolerance levels or an acceptable daily intake (ADD value have been established following toxicological evaluation of the compound. This also requires studying the metabolsim and pharmacokinetics in the target animal species, which will yield information as to whether residue analysis of the parent compound (drug administered) and(or) a metabolite is suitable for drug monitoring purposes. Thus the development of an RIA or any other JOURNAL OF ANIMAL SCIENCE, Vol. 57, No. 1, 1983

240 HOFFMANN AND BLIETZ TABLE I. COMPOSITION OF VARIOUS ANABOLIC PREPARATIONS Estrogenic Biological activity Application in Androgenic Gestagenic animal production 17#-estradiol 17#-estradiol Estradiol benzoate Estradiol benzoate Estradiol monopalmitate 17#-estradiol Diethylstilbestrol (DES) DES DES Hexestrol Dienestroldiacetate Zeranol Zeranol Testosterone Testosteronepropionate Trenbolone acetate (TBA) Trenbolone acetate (TBA) Testosterone Methyltestosterone TBA Progesterone Progesterone Melengestrol acetate (MGA) Veal calf Steer, heifer, lamb Steer, heifer, lamb Poultry Veal calf, steer, heifer Heifer, cow Heifer Steer, heifer, lamb, veal calf Steer, heifer Pig Steer, heifer, lamb Poultry Steer, heifer, lamb, veal calf Steer, heifer, lamb, veal calf nonradiometric method should be based on this knowledge. By neglecting this proper identification of parent compounds and metabolites, errors were made in the past. For example, the antiserum, and hence RIA, available for the determination of TBA and its free form, 17~-trenbolone (Hoffmann and Oettel, 1976), only cross-reacts 15 to 20% with 17c~-trenbolone (Schopper and Hoffmann, 1981), which later was identified as the major extractable metabolite of TBA in liver and kidney of cattle, where highest residue levels are found (Pottier et al., 1980; Schopper, 1981). In no case is RIA or any other method that is based on the extraction of tissues with an organic solvent able to measure the covalently bound nonextractable residues, which by far exceed the extractable ones in the case of TBA (Ryan and Hoffmann, 1978; Schopper, 1981). Regardless of the type of residue to be measured, the reliability of a method has to be established. In this respect, most of the assay problems arising are a function of the required level of sensitivity. As outlined in table 2, an adequate method for monitoring the treatment with endogenous steroids should allow analysis of the physiological tissue concentrations, while methods for residue analysis of xenobiotics should allow a safe estimate of the tolerance levels given. If the use of a pharmacologi- TABLE 2. METHODS FOR RESIDUE ANALYSIS OF ANABOLIC AGENTS: REQUIRED LEVELS OF SENSITIVITY Measurement of: Endogenous steroids Nonendogenous, synthetic compounds (xenobiotics) allowed to be used as animal drugs Compounds prohibited, from use in food animals Required sensitivity Adequate to measure physiological concentrations Adequate to measure residues in one or several tissues based on the ADl-value a Highest sensitivity available aadi = acceptable daily intake.

