University of Groningen Multi-Residue Analysis of Growth Promotors

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Multi-residue analysis of growth promotors in food-producing animals Koole, Anneke

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Download date: 01-10-2021 CHAPTER 1 INTRODUCTION

This chapter gives an overview of the subjects dealt with in this thesis. First, the history and the relevant legislation are discussed (Chapter 1.1). Next, different ways to promote growth are presented (Chapter 1.2). For the anabolic steroids (Chapter 1.2.1) and the ß- agonists (Chapter 1.2.2) the abused substances, their metabolism and available methods of analysis are discussed. Other ways to promote growth are shortly presented in Chapter 1.2.3. An introduction to Systematic Toxicological Analysis is given in Chapter 1.3. The aims, scope and overview of the rest of this thesis can be found in Chapter 1.4.

1 CHAPTER 1.1 HISTORY AND LEGISLATION

Situation in the European Union Agriculture, and in particular animal husbandry, is an important economic activity. Current practices are designed to maximize the yield at minimum costs. To achieve this, additives are incorporated into animal feeds, such as vitamins, trace elements and drugs. Medicinal compounds can be added to the feed for three main purposes: therapy, prevention of disease and growth promotion. Because residues of growth promoters and other drugs in meat may be harmful for the consumer, their use is controlled by law. In The Netherlands this control is now largely governed by European Union (EU) legislation. Growth promoters were banned here in 1961 and this example was followed in 1988 by the European Union. The most important EU directives with respect to residues of veterinary drugs and their control are given in Table 1. The oldest directive, specifying the additives that can be used in animal feeds and at which levels, dates from 1970. This directive is still being amended and updated. After numerous residue scandals the use of some growth promoters was prohibited in 1981. This was followed by a ban on the use of all hormonal substances for fattening purposes in 1985. Due to a procedural error this directive had to be reaffirmed and only became effective in 1988. Most of the other documents that appeared since 1985 deal with the control of the ban. They concern the criteria for the methods of analysis and the laboratories that execute them. In 1992 the first edition of the EU reference manual with accepted methods of analysis appeared. The directives concerning residues are revised and revoked every few years to keep them up to date [1-3]. The latest directives, 96/22/EU [4] and 96/23/EU [5], became fully effective on July 1, 1997. The first directive gives details of the substances banned and the second directive regulates the control measures. It is forbidden to trade stilbenes and ß-agonists and to feed those substances to food-producing animals. It is also not allowed to feed farm animals and fish with thyreo-statics, oestrogens, androgens, gestagens and ß-agonists or to trade meat from animals treated with those substances. However, esters of the natural steroids can be used for therapeutic purposes as intramuscular injections. Also, ß-agonists and allyltrenbolone can be

3 Chapter 1.1

Table 1. Overview of European Union legislation (CRL: community reference laboratory, NRL: national reference laboratory) (updated and modified from [2,3]) year event EU-document 1970 additives allowed in animal feeding stuffs 70/524/EEC 1980-1985 numerous debates and working and expert groups lots of documents 1981 thyreostatic and anabolic substances banned 81/602/EEC 1985 all hormonal substances banned 85/649/EEC 1986 regulations 86/469/EEC 1987 criteria routine methods organic residues 87/410/EEC 1988 annual residue monitoring programme national documents re-affirmation of 85/649/EEC 88/146/EEC 1989 CRL: powers and mandates 89/187/EEC criteria and NRL 89/610/EEC 1991 four CRLs designated 91/644/EEC 1992 EC reference manual EUR 14126 EN 1993 revoking of 87/410/EEC 93/256/EEC revoking of 89/610/EEC 93/257/EEC 1994 EC reference manual second edition EUR 15127 EN 1994-1995 revision of 86/469/EEC COM(93)441def 1996 revoking of 85/649/EEC 96/22/EU revoking of 86/469/EEC 96/23/EU used orally in horses and pets. Injections of ß-agonists are allowed for tocolysis in calving cows. In all those cases animals or meat of the animals cannot be traded for a specified period of time (withdrawal time) with the exception of expensive horses [4]. In directive 96/23/EU measures are specified to control the ban. The control should be performed by special, dedicated institutes [5]. Analysis of the samples taken is performed by routine or field laboratories (RFLs). In each member state the RFLs are coordinated and controlled by at least one national reference laboratory (NRL) designated by the national government. Finally, the NRLs are supported, advised and controlled by four community reference laboratories (CRLs), which were designated in 1991 by the EU and implemented in 1993 [6]. Annually a residue monitoring programme must be made in which the results of the controls of the previous year and the targets for control in the new year are given. People working on the farms and veterinarians are made co-responsible for the control of the ban. Samples can be taken at production plants for banned substances and animal feeds, and at

4 History and Legislation farms, slaughterhouses and butchers. There should be at least one national reference laboratory for every banned substance. Indications should be available for sampling and analysis. The main sanction on the use of banned substances is the destruction of the positive animals. The farmer has to pay for the additional controls that are performed. When meat is imported from third countries, it must also be controlled. When the products give a positive result the European Commission should be informed and additional controls are indicated. Eventually this could lead to a ban on the import from a certain country [5].

Situation in Other Countries Whereas the EU has banned the use of all hormones, other countries do allow the use of some hormones as growth promoters. For example, in the United States, Canada, Australia and New Zealand the natural hormones - testosterone, 17ß-oestradiol and progesterone - and the (semi-)synthetic hormones trenbolone, zeranol and melengestrol acetate can be used to promote growth. Preparations registered for this purpose by the American Food and Drug Administration (FDA) are listed in Table 2. The hormone ban of the EU is currently the cause of a dispute between the EU and these third countries, led by the United States and Canada. The World Trade Organisation (WTO) is trying to settle it. The reason for this dispute is that those countries want to export meat to EU nations from animals treated with in their view acceptable hormones. In their opinion the EU blocks international trade on improper grounds and against international law. The conclusions of an expert panel [7] and the first appellate body [8] were that the hormone ban of the EU is not based on a proper risk assessment conducted in accordance with WTO rules. The EU has announced that it will implement the WTO ruling and that it will carry out an additional risk assessment taking into account the conclusions of the report of the appellate body [9].

Criteria for Analytical Methods Criteria for the analytical methods used for the control of the hormone ban in the EU have been laid down in Commission Decision 93/256/EEC [10]. Analytical techniques that can be used for routine analyses are: immunoassay, thin layer chromatography (TLC), liquid chromatography (LC), gas chromatography (GC), mass spectrometry (MS), spectrometry and other methods that fulfil the criteria given in the appendix of the decision. Also, rules are given for the sampling procedure. Samples need to be representative and must be large enough to allow analysis, repeated analysis and confirmatory analysis. They should be labelled so that they can be properly identified. They should be put in a container in such circumstances that they are protected during transport and storage. Also, unauthorised people should not have access to the samples [10].

5 Chapter 1.1

Table 2. Preparations registered for use as growth promoter in the United States by the Food and Drug Administration [source Freedom of Information Summaries, FDA, as obtained from the FDA Internet site (1998)] trade name manufacturer in animal form no of anabolic dose registration pellets (mg) CALF-oid Ivy Laboratories calf sc 4 P 100 Inc E2Benz 10 ComponentTM Ivy Laboratories steer sc 6 TbAc 120 TE-S Inc E2 24 ComponentTM Ivy Laboratories steer sc 7 TbAc 140 T-S Inc ComponentTM Ivy Laboratories heifer sc 10 TbAc 200 T-H Inc Compudose 200 Elanco Animal beef sc E2 25.7* Health cattle, calf Compudose 400 Elanco Animal beef sc E2 43.9** Health cattle, calf Finaplix® Roussel-Uclaf steer sc 7 TbAc 140 heifer sc 10 TbAc 200 HEIFER-oid Ivy Laboratories heifer sc TProp 200 Inc E2Benz 20 IMPLUS-C® Ivy Laboratories calf sc 4 P 100 Inc E2Benz 10 MGA®100 The Upjohn heifer oral MGA 0.25- premix*** Company 0.5 MGA®200 The Upjohn heifer oral MGA 0.25- premix*** Company 0.5 MGA®500 The Upjohn heifer oral MGA 0.25- liquid premix*** Company 0.5 RALGRO® Pitman-Moore calf sc 3 Zer 36 Inc RALGRO® Mallinckrodt steer sc 6 Zer 72 MAGNUM Veterinary Inc

6 History and Legislation

Table 2 (continued) trade name manufacturer in animal form no of anabolic dose registration pellets (mg) REVALOR®-G Hoechst-Roussel pasture sc 2 TbAc 40 Agri-Vet cattle Company E2 8 REVALOR®-H Hoechst-Roussel heifer sc 7 TbAc 140 Agri-Vet Company E2 14 REVALOR®-S Hoechst-Roussel steer sc 6 TbAc 120 Agri-Vet Company E2 24 STEER-oid Ivy Laboratories steer sc P 200 Inc E2Benz 20 Synovex-C Fort Dodge calf sc P 200 Animal Health E2Benz 20 Synovex®-H Syntex Inc heifer sc 8 TProp 200 E2Benz 20 Synovex® Plus Syntex Animal steer sc 8 TbAc 200 Health E2Benz 28 Synovex®-S Syntex Animal steer sc 8 P 200 Health E2Benz 20 * dose for 200 days; ** dose for 400 days; *** MGA can be added to the feed in combination with lasalocid (BOVATEC®) and tylosin (TYLAN®) and is dosed in mg/head/day explanation of abbreviations: sc: subcutanous ear implant, E2: 17ß-oestradiol, E2Benz: 17ß-oestradiol benzoate, P: progesterone, TbAc: trenbolone acetate, TProp: testosterone propionate, MGA: melengestrol acetate, Zer: zeranol

According to the directive, analytical results should be reported as ‘positive’ or ‘negative’. A sample is declared ‘positive’, when the presence and identity of the analyte are proven without doubt for a substance with zero-tolerance, or when the concentration of the analyte exceeds the maximum residue limit (MRL) for that analyte. When a sample is declared ‘negative’ this can either mean that the presence and identity of the analyte can not be proven without doubt for substances with zero-tolerance, or that the concentration of the analyte is below the MRL for that analyte [10]. For substances with a zero-tolerance, in principle,

7 Chapter 1.1 qualitative methods can be used. However, usually no legal action is taken unless the concentration of the analyte is above the action limit set by the inspection and the laboratory. The detection limit of the analytical method used should be lower than this action limit. For samples with a concentration between the detection limit and the action limit the problem is encountered as to how the result should be reported. In these cases the inspection may be informed that the sample is ‘suspect’ or that it contains an illegal growth promoter at a concentration below the action limit. They may use this information for future sampling [11]. Concerning routine analysis, two types of methods are distinguished: Screening methods and confirmatory methods. Screening methods should indicate the presence of an analyte or a class of analytes at a certain concentration level in a series of samples in a short period of time. They are aimed at selecting the samples that may give a positive result and the number of false negative results should, therefore, be minimal. Confirmatory methods are aimed to give an unequivocal identification of the analyte at a certain concentration level. The number of false positives should be minimal with an acceptable number of false negatives. No definitive rules can be given for screening methods. However, the ‘specificity’ of the method should be known and the ‘limit of detection’ must be suitable for the purpose. If possible, sample throughput should be quick and the costs should be low. For confirmatory methods, rules have been defined, because the analyte must be identified unequivocally. Therefore, when two analytes give the same response, the method is not suitable as a confirmatory method. A suitable combination of a chromatographic technique with a spectrometric or immunochemical detection is considered sufficiently specific. Target values for accuracy and precision are given in the decision. The ‘limits of detection and quantification’ and the sensitivity should be adequate. Speed and costs are less important for confirmatory methods [10]. The appendix of decision 93/256/EEC gives criteria for the analytical techniques that can be used. They will be summarised below [10]. Of the techniques mentioned below TLC and infrared spectrometry generally have rather high detection limits. They should normally not be used for screening methods, but they can for example be used for the analysis of injection sites. · For immunoassays the working range of the calibration curve should be specified and at least 6 calibration standards should be used chosen over the whole working range. Quality control parameters of this calibration curve should be in line with previous determi- nations. Control samples should be analysed at zero, a low and a high level and should give normal results. · When GC or LC is used without a specific detection method, the retention time of analyte and standard should be equal. The distance between the analyte peak and the nearest peak should be at least one peak width at 10% of maximum height. Additional information can

