Boldione (1-dehydro-androstenedione) is a synthetic androgenic anabolic steroid (AAS) structurally related to androstenedione and testosterone bearing a double bound in the C1 position. Boldione is extensively metabolized to boldenone (1-dehydro-testosterone) in humans. Both boldione and boldenone are included in the WADA List of prohibited substances.The metabolism of boldione and/or boldenone is well known [1] and has been reviewed recently with the detection of new metabolites and conjugates [2]. The two compounds follow the same metabolic routes of their endogenous analogues leading to more than 20 metabolites characterized by the presence of the 1 insaturation. The presence of the double bond in the A ring produces characteristic ions at m/z 194 and 206 on the TMS-derivatives spectra of the metabolites, when analyzed by GC/MS. All the metabolites described until now by GC/MS or LC/MS techniques keep this double bond in the structure. More recently, the detection of boldenone and its main metabolite (5-androst-1-en-17-ol-3-one) naturally occurring in human urine samples, obliged the use of isotope ratio mass spectrometry (IRMS) to confirm its synthetic or natural origin [3]. The method developed in our laboratory includes two consecutive HPLC purifications, permitting to determine accurate 13C values for boldenone and 5-androst-1-en-17-ol-3-one and several endogenous reference compounds (ERC). The first HPLC purification step is the same used for the detection of the origin of pseudoendogens or its metabolites (i.e. testosterone, androsterone or androstandiols) routinely applied in the Rome Laboratory.