RESIDUE ANALYSIS OF ANABOLIC SEX HORMONES 241 cally active compound in food animals is prohibited, as it is in the case of DES in most countries, the method used for residue analysis should have the highest sensitivity possible. Methods Available and Assay Specificity The extent to which RIA is available for the measurement of anabolic sex hormones in edible animal tissues is summarized in table 3. Most methods have been published in detail and a review particularly dealing with the reliability has been given elsewhere (Hoffmann, 1978). Thus, only some general remarks on assay specificity, perhaps the most important reliability criterion, will be given. When using RIA, specificity first relates to the quality of the antiserum or antibodies used. The extent to which the antibodies reflect the stereospecificity of the compounds to be measured, largely depends on the type of functional group used to couple the steroid or anabolic agent to a protein; which of course is a necessary step to induce antigenicity (Niswender and Midgley, 1970; Pdron and Caldwell, 1970). As is shown for 17~estradiol in figure 1, an antigen made by coupling this steroid through its 17~hydroxy-group to a protein yielded, after immunization of a rabbit, an antiserum that showed almost identical cross-reactions with 17/3-estradiol, estrone and 17a-estradiol. This type of cross-reactivity was expected, because the only structural difference between these three estrogens is their functional group at carbon 17 (17~-hydroxy; 17-keto; 17r hydroxy group), which was hindered due to the coupling (antiserum I). When using a different approach by not interfering with the structure at carbon 17 and coupling 17~-estradiol to the protein through a keto-group introduced at carbon 6, antisera can be obtained, which exhibit a distinctly lowered cross-reactivity with estrone and 17cx-estradiol (antiserum II). Consequently, it is possible to obtain highly specific antisera and even structure identification has been performed by using various antisera (Kuss et al., 1973). However, assay interference due to the presence of impurities may occur. This phenomenon relates to the binding kinetics of the antibodies. Apparently the problem is negatively correlated to the percentage of high affinity binding sites in the antiserum. Nevertheless, it has been experienced during the past decade, that high quality antisera also require a certain "clean up" procedure of the sample. This procedure may vary depending on the type of tissue examined and the demands on the assay sensitivity, which directly relates to the distribution of the drug within the animal. In general residues of anabolic sex hormones are lowest in muscular tissue and highest in the excreta (Hoffmann and Karg, 1976; Karg and Vogt, 1981). For example, a similar degree of certainty in detecting a treatment with DES is obtained when the assay applied is able to detect concentrations around.02 ng/g in muscle or 5 ng/g in feces (table 4). Also, determination of DES by R1A in concentrations of less than.1 ng/g, requires quite a lengthy "clean up" of the extract (Hoffmann and Laschiitza, 1980; Vogt, 1980), while following a simple extraction, the impurities can more or less be diluted out, when DES ranges above 5 ng/g feces (Agthe, 1980). It should be noted, that in both assays the absolute amounts of DES finally quantified TABLE 3. RAD1OIMMUNOASSAY METHODS AVAILABLE FOR MEASUREMENT OF RESIDUES OF ANABOLIC AGENTS Tissue Hormone Muscle Liver Kidney Fat Reference Estradiol-17/3 + + + Estrone + + + + Testosterone + + + + Progesterone (+) (+) (+) Trenbolone + + + + DES + + + + Hexestrol/dienestrol + + Zeranol In development + + For survey of literature see Hoffmann (1978) Laschiitza and Hoffmann (1979) Hoffmann and Laschiitza (1980) Harwood et al. (1980) Dixon and Heitzman (1981)