8 History and Legislation

be obtained from co-chromatography or the use of two different columns with different polarities. · In TLC the Rf value of analyte and standard should be equal with a maximum difference of 3%. There should be no visual difference between analyte spot and standard spot. The distance between the analyte spot and the centre of the nearest spot should be at least equal to half of the sum of the two diameters of these spots. Additional information can be obtained from co-chromatography or two-dimensional TLC. · When the detection method in LC is an immunoassay, the analyte peak should be build from at least five LC fractions and the analyte peak should be present in the characteristic fraction for that analyte. The source and the characteristics of the antibody and other reagents used should be specified in the protocol. The criteria for immunoassays should be fulfilled. For identification two different LC systems and two different antibodies with different selectivity should be used. · When LC is used in combination with spectrometry, the criteria for LC should be fulfilled. The absorption maxima of analyte and standard should be equal within a margin depending on the resolution of the detector and the difference between the spectra of the analyte and standard above 220 nm should never be more than 10%. For identification co- chromatography is mandatory. Similar criteria are applicable to TLC-spectrometry, but the spectra of analyte and standard should now be visually equal. · For GC-MS the criteria for GC must be fulfilled. If possible an internal standard should be used, which is ideally a stable, labelled form of the analyte. Relative retention times of analyte and standard should be equal within 0.5% and when no internal standard is used, co-chromatography is mandatory. For screening with low resolution MS (LRMS) at least the most abundant ion should be measured and for confirmatory analysis at least four diagnostic ions including the molecular ion should be measured. When there are less than four diagnostic ions, two GC-LRMS methods both producing two or three diagnostic ions should be combined. The relative abundances of the diagnostic ions should be equal for analyte and standard within a margin of 10% in the electron impact mode or 20% in the chemical ionisation mode. · For infrared spectra ‘adequate peaks’ should be defined. These are maxima between 1800 and 500 cm-1 with a specific molar absorption of at least 40 in relation to zero and 20 in relation to the baseline, or with a relative absorption equal to 12.5% of the peak with maximum absorption when measured with reference to zero, or 5% when measured with reference to the baseline. The number of peaks that coincide with adequate peaks within 1 cm-1 should be determined. For a positive identification there should be absorption in all areas of the spectrum where adequate peaks are present. At least six adequate peaks

9 Chapter 1.1

should be present in the reference spectrum. When an adequate peak is not present in the sample spectrum, the relevant area of the spectrum may not exclude the presence of this peak. Relevant peaks in the analyte spectrum should exceed three times the peak-to-peak noise. Generally, for proper identification a combination of two methods should be used. These can be two different chromatographic techniques or the criteria given for the identification using one technique should be followed. Other analytical methods can be used as screening or confirmatory method, when similar criteria can be defined that lead to unequivocal identification of the analyte at a certain concentration.

References [1] Crosby, Determination of Veterinary Residues in Food, Ellis Horwood Series in Food Science and Technology, Ellis Horwood, Chichester, 1991, ISBN 0-747-0065- 1, Chapter 1, p 15-36 [2] Crosby, Determination of Veterinary Residues in Food, Ellis Horwood Series in Food Science and Technology, Ellis Horwood, Chichester, 1991, ISBN 0-747-0065- 1, Chapter 8, p 208-225 [3] Stephany (1993), in: N. Haagsma, A. Ruiter and P.B. Czedik-Eysenberg (ed), EuroResidue II, Conference on Residues of Veterinary Drugs in Food, May 3-5, 1993, Veldhoven, The Netherlands, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, ISBN 90-6159-016-7, p 89-98 [4] Council Directive 96/22/EU, Off J Eur Commun L125 (1996) 3-9 [5] Council Directive 96/23/EU, Off J Eur Commun L125 (1996) 10-32 [6] Stephany, L.A. van Ginkel and R.C. Schothorst, Analyst 119 (1994) 2707-2711 [7] EC measures concerning meat and meat products (hormones), panel report, World Trade Organisation, Geneva, 18-8-1997, documents WT/DS26/R/USA and WT/DS48/R/CAN [8] EC measures concerning meat and meat products (hormones), report of the appellate body, World Trade Organisation, Geneva, 16 -1-1998, documents WT/DS26/AB/R and WT/DS48/AB/R [9] Press release of the European Commission of March 13, 1998, Brussels, document IP/98/253 [10] Commission Decision 93/256/EEC, Off J Eur Commun L118 (1993) 64-74 [11] de Brabander, P. Batjoens, K. de Wasch, D. Courtheyn, G. Pottie and F. Smets, Trends Anal Chem 16 (1997) 485-489

10 CHAPTER 1.2.1 ANABOLIC STEROIDS AND RELATED SUBSTANCES

Introduction The sex hormones are important for the proper functioning of the reproductive system. They are subdivided in three groups on the basis of their effects in the human body. The oestrogens and progestagens regulate the menstrual cycle in adult women, where the former are more important in the first half of the cycle and the latter in the second half. They are responsible for the development of sexual characteristics in females. The oestrogens also increase the coagulability of the blood, resulting in an increased risk of thrombo-embolism. The androgens are responsible for the development of sexual characteristics in males and for the maintenance of spermatogenesis. All three groups have anabolic activity, but this effect is largest for the androgens. The anabolic androgenic steroids, in which the anabolic activity is enhanced relative to the other effects, have been derived from the androgens [1]. Anabolic steroids are used in replacement therapy, growth disorders, anaemias and blood disorders, cancer treatment and catabolic and debilitating states. The most important adverse effects reported are problems with reproduction, virilisation or feminisation, liver alterations, development of diabetes, disturbances of lipoprotein profiles, cardiovascular effects and cerebral dangers, musculoskeletal injuries, increased risk of prostate cancer and psychosis and schizo- phrenia [2]. However, these hormones are better known because of their abuse by athletes and farmers. Besides the steroids as such, two groups of non-steroidal substances with oestrogenic activity, the stilbenes and the resorcylic acid lactones, will be discussed. Some representative structures are given in Figure 1 and the structures of the substances that were used in our experiments are given in Appendix 1. The term ‘anabolic steroids’ will be used in this thesis to adress all the compounds belonging to the five groups together. Like it is done in most publications the common name zeranol will be used throughout this thesis instead of the chemical name ß-zearalanol. The effects of the use of anabolic steroids in food-producing ruminants have been reviewed [3]. To achieve a maximum effect on growth rate, the oestrogen concentration must be similar to that of a young female and at the same time the androgen concen-

11 Chapter 1.2.1

Figure 1. Structures of some relevant anabolic steroids and related substances

Structure 1. Androgens OH CH3 testosterone: R4 = R17 = H, R19 = methyl R17 nortestosterone: R4 = R17 = R19 = H R19 methyltestosterone: R4 = H, R17 = R19 = methyl clostebol: R4 = Cl, R17 = H, R19 = methyl

OH Structure 2. Oestrogens CH3 R 17ß-oestradiol: R = H 17 -ethynyl oestradiol: R = ethynyl

CH3 Structure 3. Progestagens C O CH progesterone: R6 = R17 = H 3 R17 medroxyprogesterone: R6 = methyl, R17 = OH CH3

OH O H CH Structure 4. Resorcylic acid lactones 3 zeranol: R = ß-hydroxy O taleranol: R = -hydroxy OH zearalenone: R = O, double bond at 1 1 R

12 Anabolic Steroids and Related Substances

Structure 5. Stilbenes OH C H diethylstilbestrol: C-C double bond 2 5 C dienoestrol: C-ethyl both double bonds C hexoestrol: as given C2 H5 OH

tration should be equivalent to that of a young male. Therefore, androgen supplementation is most useful for females and oestrogen supplementation for males. For castrated animals combined treatment with androgens and oestrogens, also in combination with progestagens, is most often used. Only a limited number of studies have been performed to determine the effect of androgens on the growth rate of entire males and only minimal effects have been reported. Oestrogens alone and even more in combination with androgens did increase growth rate in entire males. In females the effect of oestrogens was limited, but androgens alone or in combination with oestrogens resulted in an increased average daily gain. In castrated animals growth rate could be increased by treatment with androgens or oestrogens alone or in combination. However, the effectiveness was found to depend on the growth rate of the control animals. In studies where the performance of the control animals was already very good, the effect of the steroid was minimal. Several factors influence the effectiveness of anabolic steroids. Animal- related factors are sex and age, which determine the maturity of the hormonal system and the capacity to convert feed protein into muscle protein. Implants have a maximum efficacy for a limited period (usually about 100 days). Reimplantation during this period is not useful, but after about 150 days it is. With some anabolic steroids the animals should be well-nourished to achieve promotion of growth. A relation between the amount of nitrogen in the food and the growth rate has been found [3]. Anabolic steroids can exert their growth-promoting effect via different mechanisms. The effect of treatment on the concentrations of endogenous steroids depends on age and sex of the animal and is complicated by complex feedback mechanisms. The anabolic steroids increase protein synthesis and reduce proteolysis (a repartioning) via a direct effect mediated through a receptor and some may also act indirectly via a decrease of the cortisol concentration [4]. For an assessment of the risks associated with the ingestion of residues of anabolic steroids in food, the natural steroids and the synthetic compounds should be considered separately. The natural steroids - e.g. testosterone, progesterone, 17ß-oestradiol, oestrone and oestriol - are produced endogenously by both humans and cattle. Therefore, they will

13 Chapter 1.2.1 always be present in meat. Typical amounts found in muscle and liver of untreated and treated bovine animals are presented in Table 1. The residues found in treated animals fall within the physiological ranges. Therefore, meat from treated animals does in principle not present a higher risk than that from non-treated animals. Another point that has to be taken into consideration is that the natural steroids have little oral activity due to rapid deactivation. Finally, the amounts ingested by humans are well below the amounts of steroids produced endogenously (see Table 2), whether the meat is from treated or untreated animals [5].

Table 1. Concentrations of natural steroids found in muscle and liver of untreated and treated bovine animals [5]. (T: testosterone (pg/g), P: progesterone (ng/g), E2: 17ß- oestradiol (pg/g) and E1: oestrone (pg/g); steers and male calves were treated with 20 mg 17ß-oestradiol + 200 mg progesterone, heifers with 20 mg 17ß-oestradiol benzoate + 200 mg testosterone propionate, and female calves with 20 mg 17ß-oestradiol + 200 mg testosterone, all applied as subcutaneous implant). animal day after muscle liver treatment T P E2 E1 T P E2 E1 bull untreated 535 749 steer untreated 101 0.27 0.84 1.60 0.26 0.91 0.66 day 61 0.41 7.29 2.60 0.35 4.52 2.10 day 90 0.44 4.51 1.79 0.24 5.41 1.22 male calf untreated 0.247 0.269 day 70 0.515 0.325 heifer untreated 92 5.54 2.51 193 1.54 1.7 day 61 10.7 3.98 3.21 1.49 day 90 10.1 6.4 3.29 1.52 pregnant untreated 10.1 3.4 day 120 15.6 203 58.4 29.7 day 180 27.3 482 380 115 day 240 32.7 523 1027 145 female calf untreated 16 39 day 77 70 47

14 Anabolic Steroids and Related Substances

Table 2. Amounts of steroids produced endogenously by humans of different sexes and ages [5]. (T: testosterone (mg/24 h), P: progesterone (mg/24 h), E2: 17ß-oestradiol (µg/24 h) and E1: oestrone (µg/24 h)) T P E2 E1 women follicular phase 0.42 82 110 preovulatory phase 695 497 luteal phase 19.6 270 243 non-specific phase 0.24 menopausal 0.1 0.33 5.9 40 early pregnancy 2000 2000 mid pregnancy 10800 8800 late pregnancy 294 37800 26500 men 6.48 0.42 48 88 children girls 0.032 0.25 13.0 41 boys 0.065 0.15 6.5 35

For the synthetic compounds trenbolone and zeranol, a comprehensive risk assessment has been performed. This resulted in no-effect levels of 1.0 and 10 µg/kg body weight for ß- and -trenbolone, respectively, and 25 µg/kg bodyweight and 1.0 mg/kg body weight for zeranol and taleranol, respectively. This has been translated into accepted daily intakes of 0-0.01 µg/kg bodyweight for trenbolone and 0-0.5 µg/kg bodyweight for zeranol. When preparations of these steroids are used according to good agricultural practice, these levels will not be exceeded [5]. A possible risk for these two synthetic compounds and the natural steroids is posed by the ingestion of implantation or injection sites. About 10-20% of the applied amount remains at the implantation site after removal of the implant. In injection sites rather high residues may be left (up to mg amounts). These sites have to be removed after legal use, but illegal application sites will only be removed when they are discovered at the slaughterhouse. Single consumption of an injection site will result in a short-lasting suppression of endogenous hormone production. Clinical symptoms require the consumption over several days, which is rather unlikely. Only when an injection site is spread over a batch of baby food serious problems may be expected [5]. For the other synthetic steroids no proper risk assessment has been performed. From sports doping cases side effects of chronic ingestion of large doses of androgens are known: Prolonged infertility, testicular atrophy, secondary amenorrhea, virilisation in

