Hormonal growth promoting agents in food producing animals.

In contrast to the use of hormonal doping agents in sports to enhance the performance of athletes, in the livestock industry hormonal growth promoters ("anabolics") are used to increase the production of muscle meat. This leads to international disputes about the safety of meat originating from animals treated with such anabolics.As a consequence of the total ban in the EU of all hormonal active growth promoters ("hormones") in livestock production, in contrast to their legal use [e.g. of five such hormones (17beta-estradiol, testosterone, progesterone, trenbolone and zeranol) as small solid ear implants and two hormones as feed additives for feedlot heifers (melengestrol acetate) and for swine (ractopamine) in the USA], the regulatory controls also differ sharply between the EU and the USA.In the EU the treatment of slaughter animals is the regulatory offence that has to be controlled in inspection programs. In the USA testing for compliance of a regulatory maximum residue level in the edible product (muscle, fat, liver or kidney) is the purpose of the inspection program (if any).The EU inspection programs focus on sample materials that are more suitable for testing for banned substances, especially if the animals are still on the farm, such as urine and feces or hair. In the case of slaughtered animals, the more favored sample materials are bile, blood, eyes and sometimes liver. Only in rare occasions is muscle meat sampled. This happens only in the case of import controls or in monitoring programs of meat sampled in butcher shops or supermarkets.As a result, data on hormone concentrations in muscle meat samples from the EU market are very rare and are obtained in most cases from small programs on an ad hoc basis. EU data for natural hormones in meat are even rarer because of the absence of "legal natural levels" for these hormones in compliance testing. With the exception of samples from the application sites - in the EU the site of injection of liquid hormone preparations or the site of application of "pour on" preparations - the hormone concentrations observed in meat samples of illegally treated animals are typically in the range of a few micrograms per kilogram (ppb) down to a few tenths of a microgram per kilogram. In the EU dozens of illegal hormones are used and the number of active compounds is still expanding. Besides estrogenic, androgenic and progestagenic compounds also thyreostatic, corticosteroidal and beta-adrenergic compounds are used alone or in "smart" combinations.An overview is given of the compounds identified on the EU black market. An estimate is also given of the probability of consumption in the EU of "highly" contaminated meat from the application sites in cattle. Finally some data are presented on the concentration of estradiol in bovine meat from animals treated and not treated with hormone implants. These data are compared with the recent findings for estradiol concentrations in hen's eggs. From this comparison, the preliminary conclusion is that hen's eggs are the major source of 17alpha- and 17beta-estradiol in the consumer's daily "normal" diet.

Detection of anabolic androgenic steroid abuse in doping control using mammalian reporter gene bioassays.

Anabolic androgenic steroids (AAS) are a class of steroid hormones related to the male hormone testosterone. They are frequently detected as drugs in sport doping control. Being similar to or derived from natural male hormones, AAS share the activation of the androgen receptor (AR) as common mechanism of action. The mammalian androgen responsive reporter gene assay (AR CALUX bioassay), measuring compounds interacting with the AR can be used for the analysis of AAS without the necessity of knowing their chemical structure beforehand, whereas current chemical-analytical approaches may have difficulty in detecting compounds with unknown structures, such as designer steroids. This study demonstrated that AAS prohibited in sports and potential designer AAS can be detected with this AR reporter gene assay, but that also additional steroid activities of AAS could be found using additional mammalian bioassays for other types of steroid hormones. Mixtures of AAS were found to behave additively in the AR reporter gene assay showing that it is possible to use this method for complex mixtures as are found in doping control samples, including mixtures that are a result of multi drug use. To test if mammalian reporter gene assays could be used for the detection of AAS in urine samples, background steroidal activities were measured. AAS-spiked urine samples, mimicking doping positive samples, showed significantly higher androgenic activities than unspiked samples. GC-MS analysis of endogenous androgens and AR reporter gene assay analysis of urine samples showed how a combined chemical-analytical and bioassay approach can be used to identify samples containing AAS. The results indicate that the AR reporter gene assay, in addition to chemical-analytical methods, can be a valuable tool for the analysis of AAS for doping control purposes.

Determination of acetyl gestagenic steroids in kidney fat by automated supercritical fluid extraction and liquid chromatography ion-trap mass spectrometry.

Acetyl gestagenic steroids are isolated from animal tissues such as bovine kidney fat by automated supercritical fluid extraction (SFE). After the addition of internal standards and sample pretreatment, the analytes are extracted from the matrix by supercritical CO2 and trapped directly in-line on alumina placed in the extraction vessel. The samples are analysed by liquid chromatography combined with ion-trap mass selective detection (LC-MSn). For quantification, deuterated internal standards are added and single ions of the analytes and internal standards are monitored. For confirmation of the identity of the analytes, two transition ions (one MS2 and one MS3) were monitored and the ratios between the ions were calculated and compared with those of standards. The detection capability for the multi-analyte LC-MSn analysis of megestrol acetate (MA), medroxyprogesterone acetate (MPA), chlormadinone acetate (CMA) and melengestrol acetate (MGA) is 0.5 microg kg(-1). The mean within-laboratory reproducibility ranges from 16-19% (%RSD) at a concentration level of 0.5 microg kg(-1) (n = 9). Running the SFE procedure overnight allows the analysis of 24 samples of fat per day.