242 HOFFMANN AND BLIETZ 100- t"='~="~."~ I~ z z50- X 0 \, b, ', %% % 9 II M I I I 1 ~ I I t I I 1 2 3 ~ 5 1 2 3 ~ 5 STEROID, ng STEROID, ng 9 estrodiol-l"/b; 0 estrone ; = estrodiol-l"/c~ 13 non phenotic steroids I.s-Bs II oh CMO-BSA 17fl- estradioi - 17-17fl-estradio[- 6 - corboxy- hemisuccinate- 8SA methyl- oxime- BSA Figure 1. Cross-reactivity of antisera directed against 170-estradiol-17-hemisuccinate-BSA (I) and 17~-estradiol-6-carboxymethyl-oxime-BSA (1I) (see Hoffmann, 1978). TABLE 4. DETERMINATION OF DIETHYLSTILBESTROL: POSTULATED REQUIRED METHOD SENSITIVITY IN RELATION TO THE BIOLOGICAL MATRIX EXAMINED a Biological matrix - z Required lower levels of sensitivity, ng/g Muscle ~.02 Liver-kidney ~.05-. 10 Urine ~2.0 Feces ~5.O ahoffmann (1980), by RIA are in the same order of magnitude; in general the calibration curve ranges from 10 to 200 pg per tube. Due to this very high final assay sensitivity, the sample size can be limited to a maximum of 1 g tissue and hence a good practical method is still obtained even in the case of longer extraction procedures. Special Experience with the RIA for the Determination of DES Approaches to determine DES by RIA date back to the early seventies. However, in spite of

RESIDUE ANALYSIS OF ANABOLIC SEX HORMONES 243 the need to have an assay available as soon as possible, as was expressed by various regulatory agencies, and the money put into methoddevelopment, no substantial progress was made until 1978; at that time amethod was published anonymously in the Regulations of Meat Inspection Act 2 (Implementing Regulation A) of the Federal Republic of Germany prior to publication in a scientific journal. The fully validated method was not published before 1980 (Hoffmann and Laschiitza, 1980). Retrospectively, it can be said that so far the most successful approach to obtain antisera against DES proved to be the use of DEScarboxypropylether (figure 2), coupled to bovine serum albumin, as an antigen (Rombauts et al., 1973; Laschiitza, 1980). By using the same type of derivative as an antigen, antisera have also been obtained for the determination of hexestrol (Harwood et al., 1980) and dienestrol (A. Jouquey, unpublished data). Since 1981, 3H-DES (tracer used for RIA) and antisera against DES and hexestroi can be purchased in the form of a kit 3. As shown in figures 3 and 4 and table 5, the two available antisera differ markedly in their cross-reactivity between the stilbene-estrogens, while there is no difference in their existing cross-reactivity with respect to the other compounds tested. Obviously, both antisera exhibit a 100% cross-reactivity with the stilbene (DES or hexestrol) used for antigen synthesis. However, while antiserum 254 only cross-reacts 16.5 and 6.4% with hexestrol and dienestrol, respectively, antiserum 6139 cross-reacts 40 to 50% with DES and 16 to 25% with dienestrol (table 5). Hence, it was suggested in a method proposal submitted to the Commission of the European Communities 4 to use antiserum AS 254 for the specific determination of DES and antiserum AS 6139 for analysis of hexestrol and group analysis of stilbene-estrogens. How- 2Bundesgesetzblatt (Federal Register), Part I, Z 1997 A. Neufassung der Ausf(ihrungsbestimmungen A fiber die Untersuchung und gesundheitspolizeiliche Behandlung der Schlachttiere und des Fleisches bei Schlachtungen im Inland. Bonn, 1978. 3 Hoechst AG, Pharma Forschung, D-6230 Frankfurt 80 (Main). 4Commission of the European Communities; Working Document 4694/VI/81-EN, File No. 67.1. Radioimmunoassay (RIA) methods for the measurement of residues of the stilbene derivatives, diethylstilbestrol, hexestrol and dienescrol in tissues and fluids of farm animals. HO ~ " " Figure 2. DES-carboxypropylether-bovine serum albumin. I. Io 00ts o~. Ilntx o(~,~ o1(. sc on '~176 -" a _~ I*1 m............... 1~ 1 1o z 1o ] io; lo 5 StjLSs / st(aojo. P9 Figure 3. Cross-reaction of antiserum directed against DES-carboxypropylether-BSA (AS 254). Assay conditions: dilution (1:80,000), incubation (1 h at 37 C), separation (charcoal.2% in detain, water). Substances tested: HEX (hexestrol), DES (diethylstilbestrod, DIEN (dienestrol), P4 (progesterone), E: (estradio1-173), C-on (corticosterone) and Det (detergent). m,tx 0 lacg 9 oes o [z 9 aetn A C o~ (. os :,o~p~ l '"I "' 8 : 9 t... :. io I io z io 3 ~a ~ io s STaLSEN(/ sr(~o~o, pr Figure 4. Cross-reaction of antiserum directed against HEX-carboxypropylether-BSA (AS 6139; Harwood et al., 1981). Assay conditions: dilution (1: 24,000), incubation (1 h at 37 C), separation (charcoal.2% in detain, water). DES (diethylstilbestrol), DIEN (dienestrol), P4 (progesterone), E 2 (estradiol-17~), C-on (corticosterone) and Det (detergent).