15 Chapter 1.2.1 women, increased risk of coronary artery disease, liver dysfunction and hepatotoxicity resulting in hepatocellular carcinoma, changes in psychological state resulting in psychosis or violent criminal activity, and premature epiphyseal closure [6]. Also, the carcinogenic and teratogenic properties of diethylstilbestrol (DES) are notorious because of the DES daughters and sons [7]. The consumption of meat that contained residues of oestrogenic compounds, has been suggested as the cause of breast enlargement in Italy [8] and precocious puberty in Puerto Rico [9]. The synthetic steroids were banned as growth promoters in the European Union in 1988 because of their carcinogenic properties and other side effects [10]. An overview of the steroids found in Belgium in injection sites is given in Table 3 [11]. The main detected substances are the natural steroids and clostebol acetate. Stanozolol and progesterone became important in the later years, whereas the use of nortestosterone and its esters became less in recent years. These numbers are comparable to the results of the analysis of 117 injection sites collected in 1983 in The Netherlands. Of these 97, were found to contain residues of the following steroids: nortestosterone (96%), medroxyprogesterone acetate (30%), diethylstilbestrol (11%), dienoestrol (1%), methyltestosterone (6%), trenbolone (4%), testosterone (45%), 17ß-oestradiol (45%) and progesterone (2%) [12]. The Dutch meat inspection reported that 51 of 359 suspect animals from The Netherlands in 1989 showed residues of anabolic steroids, whereas of 371 imported pieces of meat with injection sites 204 contained anabolic steroids. Most of the imported meat originated from Belgium. Of 100 samples of minced meat, bought at butcher shops in The Netherlands in 1990, 3 contained residues of nortestosterone [13] and in 1991 in 5 of 99 samples medroxyprogesterone was detected [14]. However, in 1992 no anabolic steroids were found in 60 meat samples [15]. In 1994, the consumer organisations in the European Union analysed a total of 1183 steaks. The results are summarised in Table 4. In The Netherlands two samples contained residues of clostebol acetate and one residues of methylboldenone [16]. In illicit anabolic preparations analysed between 1983 and 1988 in Germany, ethynyl oestradiol, methyltestosterone, and esters of nortestosterone, 17ß-oestradiol, medroxy- rogesterone and testosterone were detected [17]. The surveillance data given above are rather old, but newer data are not publicly available.

16 Anabolic Steroids and Related Substances

Table 3. Survey of hormones found in Belgium in injection sites between 1989 and 1994 (modified from [11]). For each substance the percentage of injection sites positive for that steroid is given. steroid 1989 1990 1991 1992 1993 1994 nortestosterone + esters 69 43 19 20 6 1 17ß-oestradiol + esters 57 76 57 44 64 77 testosterone + esters 57 72 62 53 89 92 methyltestosterone 31 26 24 32 47 39 medroxyprogesterone acetate 26 21 24 15 5 megestrol acetate 11 5 6 1.6 1 ethynyl oestradiol 9 8 0.3 1.2 chlormadinone acetate 3 2 0.8 clostebol acetate 52 46 42 76 86 stanozolol 16 22 28 54 63 progesterone 9 20 44 75 77 boldenone 7 1.6 trenbolone acetate 0.4 1.3 4 6 3 17 -hydroxyprogesterone acetate 10 4 1 methylboldenone 4 4 4 4 norethisterone 0.3 17 -hydroxyprogesterone caproate 8 4 fluoxymesterone 8 39 29 delmadinone acetate 8 algestone acetophenide 5 % positive injection sites 18.8 62.2 42.4 31.4 39.7 61.7

17 Chapter 1.2.1

Table 4. Samples of steaks bought in butcher shops in the European Union analysed for the presence of residues of anabolic steroids [16]. country samples positive Netherlands 75 3 Belgium 75 5 Denmark 74 0 Germany 151 4* France 139 1 Greece 70 1 Great Britain 160 2 Ireland 75 2 Italy 145 1 Luxembourg 38 0 Portugal 75 1 Spain 107 1 total 1183 21 * one of the positive samples contained residues of two steroids

Metabolism Testosterone, 17ß-oestradiol, oestriol, oestrone and progesterone have been known as endogenous steroids for a long time and their biosynthesis, metabolism and inter- relationships have been extensively studied in both humans [18,19] and cattle [20]. In the last few years, it has become apparent that two androgens, which were previously thought to be exogenous steroids, do also occur as endogenous steroids. The major metabolite of 17ß-nortestosterone, 17 -nortestosterone, was found to be present in most urine samples taken during the last half of pregnancy in cows [21]. Also, of 20 blank bovine urines six were found to contain significant amounts of 17 -boldenone, the major metabolite of 17ß-boldenone [22]. Another case is presented by zeranol, a synthetic compound that was found in bovine bile without indications of treatment. Further investigations showed that two metabolites of the mycotoxin zearalenone, - and ß-zearalenol, were also present in high concentrations [23]. A similar report about the occurrence of zeranol in several pasture-fed animals in New-Zealand has appeared. It was suggested that zearalenone formed by mycotoxins in the mouldy silage had been metabolised to zeranol in the rumen of cows, possibly by bacteria [24]. However, a study with sheep showed that zeranol and taleranol

18 Anabolic Steroids and Related Substances were also formed after intravenous infusion of zearalenone, which implies that ruminal metabolism is not necessary [25]. Because of its natural occurrence, zeranol is now designated as a semi-synthetic steroid. The metabolism of the synthetic steroids in humans has been reviewed [26], but for many compounds the metabolism in cattle has not been published yet. A review with schemes for diethylstilbestrol, trenbolone and zeranol in several species and some notes on the metabolism of some other synthetic steroids in humans has appeared [27]. Several reactive metabolites of diethylstilbestrol were found, but their carcinogenicity could not be proven yet [27]. Trenbolone can be hydroxylated at the 1-, 2-, 6- and 16-position and the 17ß- hydroxy-group can be oxidised. Trace amounts of the possible metabolites have been detected in bile from a heifer after a single dose of trenbolone acetate. However, the main metabolite was found to be the epimer of trenbolone, 17 -trenbolone, together with small amounts of the 16-hydroxy product [27]. In calves slaughtered 50 days after implantation with tritiated trenbolone acetate, the largest amount of radioactivity was detected in bile, liver and kidney with minor amounts in muscle and fat. About three-quarters of the amounts in liver and kidney were covalently bound and could not be extracted. Main metabolites in liver and kidney were 17 -trenbolone and 17ß-trenbolone, respectively, occurring together with smaller amounts of the other epimer and trendione [28]. The metabolism of zeranol was studied in rat, rabbit, dog, monkey and man [29] and in steer [30]. In the former study, the amounts of zeranol, zearalenone and their conjugates were determined in both urine and faeces. The excretion route was found to be species dependent. In rabbit and man, more than half of the applied radioactivity was excreted in urine, whereas in rat, dog and monkey about three-quarters was recovered in faeces. In man, zeranol and zearalenone were both found in the conjugated form and about half of the radioactivity in urine could not be identified. A similar pattern was observed in the rat. The monkey and the dog mainly excreted zeranol in the unchanged form. The rabbit excreted conjugates of zeranol, free and conjugated zearalenone and unknown metabolites, including taleranol [29]. Also, a hydroxylated metabolite was found, which was not further identified [27,29]. In steers implanted or infused with zeranol, the unchanged compound together with its epimer taleranol and zearalanone were determined in several tissues. In muscle and serum mainly zeranol was found, whereas in kidney, liver, bile and urine taleranol predominated. Zearalanone was detected in all tissues, but the amounts were less than those of the other two compounds. In the implanted animals zeranol and its metabolites were mainly found in bile and

19 Chapter 1.2.1 urine, whereas in the infused animals the largest amounts were found in urine, liver and kidney [30]. The metabolism of nortestosterone was studied in female calves after oral and intramuscular administration. In none of the samples taken unchanged 17ß-nortestosterone was found. Its epimer 17 -nortestosterone was the major metabolite in urine after intramuscular injection. Other identified metabolites were 5 -estrane-3ß,17 -diol, 5ß- estrane-3 ,17ß-diol, 3 -hydroxy-5ß-estran-17-one and 3ß-hydroxy-5 -estran-17-one. After oral administration only small amounts of metabolites were detected [31]. Calves were implanted for 32-33 days or injected daily for 27 days with tritiated 17ß- oestradiol and they were slaughtered at the end of the study. Radioactivity was mainly found in liver and kidney with minor amounts in muscle and adipose tissue. Between 16% and 26% was covalently bound and could not be extracted. In liver mainly conjugates were found, whereas free metabolites dominated in kidney and muscle. Only trace amounts of unchanged 17ß-oestradiol were detected in liver and kidney, but in liver about 30% of radioactivity could be contributed to the parent compound. The major metabolites in liver and kidney were conjugated forms of the epimer 17 -oestradiol. Other unidentified, minor metabolites including oestradiol-17-esters were found [32]. In another study, male veal calves were injected with 17ß-oestradiol benzoate and testosterone propionate at an age of 19 and 23 weeks. For both compounds the two 17- epimers were analysed in urine and compared to control values (Table 5). The concentrations of 17 - and 17ß-oestradiol were elevated on days 2-7 after injection, but the result was not significantly above the decision level on day 7 for some animals. The levels of 17 - and 17ß-testosterone remained within the physiological range in all samples [33].

Table 5. Concentrations (µg/l) of natural steroids in urine of untreated male veal calves [33]. steroid concentration decision level 17ß-oestradiol < 0.05 - 0.5 1 17 -oestradiol 0.9 - 16* 20 17ß-testosterone 0.4 - 21 30 17 -testosterone 9 -420 500 * generally the level was found to be below 10 µg/l in this and previous studies [33].

20 Anabolic Steroids and Related Substances

The metabolites of clostebol acetate were determined in the urine of a heifer and a steer after a single muscular injection. 17ß-Clostebol and the acetate ester were not found, but seven metabolites were identified: 4-chloroandrost-4-ene-3 ,17ß-diol, 4- chloroandrost-4-ene-3,17,x-triol (not further identified), 17 -clostebol, 4-chloroandrost- 4-ene-3 -ol-17-one (the major metabolite in man), one of the isomers of 4-chloro- androstane-3-ol-17-one, one of the isomers of 4-chloroandrost-4-ene-3,x-diol-17-one, and 4-chloroandrost-4-ene-3,17-dione. Several of these metabolites were also detected in urine samples that were negative in the normal screening procedure and in the urine of an animal with a positive injection site [34]. In another study, a 3-year old cow was injected with clostebol acetate and urine was collected for 15 days. The major metabolite found was 17 -clostebol sulphate followed by 4-chloro-4-androstene-3 -ol-17-one glucuronide. Other metabolites were similar to those found in the previous study, but now also 17ß-clostebol was detected [35]. After oral administration of clostebol acetate to a male calf the major metabolite was 4-chloro-4-androstene-3 ,17ß-diol. Other metabolites were similar to those found in previous studies. More than 90% of the metabolites was excreted as sulphate-conjugate except 4-chloro-4-androsten-3 -ol-17-one, which glucuronidated was for more than 25% [36]. The metabolism of methyltestosterone and ethynyl oestradiol after oral administration were studied in a 5-year old cow. Methyltestosterone was not detected in urine and three metabolites were identified except for the orientation at the 5-position: 17 -methyl-5- androstan-3-one-17ß-ol, 17 -methyl-5-androstan-3 ,17ß-diol and 17 -methyl-5-androstan-3ß,17ß-diol. The metabolites were almost exclusively found in the sulphate fraction. Ethynyl oestradiol was not metabolised but only conjugated. Between 30% and 60% was found in the glucuronide fraction and the presence of mixed conjugates was suggested [36]. Methyltestosterone and ethynyl oestradiol could be detected in the hair of treated female veal calves, but the concentrations were low [37]. After subcutaneous administration of stanozolol to a cow, 16-hydroxystanozolol and 4,16-dihydroxystanozolol were found in urine, whereas after oral administration stanozolol and 16-hydroxystanozolol were detected [38]. In a male calf 3-hydroxystanozolol and 16ß-hydroxystanozolol were identified in concentrations of up to 8 µg/l after intramuscular injection with 200 mg stanozolol [39]. From these studies it becomes apparent that the main metabolites of the anabolic steroids in bovine animals are the 17-epimers and that most metabolites are excreted as conjugates.

