Objectives:To develop methods to detect boldenone metabolites in bovine urine, identify metabolites and propose definitive marker analytes to enable the detection of the abuse of boldenone or the 1,4-androstadiene-3,17-dione steroid supplement in the bovine.Policy Relevance:The use of artifical growth promoting agents in food producing animals is banned in the European Union. Boldenone (1-dehydrotestosterone) is a potent anabolic steroid the esters of which are known to provide a long duration of action. After intramuscular treatment of the bovine the parent steroid along with its 17-alpha epimer (major metabolite), androstadienedione and at least one major metabolite of unknown stereochemistry are excreted in urine. Boldenone, despite its close similarity to testosterone, has been regarded as an exogenous steroid. However, recently it has been detected in the testicles of male pigs and is reported to be present at low levels in the urine of the male horse. More worryingly both 17-alpha and 17-beta boldenone have been found in ‘blank’ bovine faeces and in a test system used to study the biodegradation of testosterone.These findings call into question the validity of using boldenone as a marker analyte for the abuse of the parent compound and its esters. In order to provide an appropriate means of controlling the abuse of boldenone in the bovine a better understanding of the nature and levels of endogenous material is required along with a more detailed knowledge if its metabolism. Steroid supplements as "over the counter" products are now readily available and have potential for abuse in livestock. Equi-bolan, 1,4-androstadiene-3,17-dione, is advertised as an oral steroidal precursor of boldenone. Such products also need to be considered in the development of an effective strategy to control boldenone use.Intended Use of results:It is intended that the proposed research program will provide an improved understanding of the metabolism of boldenone, its esters and the pro-drug 1,4-androstadiene-3,17-dione. A successful project could produce a definitive metabolic marker or markers of administration and provide possible analytical approaches. In the absence of any definitive markers assessment of the data could suggest alternative approaches to the control of boldenone use in the realisation that this may require further research studies.This proposal is intended to provide DEFRA with definitive urinary metabolic markers of the abuse of 1-dehydrotestosterone and its esters or suggest alternative strategies to controlling the illegal use of this steroid.