244 HOFFMANN AND BLIETZ ever, contrary to the RIA for the determination of DES and hexestrol, the assay for group analysis of stilbene estrogens has to be further validated and optimized. In this respect it should be noted that massive contaminations with detergent and probably other impurities interfere with the assay (figures 3 and 4). Also the absolute binding as well as the amount of cross-reactivity exhibit some variations, depending on the time of incubation (table 5). As indicated above, the need for an adequate method for the determination of DES-residues in edible animal tissues was expressed by various regulatory agencies, because the use of DES in animal production has been prohibited or strictly regulated in most countries. In 1981, the Commission of the European Communities banned the use of DES and other stilbene estrogens in all member states. Thus, in theory, no residues of DES can be tolerated and the methods to monitor for DES should enable implementation of this philosophy or regulation. Nevertheless, DES has been used illegally on a large scale in beef and veal production. As a result, in some European countries DES courd be detected even in baby food. As the major source of contamination, the high residue concentrations remaining at the site of application over extended periods of time have to be considered (Hoffmann and Karg, 1976; Karg and Vogt, 1981). This was demonstrated in our own recent experiments, which were also performed in order to provide positive and negative control samples for the regulatory agencies within the Federal Republic of Germany. The method outlined in the Regulations of Meat Inspection Act (Implementing Regulation A) of the Federal Republic of Germany (see above), requires an analysis of positive and negative tissue samples with each assay, and the field sample collected can only be estimated as DES positive if there is a significant difference between the negative control assay. As shown in table 6, DES concentrations varied between.06 and 17 ng/g at the injection site (M. triceps brachii), while residue concentrations in the other muscle sites examined were around 70 pg/g, just above the level of detection. However, previous results (Hoffmann and Laschiitza, 1980; Harwood et al., 1980; Karg and Vogt, 1981) had distinctly higher residue levels in liver and kidney, particularly if the conjugated residues were estimated. As described elsewhere (Hoffmann and Oettel, 1976), quantification of conjugated residues is performed by introducing enzymatic hydrolysis of the sample prior to the initial ether extraction. From our present experiences it can be delineated that in order to achieve adequate consumer protection, residue analysis should detect the presence of such a depot at the injection site. Because the probability of sampling an injection site when examining muscular tissue is rather low, the most reliable information can be obtained by examining excreta (urine, feces, bile). This approach has proven to be rather successful in the Federal Republic of Germany, particularly because the TABLE 5. ABSOLUTE BINDING AND CROSS-REACTION OF ANTISERA AGAINST STILBENE-ESTROGENS IN RELATION TO THE TIME OF INCUBATION AND ANTISERUM DILUTION Final Time of Absolute a assay incubation, binding, Cross-reaction (%) Antiserum dilution h % DES Hexestrol Dienestrol 254 6139 1:80,000 1 53.0 100 16.5 6.4 20 58.5 1:128,000 1 40.1 20 57.0 100 11.8 8.6 1:24,000 1 29.3 44.4 100 25.2 20 49.1 54.5 100 1:48,000 1 22.9 20 44.0 41.3 100 16.3 a40 pg 3 H-DES ~.0167 Ci.