21 Chapter 1.2.1

Analytical Methods The anabolic steroids are lipophilic compounds with log P (octanol-water) coeffi- cients of 2-4 [40]. The androgens and progestagens are generally neutral compounds. The oestrogens, stilbenes and resorcylic acid lactones posses an acidic phenol group with a pKa of 10-11 [41]. The UV spectra in methanol or ethanol are relatively unspecific with a few exceptions. The androgens and part of the progestagens have a maximum UV absorption at about 240 nm. The rest of the progestagens have a maximum between 280 and 290 nm. Trenbolone has a rather specific UV spectrum with a maximum at 350 nm. The UV spectra of the oestrogens show maxima at about 195 nm, 230 nm and 280 nm. The stilbenes have UV spectra that are similar to each other with a broad shoulder between 230 and 240 nm, but differentiation is possible. The UV spectra of the resorcylic acid lactones have three maxima at 236 nm, 274 nm and 316 nm for zearalenone and at 218 nm, 265 nm and 304 nm for zeranol [41,42]. Analytical methods for anabolic steroids in human and equine urine [6,43] and for biological samples obtained from food-producing animals [44,45] have been reviewed. As sample urine is commonly used, because it can be obtained by a non-invasive method and is freely available. Also, it contains higher concentrations of the steroids than serum [6]. As mentioned above, the anabolic steroids are mainly excreted as conjugates, which are usually hydrolysed before analysis. Helix Pomatia is often used containing glucuro- nidase and sulphatase in addition to several other enzymes. As an alternative, glucu- ronidases from E. Coli in combination with solvolysis can be performed [6]. Most methods employ solid phase extraction (SPE) or immuno-affinity chromatography (IAC) for clean up of the sample and GC-MS for the detection of the steroids [6,43-45]. How- ever, other extraction and detection techniques have also been used. Liquid-liquid extraction has been performed as a pre-extraction before SPE for urine and meat samples [46]. XAD-2 was used for the extraction of metabolites and conjugates from urine, and Sephadex LH-20 for the separation of glucuronides and sulphates [47,48]. For SPE of steroids from urine, most often reversed phase sorbents have been used, including C2 [49] and C18 [50-59] bonded silica. Polar sorbents, like silicagel [54,58,60], alumina [56,58] and aminopropyl-bonded silica [51,55,59], were used as additional clean up steps. These extraction techniques are suitable for both single- and multi-residue procedures. For single-residue methods the extraction parameters can be optimised to obtain cleaner extracts. Nortestosterone has been extracted from urine with IAC columns alone [61] or in combination with C18 SPE [52]. Other applications of IAC for the analysis of anabolic steroids in biological matrices have been reviewed [62]. IAC

22 Anabolic Steroids and Related Substances procedures can be selective when an antibody for a characteristic part of the molecule is used. Antibodies with good cross-reactivities for several structurally related steroids or combinations of different antibodies can be used for multi-residue methods [62]. An HPTLC system for the analysis of anabolic compounds in injection sites has been described [63]. HPLC has been used for the analysis of anabolic steroids in preparations of illegal growth promoters [64-66] and as a clean up step for biological samples [67,68]. GC-MS requires derivatisation of the steroids. For multi-residue procedures, TMS enol ethers are formed [69,70], but in single-residue procedures a more selective derivate may result in lower detection limits [54]. When a confirmatory analysis is performed, both a retention parameter and a full scan mass spectrum are needed, but for screening single ion monitoring at the most abundant peaks in the spectrum is sufficient [6]. Immuno- assays are used in screening procedures, because they are rapid and require minimal sample preparation [60]. However, problems can be encountered due to cross-reactivity with endogenous hormones and low cross-reactivities of antibodies with some synthetic hormones. They have also been used as a detector in HPLC [67,71].

References [1] The reproductive system, in: H.P. Rang and M.M. Dale, Pharmacology, Churchill Livingstone, Edinburgh, 1987, ISBN 0-443-03407-9, p 413-432 [2] Hickson, K.L. Ball and M.T. Falduto, Med Toxicol Adverse Drug Exp 4 (1989) 254-271 [3] P. Schmidely, Ann Zootech 42 (1993) 333-359 [4] P. Schmidely, Reprod Nutr Develop 33 (1993) 297-323 [5] B. Hoffmann (1996), in: European Commission, Directorate-General VI (Agri- culture), Proceedings of the Scientific Conference on Growth Promotion in Meat Production, Brussels, November 29-December 1, 1995, Office for Official Publications of the European Communities, Luxembourg, ISBN 92-827-6321-8, p 271-296 [6] D.B. Gower, E. Houghton and A.T. Kicman, Chapter 8. Anabolic Steroids: Meta- bolism, Doping and Detection in Equestrian and Human Sports, p 468-526 in: H.L.J. Makin, D.B. Gower and D.N. Kirk (ed), Steroid Analysis, Blackie Academic & Professional, Glasgow, first edition, 1995, ISBN 0-7514-0128-5 [7] R.M. Giusti, K. Iwamoto and E.E. Hatch, Ann Intern Med 122 (1995) 778-788 [8] G.M. Fara, G. Del Corzo, S. Bernuzzi, A. Bigatello, C. Di Pietro, S. Scaglioni and G. Chiumello, Lancet ii (1979) 295-297

23 Chapter 1.2.1

[9] C.A. Saenz de Rodriguez, A.M. Bongiovanni and L Conde de Borrego, J Pediatr 107 (1985) 393-396 [10] Council directive 88/146/EEC, Off J Eur Commun L70 (1988) 16-18 [11] K. Vanoosthuyze, E. Daeseleire, A. van Overbeke, C. van Peteghem and A. Ermens, Analyst 119 (1994) 2655-2658 [12] E.H.J.M Jansen, H. van Blitterswijk and R.W. Stephany, Vet Q 6 (1984) 60-65 [13] Dutch consumer organisation, Consumentengids, March 1990, 150-155 [14] Dutch consumer organisation, Consumentengids, May 1991, 296-300 [15] Dutch consumer organisation, Consumentengids, June 1993, 348-351 [16] Dutch consumer organisation, Consumentengids, February 1995, 104-107 [17] H.H.D. Meyer, Ann Rech Vet 22, (1991) 299-304 [18] H.J. Degerhart (1974), Steroid Hormones - Introduction, in: H.Ch. Curtius and M. Roth (ed), Clinical Biochemistry - Principles and Methods, Walter de Gruyter, Berlin, ISBN 3-11-001622-2, p 668-672 [19] H. Breuer and L. Nocke-Finck (1974), Oestrogens, in: H.Ch. Curtius and M. Roth (ed), Clinical Biochemistry - Principles and Methods, Walter de Gruyter, Berlin, ISBN 3-11-001622-2, p 790-830 [20] F. Döcke (1994), Keimdrüsen, in: F. Döcke (ed), Veterinärmedizinische Endo- krinologie, Gustav Fischer Verlag, Jena, 3rd edition, ISBN 3-334-60432-2, p 399- 508 [21] H.F. De Brabander, J. van Herde, P. Batjoens, L. Hendriks, J. Raus, F. Smets, G. Pottie, L. van Ginkel and R.W. Stephany, Analyst 119 (1994) 2581-2585 [22] C.J.M. Arts, R. Schilt, M. Schreurs and L.A. van Ginkel (1996), in: N. Haagsma and A. Ruiter (ed), EuroResidue III Conference on Residues of Veterinary Drugs in Food, Veldhoven, The Netherlands, May 6-8, 1996, University of Utrecht, Faculty of Veterinary Medicine, Utrecht, The Netherlands, ISBN 90-6159-023-X, p 212-217 [23] D.G. Kennedy, J.D.G. McEvoy, W.J. Blanchflower, S.A. Hewitt, A. Cannavan, W.J. McCaughey and C.T. Elliott, J Vet Med B 42 (1995) 509-512 [24] A.F. Erasmuson, B.G. Scahill and D.M. West, J Agric Food Chem 42 (1994) 2721- 2725 [25] C.O. Miles, A.F. Erasmuson, A.L. Wilkins, N.R. Towers, B.L. Smith, I. Garthwaite, B.G. Scahill and R.P. Hansen, J Agric Food Chem 44 (1996) 3244-3250 [26] W. Schänzer, Clin Chem 42 (1996) 1001-1020 [27] M. Metzler, J Chromatogr 489 (1989) 11-21 [28] P. Evrard, G. Maghuin-Rogister and A.G. Rico, J Anim Sci 67 (1989) 1489-1496

24 Anabolic Steroids and Related Substances

[29] B.H. Migdalof, H.A. Dugger, J.G. Heider, R.A. Coombs and M.K. Terry, Xenobiotica 13 (1983)209-221 [30] T.M.P. Chichilla, D. Silvestre, T.R. Covey and J.D. Henion, J Anal Toxicol 12 (1988) 310-318 [31] E. Daeseleire, A. De Guesquière and C. van Peteghem, Anal Chim Acta 275 (1993) 95-103 [32] A. Paris, L. Dolo, D. Rao and M. Terqui (1993), in: N. Haagsma, A. Ruiter, P.B. Czedik-Eysenberg (ed), Residues of Veterinary Drugs in Food, Proceedings of the EuroResidue II Conference, Veldhoven, The Netherlands, May 3-5, 1993, ISBN 90- 6159-0-16-7, p 518-522 [33] C.J.M Arts, M.J. van Baak, J. Huisman, I.J.R. Visser, M.J Groot and J.M.P den Hartog, Arch Lebensmittelhyg 41 (1990) 31-37 [34] L. Leyssens, E. Royackers, B. Gielen, M. Missotten, J. Schoofs, J. Czech, J.P. Noben, L. Hendriks and J. Raus, J Chromatogr B 654 (1994) 43-54 [35] F. André, B. le Bizec, M.-P. Montrade, D. Maume, F. Monteau and Ph. Marchand, Analyst 119 (1994) 2529-2535 [36] B. le Bizec, M.-P. Montrade, F. Monteau and F. André (1996), in: N. Haagsma and A. Ruiter (ed), EuroResidue III Conference on Residues of Veterinary Drugs in Food, Veldhoven, The Netherlands, May 6-8, 1996, University of Utrecht, Faculty of Veterinary Medicine, Utrecht, The Netherlands, ISBN 90-6159-023-X, p 248-252 [37] A. Gleixner, H. Sauerwein and H.H.D. Meyer, Food Agric Immunology 9 (1997) 27-35 [38] V. Ferchaud, B. le Bizec, M.-P. Montrade, D. Maume, F. Monteau and F. André, J Chromatogr B 695 (1997) 269-277 [39] Ph. Delahaut, X. Taillieu, M. Dubois, K. de Wasch, H.F. de Brabander and D. Courtheyn, Arch Lebensmittelhyg 49 (1998) 3-7 [40] B. Lundberg, Acta Pharm Suec 16 (1979) 151-159 [41] A.C. Moffat, J.V. Jackson, M.S. Moss, B. Widdop and E.S. Greenfield (ed) (1986), Clarke’s Isolation and Identification of Drugs in Pharmaceuticals, Body Fluids, and Post-mortem Materials, The Pharmaceutical Press, London, ISBN 0-85369-166-5 [42] S. Budavari, M.J. O’Neil and A. Smith (ed) (1989), The Merck Index, Merck, Rahway, NJ, USA, 11th edition, ISBN 0-911910-28-X [43] C. Ayotte, D. Goudreault and A. Charlebois, J Chromatogr B 687 (1996) 3-25 [44] F. André, in: N. Haagsma and A. Ruiter (ed), EuroResidue III Conference on Residues of Veterinary Drugs in Food, Veldhoven, The Netherlands, May 6-8, 1996,

25 Chapter 1.2.1

University of Utrecht, Faculty of Veterinary Medicine, Utrecht, The Netherlands, ISBN 90-6159-023-X, p 53-61 [45] M. O’Keeffe (1996), Strategies for the detection of veterinary drug residues, in: G. Enne, H.A. Kuipers and A. Valentini (ed), Residues of Veterinary Drugs and Mycotoxins in Animal Products - New Methods for Risk Assessment and Quality Control, Proceedings of the Teleconference held on Internet from April 15-August 31, 1994, Wageningen Press, Wageningen, ISBN 90-74134-27-0, p 31-40 [46] W.M. Muck and J.D. Henion, Biom Environ Mass Spectrom 19 (1990) 37-51 [47] B.C Chung, H.Y. Choo, T.W. Kim, K.D. Eom, O.S. Kwong, J. Suk, J. Yang and J. Park, J Anal Toxicol 14 (1990) 91-95 [48] M.C. Dumasia and E. Houghton, Xenobiotica 14 (1984) 647-655 [49] D. Barrón, J. Barbosa, J.A. Pascual and J. Segura, J Mass Spectrom 31 (1996) 309- 319 [50] C.H.L. Shackleton and J.O. Whitney, Clin Chim Acta 107 (1980) 231-243 [51] N. A. Schmidt, H.J. Borburgh, T.J. Penders and C.W. Weykamp, Clin Chem 31 (1985) 637-639 [52] A. Farjam, G.J. de Jong, R.W. Frei, U.A.Th. Brinkman, W. Haasnoot, A.R.M. Hamers, R. Schilt and F.A. Huf, J Chromatogr 452 (1988) 419-433 [53] P. Teale and E. Houghton, Biol Mass Spectrom 20 (1991) 109-114 [54] B. le Bizec, M.-P. Montrade, F. Monteau and F. Andre, Anal Chim Acta 275 (1993) 123-133 [55] G. van Vyncht, P. Gaspar, E. DePauw, G. Maghuin-Rogister, J Chromatogr A 683 (1994) 67-74 [56] S. Calvarese, P. Rubini, G. Urbani, N. Ferri, V. Ramazza and M. Zucchi, Analyst 119 (1994) 2611-2615 [57] M.P. Oriundi, R. Angeletti, E. Bastiani, C. Nachtmann, K.E. Vanoosthuyze and C. van Peteghem, Analyst 120 (1995) 577-579 [58] G. Casademont, B. Pérez and J.A. García Regueiro, J Chromatogr B 686 (1996) 189-198 [59] H.A. Herbold, S.S. Sterk, R.W. Stephany and L.A. van Ginkel (1996), in: N. Haagsma and A. Ruiter (ed), EuroResidue III Conference on Residues of Veterinary Drugs in Food, Veldhoven, The Netherlands, May 6-8, 1996, University of Utrecht, Faculty of Veterinary Medicine, Utrecht, The Netherlands, ISBN 90-6159-023-X, p 491-495 [60] M.E. Ploum, W. Haasnoot, R.J.A. Paulussen, G.D. van Bruchem, A.R.M. Hamers, R. Schilt and F.A. Huf, J Chromatogr 564 (1991) 413-427