RESIDUE ANALYSIS OF ANABOLIC SEX HORMONES 245 TABLE 6. FREE AND CONJUGATED DIETHYLSTILBESTROL (DES) IN TISSUES OF A VEAL CALF 7 DAYS AFTER INTRAMUSCULAR INJECTION OF 200 MG DES IN OILY SOLUTION Tissue Injection site a DES Muscle Liver Kidney "Edge" Range Free 70 233 75 830 60-16.660 Conjugated 32 517 543 48 alnjection given into the M. triceps brachii. About 500 g of tissue were removed at slaughter and segments of 10 to 20 g were examined individually. examination of feces allowed the control of the live animal already at the production unit (Agthe et al., 1979; Agthe, 1980). The effectiveness becomes apparent from unofficial statistics from various Veterinary Control Laboratories that show that within 1 yr following the introduction of this method, the number of veal calves identified as treated with DES dropped from approximately 40% to less than 1.0% (Hoffmann, 1982). Due to the export-import business of carcasses, tissue methods also have to be available and applied. As is shown for muscle in table 7, 2 yr after introduction of the RtA for DES, an inter-laboratory validation yielded quite acceptable results. Finally, it should be stressed that confirmatory methods based on a different technique of TABLE 7. RESULTS (JULY, 1981), INTER-LABORATORY VALIDATION: RADIOIMMUNOASSAY FOR DES IN MUSCULAR TISSUE OF A CALF INJECTED (INTRAMUSCULAR) WITH 150 MG DES- DIPROPRIONATE AS A CRYSTAL SUSPENSION 3 WEEKS BEFORE SLAUGHTER Laboratory No. a pg/gb CV c, % 1 23 170 53 31 2 10 165 58 35 3 3 156 28 17 4 15 174 50 25 5 d 3 157 a Number of duphcates or triplicates. bmean SE. Ccoefficient of variation. dmodified extraction procedure. measurement will be necessary. Problems in this respect particularly concern the determination of the low residue concentrations observed in muscular tissue outside the injection site. However, due to recent progress with gas chromatography-mass-spectrometry (Bergner- Lang and K~ichele, 1981) this gap should soon be closed. Literature Cited Agthe, O. 1980. Die anwendung des radioimmunoassay f(ir Di~ithylstilb6strol auf kotproben von mastk/ilbern. Arch. f. Lebensmittelhyg. 31 : 102. Agthe, O., D. de Frenne and C. M. Sperveslage. 1979. 0her die verbreitung der anwendung yon 6strogenen in der k/ilbermast im raum weser eros. Arch. f. Lebensmittelhyg. 30:67. Bergner-Lang, B. and M. K~chele. 1981. Anabotica in kalbfleisch. Nachweis und bestimmung, befunde und beobachtungen. Deutsche Lebensmittel- Rundschau 77: 305. Dixon, S. N. and R. J. Heitzman. 1981. Residues of exogenous anabolic agents in beef cattle and sheep, in: Anabolic agents in beef and veal production, pp 58-69. Commission of the European Communities. Harwood, D. J., R. J. Heitzman and A. Jouquey. 1980. A radioimmunoassay method for the measurement of residues of the anabolic agent, hexoestrol, in tissues of cattle and sheep. J. Vet. Pharmacol. Therap. 3:245. Henricks, D. M. and A. K. Torrence. 1978. Endogenous estradiol-173 in bovine tissues. J. Assoc. Official Anal. Chem. 61:1280. Hoffmann, B. 1978. Use of radioimmunoassay (RIA) for monitoring hormonal residues in edible animal products. J. Assoc. Official Anal. Chem. 61:1263. Hoffmann, B. 1980. Analytischer stand des 6strogennachweises. In: Proc. 21. Arbeitstagung des Arbeitsgebietes Lebensmittel-hygiene. pp 89-98. DVG, Frankfurter Str. 87, D-6300 Gie~en. Hoffmann, B. 1981. Levels of endogenous anabolic sex hormones in farm animals. In: Anabolic Agents in Beef and Veal Production. pp 96-112. Commission of the European Communities.

246 HOFFMANN AND BLIETZ Hoffmann, B. 1982. Derzeitiger stand des stilbennachweises und gesetzliche grundlagen. Fleischwirtschaft 62:95. Hoffmann, B. and H. Karg. 1976. Metabolic fate of anabolic agents in treated animals and residue levels in their meat. In: F. Coulston and F. Corte (Ed.). Environmental Quality and Safety. Suppl. Vol. 5. Anabolic Agents in Animal Production. pp 181-191.Georg Thieme Verlag, Stuttgart, Germany. Hoffmann, B. and W. Lasch~itza. 1980. Entwicklung eines radioimmunotests zur bestimmung von di~thylstilbisstrol im blutplasma and e~baren geweben vom rind. Arch. f. Lebensmittelhyg. 31:105. Hoffmann, B. and G. Oettel. 1976. Radioimmunoassay for free and conjugated trienbolone and for trienbolone acetate in bovine tissue and plasma samples. Steroids 27: 509. Karg, H. and K. Vogt. 1981. Residues of diethylstilboestrol (DES) in veal calves. In: Anabolic Agents in Beef and Veal Production. pp 70-88. Commission of the European Communities. Kuss, E., R. Goebel and H. Enderle. 1973. Influence of oxo-, and/or hydroxy-groups at C-16/C-17 of estrogens and affinity to anti-estrone-, antiestradiol-17~- and anti-estradiol-17~-antisera. Hoppe-Seyler's Z. Physiol. Chem. 354: 347. Laschiitza, W. 1980. Entwicklung eines radioimmunologischen verfahrens zur bestimmung der anabol wirksamen substanz di~ithylstilb6strol (DES) im blutplasma und in ea3baren geweben vom rind. Dissertation Rer. Nat. Miinchen, Technische Universit/it Miinchen. LaschiJtza, W. and B. Hoffmann. 1979. Radioimmunoassay of diethylstilboestrol (DES) in muscular tissue of calves. Acta Endocrinol. Suppl. 225:98. Niswender, G. D. and A. R. Midlgey, Jr. 1970. Haptenradioimmunoassay for steroid hormons. In: F. G, P~ron and B. V. Caldwell (Ed.). Immunological Methods in Steroid Determination. pp 149--173. Appleton Century Crofts, New York. P~ron, F. G. and B. V. Caldewell. 1970. Immunologic Methods in Steroid Determination. Appleton Century Crofts, New York. Pottier, J., D. Coussediere and R. J. Heitzman. 1980. Levels of 17a and 17~3-trienbolone in tissues and bile of heifers implanted with trenbolone acetate. J. Anita. Sci. 51(Suppl. 1):316. Reid, J.F.S. 1980. Significance of natural estrogenimplanted beef to human health. In: The Use, Residues and Toxicology of Growth Promoters. pp 24-30. An Foras Taluntais, Dublin. Rombauts, P., A. Pierdet and A, Jouquey. 1973. Preparation et propri6ties d'immun-s~rum antidi~tylstilboestrol. C. R. Acad. Sci., Paris. S~r. D. 277:1921. Ryan, J. J. and 13. Hoffmann. 1978. Trenbolone Acetate (TBA): Experiences with bound residues in cattle tissue. J. Assoc. Official Anal. Chem. 61:1274. Schopper, D. 1981. Messungen yon rijckst~inden yon ostradiol- 17~3, testosteron und progesteron im plasma und teilweise im urin yon mastrindern nach anabolikabehandlung sowie untersuchungen zum stoffwechsel yon trenbolonacetat. Dissertation Med. Vet., MiJnchen, Ludwig-Maximilians- Universit~/t. Schopper, D. and 13. Hoffmann. 1981. Identifizierung yon 17c~- Trenbolon als hauptausscheidungsprodukt des trenbolonacetatstoffwechsels beim Kalb und sich daraus ergebende konsequenzen f/jr die riickstandsanalytik. Arch. f. Lebensmittelhyg. 32.:141. Vogt, K. 1980. Vereinfachtes extrakhons-und reiniguhgsverfahren fur die radioimmunologlsche bestimmung yon DES in fleisch, leber und neire. Arch. f. Lebensmittelhyg. 31:138.