26 Anabolic Steroids and Related Substances

[61] L.A. van Ginkel, R.W. Stephany, H.J. van Rossum, H. van Blitterswijk, P.W. Zoontjes, R.C.M. Hooijschuur and J. Zuydendorp, J Chromatogr 489 (1989) 95-104 [62] L.A. van Ginkel, J Chromatogr 564 (1991) 363-384 [63] F. Smets, H.F. de Brabander, P.J. Bloom and G. Pottie, J Planar Chromatogr 4 (1991) 207-212 [64] M.J. Walters, R.J. Ayers and D.J. Brown, J AOAC 73 (1990) 904-926 [65] J.O. de Beer, J Chromatogr 489 (1989) 139-155 [66] I.S. Lurie, A.R. Sperling and R.P. Meyers, J Forensic Sci 39 (1994) 74-85 [67] E.H.J.M. Jansen, L.A. van Ginkel, R.H. van den Berg and R.W. Stephany, J Chromatogr 580 (1992) 111-124 [68] L.A. van Ginkel, E.H.J.M Jansen, R.W. Stephany, P.W. Zoontjes, P.L.W.J. Schwillens, H.J. van Rossum and T. Visser, J Chromatogr 624 (1992) 389-401 [69] H.O. Günther, Fres Z Anal Chem 290 (1978) 389-416 [70] P. Teale and E. Houghton, Biol Mass Spectrom 20 (1991) 109-114 [71] P.M. Krämer, Q.X. Li and B.D. Hammock, J AOAC Int 77 (1994) 1275-1287

27 CHAPTER 1.2.2 BETA-AGONISTS

Introduction

The ß-agonists, or more properly ß2-agonists, have been derived from the endogenous catecholamine adrenaline. By increasing the bulkiness of the substituent on the N-atom and substitution of the catechol hydroxyl groups by similar electron-withdrawing groups or transfer of these groups to different ring positions, substances with greater ß-selectivity and less susceptibility to metabolic degradation were obtained. The structures of the most important ß-agonists are given in Figure 1 and those of the substances that were used in our experiments are given in Appendix 1. They virtually all have at least one asymmetric carbon atom. Although the (-)-enantiomer is the pharmacologically active form, they are most often used as racemates. The two enantiomers were found to have different pharmacokinetics. The synthetic ß-agonists are divided in the phenol- and aniline-type ß-agonists on the basis of their chemical structure. Stimulation of the ß2- receptor leads to relaxation of smooth muscle, which makes the ß-agonists useful in the treatment of asthma and premature labour. The main side effects are muscle tremor and peripheral vasodilatation. Also, because the ß2-agonists all show some ß1-activity, disturbances of the cardiac rhythm, like tachycardia, are observed. This effect is made worse by the occurrence of hypokalaemia. Another important effect of long-term use of ß-agonists is tachyphylaxis or desensitisation, caused by receptor downregulation. Although a tolerance to the systemic responses does occur, the bronchodilator effect is not affected [1-3]. Another side effect, which became apparent at a later stage, was that some ß- agonists, e.g. clenbuterol at about 10 times the therapeutic dose, caused an increase in muscle mass and a decrease in adipose tissue. The exact way in which this repartitioning effect is accomplished is not known, because many systems are affected by the ß- agonists. The main representative compound of this group of compounds, clenbuterol, can pass the blood-brain-barrier and may influence all hormonal systems that regulate growth. It was found to be an effective growth promoter in pigs and birds, but most spectacular results were obtained in ruminants. Treatment of young animals resulted in a 10-20% higher growth rate and better food conversion. Fat depots were reduced by more than 10% and muscle percentage increased by 10-25%, depending on the muscle studied.

29 Chapter 1.2.2

Figure 1. Formulas of the most important ß-agonists

R1 H H

R2 C C N R4 OH H H

R1 R2 R3 R4 endogenous compounds adrenaline OH H OH H noradrenaline OH H OH methyl phenolic-type ß-agonists

salbutamol H OH CH2OH t-butyl terbutaline OH H OH t-butyl fenoterol OH H OH 4-hydroxy- -methylphenethyl aniline-type ß-agonists

clenbuterol Cl NH2 Cl t-butyl

clenproperol Cl NH2 Cl i-propyl

clenpenterol Cl NH2 Cl t-pentyl

brombuterol Br NH2 Br t-butyl

mabuterol Cl NH2 CF3 t-butyl

mapenterol Cl NH2 CF3 t-pentyl

cimbuterol CN NH2 H t-butyl

cimaterol CN NH2 H i-propyl

As a result, the carcasses were graded 2-4 points higher at slaughter. The effect was found to be dose-dependent, but above a certain optimum dose no additional growth promotion was obtained [4]. It has not yet been reported whether a tolerance to the growth promoting effect occurs, but it may be expected on the basis of earlier findings [3]. The repartitioning effect has led farmers to use the ß-agonists during fattening of cattle. Clenbuterol is a very potent compound and pharmacological effects in humans have been observed with doses of 2.5 µg [5]. Since 1990, already 6 reports of clenbuterol poisonings by food products have been described in the medical literature [6-11]. The reported cases are summarised in Table 1. The main symptoms found were tremors,

30 Beta-Agonists

Table 1. Reported cases of clenbuterol poisoning by food products year place cases source concentration ref 1990 Spain (north) 135 bovine liver 160-291 µg/kg 6 1990 France (Lyon area) 22 veal liver 375-500 µg/kg 7 1991 Spain (Aragon) 59 cinnamon (accidental 8 contamination) 1992 Spain (Catalonia) 232 veal liver, veal 19-5395 µg/kg 8 tongue, cannelloni 1995 Italy (Vicenza) 16 fillet, rump steak 500 µg/kg 9 1996 Spain (Madrid) 15 veal liver 500 µg/kg 10 1996 Italy (Caserta) 62 beef 4500 µg/kg 11 palpitations and tachycardia, nervousness and general malaise. No fatalities have been reported until now. The risks associated with the presence of residues of ß-agonists in meat have led to a ban on the use of these compounds as growth promoters in the European Union [12]. Although clenbuterol has been registered in some countries for therapeutic purposes, this is now restricted to oral use in pets and horses and injections for tocolysis in calving cows. Meat from animals cannot be traded during treatment and during a withdrawal period [12]. The findings on the control of the ban on the use of ß-agonists in food-producing animals in the European Union have been reviewed recently [13]. The data are rather old, but newer data are not publicly available. The results of the analysis of random samples taken under national residue plans are summarised in Table 2. In The Nether- lands ß-agonists have been detected in samples from veal calves, young cattle and cows, but not in samples from pigs, sheep and horses. The number of positive samples found during random sampling decreased from 3-10% in 1989 and 1990 to less than 0.5% in 1994 and 1995. In most of these cases clenbuterol was detected. During targeted sampling, 30% of the urine samples were positive in 1993 and this decreased to 5% in 1995. However, ß-agonists were detected in these years in 11-15% of the feed samples and preparations taken at suspected farms. In those samples mainly clenbuterol was found, but also mabuterol, brombuterol, salbutamol and clenproperol were found. Also, combinations of clenbuterol and mabuterol and combinations with steroids occurred. In Germany, the number of positive urine samples in the farm phase decreased from 16% in 1994 to 10% in 1995. A somewhat larger decrease was seen in the slaughter phase with

31 Chapter 1.2.2

Table 2. Beta-agonists detected in bovine animals from random sampling in the farm phase and the slaughter phase in 1992 and 1993 in the European Union (modified from [13]). Empty boxes mean that data were not available. country farm phase slaughter phase 1992 1993 1992 1993 total % pos total % pos total % pos total % pos Belgium 798 7.6 806 17.6 99 7.1 168 5.4 Denmark 107 0.0 108 0.0 300 0.0 301 0.0 Germany 1.1 Greece 19 20 0.0 20 20 0.0 Spain 5294 1.1 3988 0.9 6515 4.1 7040 4.9 France 1013 0.1 3.8 953 4.2 Ireland 0 69 0.0 781 1.3 210 0.0 Italy 0 0 7121 3.7 5883 6.8 Luxembourg 0 0 24 20 0.0 Netherlands 848 0.7 920 0.8 2069 1.0 2091 2.0 Portugal 132 0.0 97 0.0 1112 5.4 821 2.6 United Kingdom 156 0.0 175 0.0 509 0.4 1242 0.0

15% positives in 1994 and 5% in 1995. However, in some cases eyes were collected at slaughter and when these were analysed the ß-agonists were detected in 15% and 25% of the samples for 1994 and 1995, respectively. In Northern Ireland random liver samples taken from cattle revealed only 3 positives of 151 samples in 1992 and 1 of 219 samples in 1994. However, targeted sampling in the slaughter phase resulted in 6% positive retina and liver samples in 1992 and 1993 and 1.5% in 1994, which is much better than the 36% and 49% positive bile and liver samples in 1990 and 1991, respectively. Urine samples taken at suspected farms revealed 19% positives in 1991 and this decreased to 0% in 1994. In 1994 also hair samples were taken during targeted sampling on the farm and of these samples 28% were positive. This indicates that although the use of ß- agonists may not be detected by the authorities, the ß-agonists are still used extensively [13]. In 1994, European consumer organisations performed their own survey on the use of clenbuterol. They bought bovine livers in butcher shops and had them analysed for the presence of clenbuterol. The results are summarised in Figure 2 [14]. In total 92 out of 936 positive samples were found positive. In The Netherlands 6 of 60 livers contained residues of clenbuterol, whereas in 1993 only 1 of 43 livers was positive [14,15].

32 Beta-Agonists

Figure 2. Percentage of livers bought at butcher shops found to contain clenbuterol in European countries (modified from [14]).

40 36 35

30 25 23 20

15 12 10 10 10 8 7 percentage positive 5 3 5 2 2 0 0 Italy Spain Ireland France Greece United Belgium Portugal Kingdom Denmark Germany Netherlands Luxembourg country

According to the Dutch veterinary inspection, in 1992 5% of beef cattle and 3% of culled cows were illegally treated with clenbuterol and on 12% of suspected farms clenbuterol was found [15]. Recently, it was reported that clenbuterol had also been detected in show animals and in meat-producing animals in the United States. Also, an animal feed supplier who illegally imported unapproved drugs including clenbuterol was convicted [16]. In Canada in 1993-1995 between 0.3% and 0.8% of the compliance samples were found to contain residues of ß-agonists [17].

Residues and Metabolism An overview of the pharmacokinetics, metabolism and residues of the ß-agonists, paying specific attention to the use in food-producing animals, has appeared recently [18]. The overview given here will mainly deal with clenbuterol as this is the substance detected in most samples. The distribution of 14C-clenbuterol in the body after oral administration was studied in rat [19] and in calf [20]. Three hours after an oral dose of 2 mg/kg in the rat, tissues with more than 1% of total radioactivity were stomach (41%), small intestine (10%),

33 Chapter 1.2.2 liver (8%), urine (5%), kidney (4%), blood (2%) and lung (1%). After 24 hours 58% of the total radioactivity had been excreted in urine and 14% in faeces. Significant amounts were then also present in the stomach (13%), small (3%) and large (6%) intestine and liver (2%). Two days after dosing 75% of the total radioactivity had been excreted in urine and 20% in faeces and residues were only found in the large intestine (1%) and liver (0.5%) [19]. In calves the residues in the tissues remaining two days after a 3 mg/kg dose were much larger. Only 42% of the total radioactivity had been excreted in urine and 2% in faeces. Large amounts of clenbuterol were still present in the gastrointestinal tract: stomach (10%), small intestine (4%) and large intestine (4%). Significant residues were found in liver (5%), lung (4%), blood (1%) and kidney (1%). The carcass, including adipose tissue, muscle and skin, still contained 23% of the total dose of radioactivity that had been administered. High concentrations were found in iris (260 ppm), retina/ choroid (85 ppm), bile (13 ppm), lung (8 ppm), kidney (6 ppm) and liver (5 ppm). Of the total amount of radioactivity detected in heart, spleen and kidney, only 60% originated from unchanged clenbuterol. In lung and liver this was 80% and 45%, respectively. This indicates that clenbuterol undergoes metabolic changes [20]. It was found that clenbuterol was also excreted in milk [21]. Although clenbuterol has a relatively long half life, its use cannot be detected in urine after a withdrawal period of about one week [22,23]. Therefore, alternatives were sought, which would allow detection for a longer period of time. Liver was found to contain detectable residues for up to 56 days after withdrawal using an immunoassay, but it was doubted if the low concentrations at that time could be confirmed by GC-MS [24,25]. Liver is also suggested to be a good indicator for the presence of residues in edible tissues and therefore, as a suitable tissue for surveillance analysis [26]. Clenbuterol abuse can even be detected for a much longer time by the analysis of choroid and pigmented retinal tissues. In this tissue very high concentrations of clenbuterol were found [20,23-26] and abuse could be detected for at least 140 days after stopping of treatment [24]. Also, in a study where liver and retina samples from 703 animals suspected to be treated with clenbuterol were taken, clenbuterol was detected in both tissues in only 46 cases, but retina revealed another 50 positive samples [25]. However, a main disadvantage of the use of eye tissues is that they are only available in small amounts at slaughter. Clenbuterol was found to be excreted in hair [27-31]. Because it takes about 10 days before a new hair has grown through the skin, it can generally not be detected in the first week of treatment [30,31]. However, after this lag time, use at a therapeutic dose of 0.8 µg/kg twice daily in calves could be detected for at least 60 days after stopping of treatment [30]. Also, 80 days after 15 days at a growth promoting dose 3.33 mg/day

34 Beta-Agonists

(about 30 µg/kg) twice daily in calves high levels of clenbuterol were present in the hair [31]. Concentrations in black hair were found to be higher than those in white and brown hair of guinea pigs [28,29]. Also, in a study with male veal calves of different breeds with different hair colours treated with a therapeutic dose of clenbuterol larger amounts were found in dark grey and black hair. At 25 and 35 days the concentration in all coloured hair samples was higher than that in white hair [30]. In studies with guinea pigs it was found that treatment with salbutamol at high doses can also be detected in hair samples. However, its concentrations were lower than those of clenbuterol, probably due to its more polar nature [28,29]. The first studies on the metabolism of clenbuterol were performed at Karl Thomae GmbH [19,32,33] in rat, rabbit, dog and man. It was found that clenbuterol was mainly excreted unchanged in urine (Table 3). Eight metabolites were found, which were not identified. In an abstract from the same source the metabolic pattern observed in dog was given [34] (see Figure 3). The same scheme was suggested to be applicable to all mammalian species [5]. In dog, man, rat and rabbit the same metabolites were found, but

Table 3. Amounts of the metabolites of clenbuterol (expressed as % of radioactivity excreted in urine) detected in the urine of rats (n=5, dose 2 mg/kg), rabbits (n=5, dose 2.5 mg/kg), dogs (n=1, dose 2.5 mg/kg) and human volunteers (n=6, dose 20 µg) after oral administration [19,32,33] rat rabbit dog man urine collected for 0-72 h 0-24 h 0-24 h 0-24 h % of dose in urine 55.6 82.8 69.1 65.0 metabolite nature M0 clenbuterol 69.7 67.1 41.1 66.4 M1 neutral 0.6 2.0 1.0 0.8 M2 basic 0.3 0.2 M3 basic M4 acidic 0.9 2.2 6.2 3.5 M5 acidic 1.6 0.5 1.3 M6 neutral 0.2 0.5 0.2 1.2 M7 acidic 0.9 5.6 15.4 5.4 M8 acidic 5.4 1.4 3.9 2.7 residual radioactivity 7.8 9.3 5.2 6.2 undefined radioactivity 12.9 11.1 25.5 13.8

35 Chapter 1.2.2

Figure 3. Metabolites detected in the urine of a dog after administration of clenbuterol (modified from [34]).

Cl Cl H H CH H H CH3 3 NH C C N C CH NH2 C C N C CH3 2 3 OH H GlucCH O H H CH3 3 Cl Cl Gluc

Cl Cl H H CH3 - H H CH3 SO3 N C C N C CH3 NH2 C C N C CH3 H OH H H CH3 O H H CH3 Cl Cl CH2 Cl H H CH3 CH3

NH2 C C N C CH3

OH H H CH3 Cl

Cl Cl H H CH H 3 NH2 C C N C CH3 NH2 C COOH OH H H CH2 OH OH Cl Cl

Cl Cl H H CH3

NH2 C COOH NH2 C C N C CH3 O OH H H COOH Cl Cl

Cl Cl H

NH2 COOH NH2 C N C COOH O H H Cl Cl

Cl H H CH O 3 + NH2 OH C C N C CH3 O- H H CH3 Cl

36 Beta-Agonists in different amounts. The amount of unchanged drug in urine were now reported to be 61% in man, 37% in rabbit, 26% in rat and 24% in dog [34]. In cattle, clenbuterol was mainly excreted in urine as the unchanged drug (28-52%). In addition, small amounts of 4-amino-3,5-dichlorobenzoic acid in urine and liver and of 4-amino-3,5-dichlorohippuric acid in urine were observed [5]. Recent studies [35-39] have shown that other metabolites are also formed by oxidation of the primary aromatic amine function. The resulting arylhydroxylamine metabolite is labile due to oxidoreductive reactions and was therefore probably not identified in earlier studies. In rats dosed 200 µg/kg 3H-clenbuterol 38% of the administered dose was recovered in urine within 7 days. At least 9 metabolites were found. Unchanged clenbuterol formed 20-25% of the urinary radioactivity and the arylhydroxylamine 30-40%. Three other metabolites comprised 5-10% of urinary radioactivity on days 1-2 and 15-20% on days 3-4. Of these three only clenbuterol N- sulfamate was identified. Also, a product formed through hydroxylation of the t-butyl group was reported [35,36]. In a calf that had received a dose of 5 µg/kg 3H-clenbuterol more than 45% of the dose was recovered in urine within 48 hours. The products identified were unchanged clenbuterol (40% of urinary radioactivity), the arylhydroxylamine metabolite (40%) and clenbuterol N-sulfamate (15%) [36]. A more detailed study in rats showed that the amount of unchanged clenbuterol and of the arylhydroxylamine metabolite depended on the administered dose: They increased from 6% and 6.5% at a dose of 4.5 µg/kg to 30% and 27% at a dose of 45 mg/kg, respectively. The arylhydroxylamine metabolite was found to be reduced to clenbuterol easily and it could also be oxidised to nitroso-clenbuterol and nitro-clenbuterol. Both compounds were found in samples that had been stored for some time, but the latter compound was also detected when samples were analysed directly [37]. In vitro studies confirmed the formation of the arylhydroxylamine metabolite as the major metabolite of clenbuterol. Trace amounts of nitro-clenbuterol were also formed. Minor metabolites detected in some experiments were 4-amino-3,5-dichlorobenzoic acid, 4-amino-3,5-dichlorohippuric acid, clenbuterol N-sulfamate and 4-amino-3,5-dichloro- -(2-hydroxy-1,1-dimethyl)ethylaminomethyl- benzyl alcohol (this is the metabolite with a hydroxyl in the t-butyl group) [38]. There are indications that the arylhydroxylamine metabolite may bind to DNA, hemoglobin and other proteins. This may result in several toxicological effects [37]. However, very recently it was reported that the arylhydroxyl metabolite could not be detected in the tissues of calves that had received a single dose or repeated doses of 14C-clenbuterol (5 µg/kg body weight). Furthermore, more than 90% of the tissue residues were found to consist of the unchanged parent compound [39].

37 Chapter 1.2.2

Analytical Methods As mentioned above, the ß-agonists are divided in a phenolic and an anilinic group. The chemical properties of these two groups are different, because of the presence of the phenol or the aromatic amine function. The phenolic ß-agonists have both acidic and basic functions with the phenol and the aliphatic amine, respectively, whereas the anilines only have basic functional groups. The pKas of the endogenous and phenolic ß- agonists have been published to vary between 8.5 and 9.3 for the basic aliphatic amine and are 9.8-10.3 and 11.0-12.0 for the first and second acidic phenol, respectively [40,41]. As a result for analytical purposes this group of substances is considered to be ionised under all pH conditions. For the anilines only the pKas of clenbuterol have been published. They are 9.6 and < 1 for the aliphatic and aromatic amine, respectively [42]. The other anilines will most probably show a similar behaviour. The UV spectra of the phenolic ß-agonists show a maximum at 275-280 nm under acidic conditions and at 295-297 nm under alkaline conditions. However, the absorbance at wavelengths below 230 nm is much larger [41]. This is also the case for clenbuterol, for which maxima at 240 and 300 nm have been reported [42]. An extensive review covering sample pre-treatment and detection methods for the analysis of the ß-agonists in biological matrices has appeared [43]. The amount of conjugates formed is relatively low for clenbuterol (see above). However, for the phenolic ß-agonists, substantial conjugate formation has been reported [18,44]. Therefore, a broad screening procedure should include an enzymatic or solvolytic deconjugation step. Enzy- matic digestion of tissue proteins was found useful because of protein binding of the ß- agonists [45,46]. After this pre-treatment, the primary extract or sample is further cleaned up by liquid-liquid extraction (LLE), solid phase extraction (SPE), matrix-solid phase dispersion (MSPD) or immunoaffinity chromatography (IAC). For LLE the sample should be extracted either at an alkaline pH to ensure that the ß-agonists are in the unprotonated form or an ion-pair extraction should be performed [47,48]. Also, on- column liquid-liquid partitioning using hydrophilic packing materials, like diatomaceous earth, has been used [49]. Again an alkaline pH is required and phenolic ß-agonists were not eluted using non-polar or semi-polar solvents. Different SPE sorbents have been used to extract the ß-agonists. Reversed phase materials [50-53] require an alkaline sample pH, which may damage the sorbent. Adsorption materials like silica, can act as adsorbent and ion exchanger with basic analytes and have been used to extract clenbuterol from animal feed [54]. Mixed phases, containing both reversed phase and cation exchange functional groups [45,55], can be used for more multi-residue procedures, but these are difficult to optimise for both phenol- and aniline-type compounds. For MSPD the sample

38 Beta-Agonists is blended with C18 material and then poured in a column, after which the analyte is eluted [56]. Wash steps to achieve further clean up can be used. IAC [57,58] uses antibodies against the target molecule, which may result in high specificity and sample clean up efficiency. An antibody against the N-t-butyl group provides good cross- reactivity with various ß-agonists of interest [58]. On-line extraction procedures using SPE sorbents or immunosorbents have been reported [53,57]. Recently, the ion-pair supercritical fluid extraction of clenbuterol from different food samples has been reported [59]. For the detection of the ß-agonists in the extract, immunoassays can be used, or a chromatographic separation followed by detection can be performed. Immunoassays have very low detection limits (0.5 ng/ml and better). Both radio immunoassays and enzyme immunoassays are used. However, because antibodies are never substance-specific, the detected substance cannot be identified. Therefore, immunoassays are mainly used as screening methods [50,60,61] or as detection method after an HPLC separation [51,62]. Also, a radioreceptor assay has been reported [47]. The advantage of the use of a radioreceptor assay for screening purposes is that all substances that have an affinity for the receptor can be detected. However, the detected compounds are normally not identified. For HPLC, mainly reversed phase columns have been used with buffered mobile phases, either with [49,52,57] or without [51,53] an ion-pair reagent. For detection UV with [63] or without derivatisation [49,52,53,57], fluorescence for ß-agonists with a catechol or resorcinol group [42], electrochemical detection [52] and MS [64] can be considered. The use of GC requires derivatisation, for which different reagents [58,65] have been suggested. GC is mostly combined with an MS detector. TLC and CE are suitable for the separation of the ß-agonists, but detection limits may present problems for residue analysis [43]. Most reported methods have been developed for single compounds or for a small group of structurally related substances. With the exception of IAC extraction of both phenolic and aniline ß-agonists in one step is complicated by differences in polarity and by the fact that the phenols are ionised under all pH conditions. Another difficult point is that the concentrations of the ß-agonists in urine may vary considerably. This is caused by differences in dosing regimen and by the rapid clearance of the compounds. There- fore, detection of abuse in urine samples is not possible from about one week after treatment has stopped.

39 Chapter 1.2.2

References [1] Adrenergic transmission, in: H.P. Rang and M.M. Dale, Pharmacology, Churchill Livingstone, Edinburgh, 1987, ISBN 0-443-03407-9, p 146-176 [2] D.J. Morgan, Clin Pharmacokinet 18 (1990) 270-294 [3] B.J. Lipworth, Drug Safety 7 (1992) 54-70 [4] M. Lafontan, M. Berlan and M. Prud’hon, Reprod Nutr Develop 28 (1988) 61-84 [5] K.N. Woodward, Clenbuterol, in: The forty-seventh meeting of the joint FAO/WHO expert committee on food additives (JECFA), Toxicological evaluation of certain veterinary drug residues in food, WHO food additives series 38, ICPS, WHO, Geneva, 1996, ISBN 92-4-166038-4, p 3-43 [6] J.F. Martinez-Navarro, Lancet 336 (1990) 1311 [7] C. Pulce, D. Lamaison, G. Keck, C. Bostvironnois, J. Nicolas and J. Descotes, Vet Hum Toxicol 33 (1991) 480-481 [8] L. Salleras, A. Domínguez, E. Mata, J.L. Taberner, I. Moro and P. Salvà, Public Health Rep 110 (1995) 338-342 [9] S. Maistro, E. Chiesa, R. Angeletti and G. Brambilla, Lancet 346 (1995) 180 [10] J. Bilbao-Garay, J.F. Hoyo-Jimenez, M. Lopez-Jimenez, M. Viruesa-Sebastian, J. Perianes-Matesanz, P. Munoz-Moreno and J. Ruiz-Galiana, Rev Clin Esp 197 (1997) 92-95 via Medline [11] G. Brambilla, A. Loizzo, L. Fontana, M. Strozzi, A. Guarino and V. Soprano, J Am Med Assoc 278 (1997) 635 [12] Council directive 96/22/EU, Off J Eur Commun L125 (1996) 3-9 [13] H.A. Kuiper, M.Y. Noordam, M.M.H. van Dooren-Flipsen, R. Schilt and A.H. Roos, J Anim Sci 76 (1998) 195-207 [14] Dutch Consumer Organisation, Consumentengids, February 1995, 104-107 [15] Dutch Consumer Organisation, Consumentengids, June 1993, 348-351 [16] G.A. Mitchell and G. Dunnavan, J Anim Sci 76 (1998) 208-211 [17] E. Neidert and P. W. Saschenbrecker (1996), in: N. Haagsma and A. Ruiter (ed), EuroResidue III Conference on Residues of Veterinary Drugs in Food, Veldhoven, The Netherlands, May 6-8, 1996, University of Utrecht, Faculty of Veterinary Medicine, Utrecht, The Netherlands, ISBN 90-6159-023-X, p 185-190 [18] D. J. Smith, J Anim Sci 76 (1998) 173-194 [19] Z. Kopitar and A. Zimmer, Arzneim Forsch 26 (1976) 1435-1441 [20] D.J. Smith and G.D. Paulson, J Anim Sci 75 (1997) 454-461 [21] B. Stoffel and H.H.D. Meyer, J Anim Sci 71 (1993) 1875-1881

40 Beta-Agonists

[22] A. Papillon, B. Turck and G. Briantais, Revue de l’Alimentation Animale, Décembre 1988, 63-64 [23] H.H.D. Meyer and L.M. Rinke, J Anim Sci 69 (1991) 4538-4544 [24] C.T. Elliott, S.R.H. Crooks, J.D.G. McEvoy, W.J. McCaughey, S.A. Hewitt, D. Patterson and D. Kilpatrick, Vet Res Commun 17 (1993) 459-468 [25] C.T. Elliott, J.D.G. McEvoy, W.J. McCaughey, D.H. Shortt and S.R.H. Crooks, Analyst 118 (1993) 47-448 [26] M.J. Sauer, R.J.H. Pickett, S. Limer and S.N. Dixon, J vet Pharmacol Therap 18 (1995) 81-86 [27] A. Adam, N. Gervais, A. Panoyan, H. Ong, L. Beliveau, C. Ayotte and Ph. Delahaut, Analyst 119 (1994) 2663-2666 [28] A. Polettini, J. Segura, G. Gonzalez, X. de la Torre and M. Montagna, Clin Chem 41 (1995) 945-946 [29] A. Polettini, M. Montagna, J. Segura and X. de la Torre, J Mass Spectrom 31 (1996) 47-54 [30] A. Gleixner, H. Sauerwein and H.H.D. Meyer, Arch Lebensmittelhyg 47 (1996) 131-135 [31] Y. Gaillard, A. Balland, F. Doucet and G. Pépin, J Chromatogr B 703 (1997) 85-95 [32] A. Zimmer, Arzneim Forsch 26 (1976) 1442-1445 [33] A. Zimmer, Arzneim Forsch 26 (1976) 1446-1450 [34] J. Schmid, A. Prox, Z. Zimmer, J. Keck, S. Kaschke, Fresenius J Anal Chem 337 (1990) 121 [35] L. Debrauwer, D. Zalko and J. Tulliez (1996) in: N. Haagsma and A. Ruiter (ed), EuroResidue III Conference on Residues of Veterinary Drugs in Food, Veldhoven, The Netherlands, May 6-8, 1996, University of Utrecht, Faculty of Veterinary Medicine, Utrecht, The Netherlands, ISBN 90-6159-023-X, p 367-371 [36] D. Zalko, G. Bories and J. Tulliez (1996) in: N. Haagsma and A. Ruiter (ed), EuroResidue III Conference on Residues of Veterinary Drugs in Food, Veldhoven, The Netherlands, May 6-8, 1996, University of Utrecht, Faculty of Veterinary Medicine, Utrecht, The Netherlands, ISBN 90-6159-023-X, p 993-997 [37] D. Zalko, L. Debrauwer, G. Bories and J. Tulliez, Chem Res Toxicol 10 (1997) 197- 204 [38] D. Zalko, E. Perdu-Durand, L. Debrauwer, M.-P. Bec-Ferte and J. Tulliez, Drug Metab Dispos 26 (1998) 28-35 [39] D. Zalko, G. Bories and J. Tulliez, J Agric Food Chem 46 (1998) 1935-1943

41 Chapter 1.2.2

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42 Beta-Agonists

[58] L.A. van Ginkel, R.W. Stephany, H.J. van Rossum and J. Farla, in: J.H.M. Metz and C.M. Groenestein (ed), New Trends in Veal Calf Production, EAAP publication 52, ISBN 90-220-1016-3, 1991, p. 192-197 [59] M.M Jimenez-Carmona, M.T. Tena and M.D. Luque de Castro, J Chromatogr A 711 (1995) 269-276 [60] Ph. Delahaut, M. Dubois, I. Pri-Bar, O. Buchman, G. Degand and F. Ectors, Food Addit Contam 8 (1991) 43-53 [61] G. Degand, A. Bernes-Duyckaerts and G. Maghuin-Rogister, J Agric Food Chem 40 (1992) 70-75 [62] P.M. Krämer, Q.X. Li and B.D. Hammock, J AOAC Int 77 (1994) 1275-1287 [63] D. Courtheyn, C. Desaever and R. Verhe, J Chromatogr 564 (1991) 537-549 [64] A. Polettini, J Chromatogr B 687 (1996) 27-42 [65] M.-P. Montrade, D. Maume, B. le Bizec, K. Pouponneau and F. Andre, J Mass Spectrom 32 (1997) 626-644

43 CHAPTER 1.2.3 OTHER WAYS TO PROMOTE GROWTH

Apart from the anabolic steroids and the beta-agonists several other techniques can be used to improve the growth rate of cattle: Addition of subtherapeutic doses of antibiotics to the feed, injections with growth hormone, immunomodulation, and photoperiodic regulation. Growth can also be increased by gene transfer, but this has rarely been tried because of the tremendous cost [1]. Antibiotics administered in subtherapeutic doses exert their growth promoting effect in two ways: · They increase beneficial micro-organisms in the microflora normally present in the gastrointestinal tract, resulting in an improvement of the digestion and absorption of nutrients; · They improve the efficiency of the digestion of cellulosic and fibrous material by the microflora in the rumen resulting in more beneficial volatile fatty acids and less methane. They typically increase the average daily gain by about 10% in comparison with control animals, but are much less effective in ruminants than in single-stomach animals [2]. The following antibiotics are allowed as feed additives for bovine animals in both the European Union and the United States: Avoparcin, bambermycin, monensin, mupirocin, spiramycin, virginiamycin, and zinc bacitracin [2,3]. Previously, nitrovin was also allowed, but it has now been withdrawn in the European Union. Spiramycin and zinc bacitracin should not be used in adult animals. The antibiotics are usually added at concentrations below 50 mg/kg to the feed [2]. Their main advantages over other growth promoters are that they can be administered orally and that absorption from the gastrointestinal tract is not necessary. Also, when they are absorbed, they are rapidly and almost completely metabolised with the exception of avoparcin. Therefore, no residues will be found in the meat and no withdrawal time is necessary. However, the unchanged drug is excreted into the environment and the gut flora of the animal is changed. This may result in resistant strains of bacteria and in shedding of microbial pathogens. There- fore, only those products with little or no application as therapeutic agents in man or animals have been permitted as growth promoters [3].

45 Chapter 1.2.3

Analytical methods used for the detection of residues of antibiotics in meat can be divided into microbiological and physicochemical methods. Microbiological screening procedures give little information about the identity of the detected substance and non- active metabolites are not detected. However, they can be performed rather quickly and no sample pre-treatment is necessary. The four-plate test is widely used in the European Union for samples of fresh meat. Three agar plates are incubated for 18-24 h at 30 ° C with Bacillus subtilis at a pH 6.0, 7.2 or 8.0 and a fourth plate for 18-24 h at 37 ° C with Micrococcus luteus at pH 8.0. To the medium of the plate at pH 7.2 trimethoprim is added to increase the sensitivity for sulphonamides. The Swab Test On Premises (STOP) is used in the United States. A swab is soaked in the exsudate of a carcass and is then laid on special agar medium and incubated for 16-18 h at 30 ° C with Bacillus subtilis at pH 7.9. These two screening methods can be used for the detection of a large number of antibiotics [4,5]. The Charm II Test utilises a receptor or antibody, for which the analyte competes with a labelled drug. Test kits are now available for all common antimicrobial drug families. The test takes about 15 minutes to perform and has been used with dairy products, tissues, eggs and feed [6]. Chromatographic methods allow identification and quantitation of the detected antibiotic. GC has only limited application as the substances are rather polar, non-volatile and sometimes heat-sensitive. Therefore, HPLC with a reversed phase stationary phase is most often used. In some cases an ion-pair reagent is added to the mobile phase. Detection is performed by UV or fluorescence, or after post- column derivatisation [7]. Recently, extraction methods for antibiotics from biomatrices [8], LC-MS of antibiotics [9] and extraction and analysis with LC-MS of antibiotics in meat and milk [10] with particular attention to residue analysis were reviewed. The corticosteroids are, or have been derived from endogenous hormones excreted by the adrenal cortex. They are divided in the mineralocorticoids with an effect on the excretion of sodium and potassium by the kidneys and the glucocorticoids which alter intermediary metabolism, but they all exhibit both effects to some degree. They are used in physiological doses in replacement therapy. In larger, pharmacological doses, the glucocorticoids decrease inflammation, suppress the immune system, increase protein catabolism, promote gluconeogenesis, redistribute fat and produce a negative calcium balance [11]. This may result in reduced growth rates and muscle atrophy. However, in low doses of about 0.1-0.5 ppm in feed dexamethasone increased live weight gain and reduced the feed conversion ratio [12]. They may also increase the efficacy of ß-agonists [13]. Between 1994 and 1996 2-6% of analysed samples in Belgium were found to contain corticosteroids. Dexamethasone was found in most of these samples [14]. Various sample preparations techniques and HPLC methods for the analysis of cortico-

46 Other Ways to Promote Growth steroids in biomedical samples have been reviewed [15]. Other methods have been specifically developed for multi-residue analysis of corticosteroids in the urine of cattle or horses, including IAC-GC-MS [16], solid phase micro-extraction (SPME) with LC-MS detection [17] and IAC-LC-MS [18] to mention a few. An HPTLC method that can be used to screen injection sites for the presence of 14 corticosteroids and 12 esters has also been reported [19]. Injections with bovine growth hormone can increase average daily gain in heifers by about 10%. Yet, in entire males and steers, the response is variable, ranging from an increase of 18% to zero-effect or a reduction of 38% [20]. Feed efficiency is generally improved. Fat content of the carcass is 10-20% lower than in control animals. The optimum dose has been estimated to be between 41 and 64 µg/kg bodyweight/day and injections 4 times a day seem to be more effective because they more closely resemble the normal secretion pattern [20]. Growth hormone is also used to increase milk production in cows. No changes in the composition of the milk have been observed after treatment. Under normal conditions growth hormone is not detectable in milk and meat from treated animals. Also, because it is a protein, it will be digested in the human gastrointestinal tract after consumption. Therefore, according to the author no risks are expected from the use of growth hormone in meat or milk production [21]. In immunomodulation the animal is immunised against one of its own endogenous hormones. This hormone is made antigenic by coupling it to a larger foreign carrier molecule, like human serum albumin, and is then injected together with an immuno- stimulant. Several possible applications of this technique have been reported. To reduce aggressive behaviour in bulls they are normally castrated, but this reduces efficiency and increases the fat content of the meat. Immunisation against gonadotrophin-releasing hormone in bull calves resulted in reduced aggressive behaviour with a normal growth rate. This ‘immunocastration’ is supposed to be a good alternative to surgical castration and it will be acceptable to the consumer [22]. Duration of the daylight cycle (photoperiod) affects seasonal reproductive cycles and growth in livestock. A long-day photoperiod stimulated live weight gain by 11-17% in heifers. It was found that daylight should be present 14-17 hours after dawn to obtain an optimum effect. However, it is not necessary to have the light on during all of these 16 hours as a cycle of 7 h light - 9 h dark - 1 h light - 7 h dark was found to be equally effective. This treatment tended to increase the protein percentage and reduce the fat percentage in heifers. Animal health seems to be unaffected by the longer photoperiod [23].

47 Chapter 1.2.3

References [1] M. Müller and G. Brem (1996), in: European Commission, Directorate-General VI (Agriculture), Proceedings of the Scientific Conference on Growth Promotion in Meat Production, Brussels, November 29-December 1, Office for Official Publications of the European Communities, Luxembourg, ISBN 92-827-6321-8, p 213-232 [2] N.T. Crosby, Determination of Veterinary Residues in Food, Ellis Horwood Series in Food Science and Technology, Ellis Horwood, Chichester, 1991, ISBN 0-747- 0065-1, Chapter 6, p 148-176 [3] F.R. Ungemach (1996), in: European Commission, Directorate-General VI (Agri- culture), Proceedings of the Scientific Conference on Growth Promotion in Meat Production, Brussels, November 29-December 1, Office for Official Publications of the European Communities, Luxembourg, ISBN 92-827-6321-8, p 333-346 [4] G. Schramm, L. Ellerbroek, E. Weise and G. Reuter (1993), in: N. Haagsma, A. Ruiter and P.B. Czedik-Eysenberg (ed), EuroResidue II, Conference on Residues of Veterinary Drugs in Food, May 3-5, 1993, Veldhoven, The Netherlands, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, ISBN 90-6159-016-7, p 632- 636 [5] N.T. Crosby, Determination of Veterinary Residues in Food, Ellis Horwood Series in Food Science and Technology, Ellis Horwood, Chichester, 1991, ISBN 0-747- 0065-1, Chapter 4, p 81-122 [6] E. Zomer, B. Salter, D. Legg, J. Lawton Scheemaker, L. Plumley and S.E. Charm (1993), in: N. Haagsma, A. Ruiter and P.B. Czedik-Eysenberg (ed), EuroResidue II, Conference on Residues of Veterinary Drugs in Food, May 3-5, 1993, Veldhoven, The Netherlands, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, ISBN 90-6159-016-7, p 706-709 [7] B. Shaikh and W.A. Moats, J Chromatogr 643 (1993) 369-378 [8] R.W. Fendiuk and P.J. Shand, J Chromatogr A 812 (1998) 3-15 [9] W.M.A. Niessen, J Chromatogr A 812 (1998) 53-75 [10] D.G. Kennedy, R.J. McCracken, A. Cannavan and S.A. Hewitt, J Chromatogr A 812 (1998) 77-98 [11] H.P. Rang and M.M. Dale, in: Pharmacology, Churchill Livingstone, Edinburgh, 1987, ISBN 0-443-03407-9, p 393-403 [12] D. Courtheyn, J. Vercammen, H. de Brabander, I. Vandenreyt, P. Batjoens, K. Vanoosthuyze and C. van Peteghem, Analyst 119 (1994) 2557-2564

48 Other Ways to Promote Growth

[13] S. Calvarese, P. Rubini, G. Urbani, N. Ferri, V. Ramazza and M. Zucchi, Analyst 119 (1994) 2611-2615 [14] Data from ‘Instituut voor Veterinaire Inspectie’, Belgium obtained via the OSTC database (http://cemu10.ulg.ac.be/ostc) on Internet, 1998 [15] P. Volin, J Chromatogr B 671 (1995) 319-340 [16] Ph. Delahaut, P. Jacquemin, Y. Colemonts, M. Dubois, J. de Graeve and H. Deluyker, J Chromatogr B 696 (1997) 203-215 [17] D.A. Volmer and J.P.M. Hui, Rapid Commun Mass Spectrom 11 (1997) 1926-1934 [18] C.S. Creaser, S.J. Feely, E. Houghton and M. Seymour, J Chromatogr A 794 (1998) 37-42 [19] K.E. Vanoosthuyze, L.S.G. van Poucke, A.C.A. Deloof and C.H. van Peteghem, Anal Chim Acta 275 (1993) 177-182 [20] K. Sejrsen, N. Oksbjerg, M. Vestergaard and M.T. Sorensen (1996), in: European Commission, Directorate-General VI (Agriculture), Proceedings of the Scientific Conference on Growth Promotion in Meat Production, Brussels, November 29- December 1, Office for Official Publications of the European Communities, Luxembourg, ISBN 92-827-6321-8, p 87-119 [21] O. Butenandt (1996), in: European Commission, Directorate-General VI (Agri- culture), Proceedings of the Scientific Conference on Growth Promotion in Meat Production, Brussels, November 29-December 1, Office for Official Publications of the European Communities, Luxembourg, ISBN 92-827-6321-8, p 325-332 [22] W.J. Enright and J.F. Roche (1996), in: European Commission, Directorate-General VI (Agriculture), Proceedings of the Scientific Conference on Growth Promotion in Meat Production, Brussels, November 29-December 1, Office for Official Publi- cations of the European Communities, Luxembourg, ISBN 92-827-6321-8, p 169- 195 [23] H.A. Tucker (1996), in: European Commission, Directorate-General VI (Agri- culture), Proceedings of the Scientific Conference on Growth Promotion in Meat Production, Brussels, November 29-December 1, Office for Official Publications of the European Communities, Luxembourg, ISBN 92-827-6321-8, p 197-212

49 CHAPTER 1.3 SYSTEMATIC QUALITATIVE ANALYSIS

The aim of qualitative analysis is to detect whether substances of interest are present in the sample, to identify them correctly within a reasonable amount of time and to establish the absence of other relevant substances within reasonable limits. This task is complicated by the large number of substances that may be of interest and by the limited amount of the complex specimen that is available for analysis. In our case the substances of interest are the growth promoters. A large number of anabolic substances and ß- agonists have been synthesised already. A list of these substances with some relevant chemical properties is given in Appendix 1. This list is not intended to be complete, as new compounds are continuously synthesised to circumvent detection by the current analytical methods. The large number of relevant substances and the appearance of new compounds that should be detected in the analytical systems makes a systematic approach necessary. Such an approach has been worked out by our group for samples in clinical and forensic toxicology and is called ‘systematic toxicological analysis’ (STA). STA is defined as ‘the logical chemical-analytical search for potentially harmful substances, whose presence are uncertain and their identity unknown’ [1]. Three key steps are recognised in the analytical procedure: a) sample work up, isolation and concentration, b) differentiation and detection, and c) identification (database retrieval). Pre- requisites for STA are: · Retain all relevant substances and remove non-relevant ones (matrix components); · Obtain maximum differentiation in a minimum amount of time and optimum universality and sensitivity in detection, but also differentiation during the detection step; · Have comprehensive and updated databases available, which also include data on metabolites, endogenous interferences and contaminants. Until now no analytical method has been found that is able to identify all relevant toxicological substances unambiguously. Therefore, a combination of the results of two or more techniques is usually needed [1]. Several analytical methods have been developed for STA: TLC with colour codes, HPLC with diode array detection, and GC with MS detection. For the selected systems large databases with toxicologically relevant substances have been published [2-5]. By comparison of the retention and detection

51 Chapter 1.3 parameters obtained for the unknown substance(s) in the analysis with those of the reference data in the database, similarity indices (SI) are obtained, which can easily be combined when multiple analytical techniques are used. A computer program has been developed to perform all the necessary calculations [2]. When the analytical data obtained with an unknown sample are entered, the software produces a list of candidates with their overall SI. Ideally, this list contains only one substance, but it is usually longer. In the latter case the number of possible candidates can be reduced by the determination of analytical data in other systems [1].

References [1] R.A. de Zeeuw, J Chromatogr B 689 (1997) 71-79 [2] J. Hartstra (1997), Computer Aided Identification of Toxicologically relevant Substances by Means of Multiple Analytical Methods, PhD Thesis, State University of Groningen, ISBN 90-367-0831-1 [3] R.A. de Zeeuw, J.P. Franke, H.H. Maurer and K. Pleger (1992), Gas Chromatographic Retention Indices of Toxicologically Relevant Substances on Packed or Capillary Columns with Dimethylsilicone Stationary Phases, VCH, Weinheim, third edition, ISBN 3-527-27396-4 [4] R.A. de Zeeuw, J.P. Franke, G. Machata, M.R. Möller, R.K. Müller, A. Graefe, D. Tiess, K. Pfleger and M. Geldmacher-von Malinckrodt (1992), Gas Chromatographic Retention Indices of Solvents and Other Volatile Substances for Use in Toxicological Analysis, VCH, Weinheim, ISBN 3-527-27395-6 [5] R.A. de Zeeuw, J.P. Franke, F. Degel, G. Machbert, H. Schütz and J. Wijsbeek (1992), Thin-Layer Chromatographic Rf Values of Toxicologically Relevant Substances on Standardized Systems, VCH, Weinheim, second edition, ISBN 3- 527-27397-2

52 CHAPTER 1.4 AIMS, SCOPE AND OVERVIEW OF THESIS

The use of anabolic steroids and ß-agonists as growth promoters is prohibited in the European Union, because residues of these substances may be harmful for the consumer. For the control of this ban samples obtained at farms and in slaughterhouses are analysed. At this moment mainly GC-MS techniques are used for the final identification and quantitation steps. They require extensive clean up of the sample and the analytes must be derivatised. Also, deuterated compounds are used as internal standards. All this results in expensive and time-consuming procedures. The aim of the project was to develop more rapid and cost-effective multi-residue methods for the detection of growth promoters in the urine of food-producing animals. This was undertaken as a multi-centre endeavour and financed by Directorate VI of the European Commission under contract AIR3-CT94-1511. A system based on the principles of systematic qualitative analysis and similar to the one used for toxicological samples was envisaged (see chapter 1.3). Ideally, such a system may have sufficient potential and identification power that it can serve not only for screening, but also for identification and quantitation of a large number of relevant growth promoters. Together with our partners (Laboratoire d’Hormonologie, Centre d’Economie Rurale, Marloie, Belgium; Lab Chrom 1, Merck KGaA, Darmstadt, Germany; and Laboratory for Residue Analysis/Community Reference Laboratory, RIVM, Bilthoven, The Netherlands), TLC, HPLC and GC systems and sample pre- treatment procedures were designed and evaluated. As the system should be used in surveillance, the detection limit aimed for was 0.5 ng/ml, as specified by the EU. How- ever, if it were to turn out that we were unable to reach the above detection limit, it was hoped that the developed methodologies would still be suitable for ‘on farm’ screening during fattening, when expected concentrations of the growth promoters are higher. For the latter purpose, detection limits of several ng/ml would also be acceptable. The main task for our group in Groningen was to develop HPLC methods and SPE procedures. The research was limited to the anabolic steroids including related substances and ß-agonists. In Chapter 2 the development of an SPE-HPLC-DAD method for the analysis of the anabolic steroids and related substances in calf urine is described. For these studies 20

53 Chapter 1.4 anabolic substances, belonging to the androgens, oestrogens, progestagens, stilbenes and resorcylic acid lactones, were used. Chapter 3 deals with the ß-agonists. We evaluated the Ionscan 350, an ion mobility spectrometer, used for the detection of drugs of abuse and explosives, for our project. This apparatus allows for very rapid analysis and has low detection limits. The technique is introduced in Chapter 3.1.1. The use for hair analysis (Chapter 3.1.2), the repro- ducibility (Chapter 3.1.3) and the problems with solvent residues (Chapter 3.1.4) are described. In the next two subchapters an HPLC-UV system was developed for the analysis of clenbuterol. It was extracted from urine with ultra-rapid extraction disks (Chapter 3.2) and mixed-mode SPE columns (Chapter 3.3). The latter column was also used to obtain additional clean up after immuno-affinity chromatography in a multi- residue procedure for the beta-agonists with HPLC-ECD analysis (Chapter 3.4). The methods developed are reviewed in Chapter 4.1. A general discussion on all activities in the AIR project and on the status of the envisaged system for multi-residue analysis is given in Chapter 4.2. Finally, appendices with data on the anabolic steroids and the ß-agonists (Chapter 5.1), a glossary of bovine animals (Chapter 5.2) and a list of the abbreviations used in this thesis (Chapter 5.3) are given.