Biomarker Approach for Differentiating Boldenone Administration From Ex Vivo Formation in Colt Urine

Boldenone is a prohibited anabolic-androgenic steroid in equine sports, but also endogenous in colts with an International Threshold of 15 ng/mL in urine. Recently, its presence was demonstrated at very low concentrations in the urine of other genders, resulting in the establishment of biomarker approaches, based on the detection of Δ¹-progesterone and 20α-dihydro-Δ¹-progesterone, to distinguish drug administrations from microbial sources (altered feeds and ex vivo formation in urine). In the current study, the applicability of these biomarkers in colt urine was assessed by quantitative analysis of 121 samples. Boldenone was detected in all samples (24-8420 pg/mL), Δ¹-progesterone was only detected in one sample (< LLOQ of 20 pg/mL) and 20α-dihydro-Δ¹-progesterone in seven samples (only one ≥ LLOQ of 50 pg/mL at 66.7 pg/mL). Analysis of administration samples (boldione and boldenone undecylenate) showed boldenone concentrations exceeding the International Threshold post-administration, but Δ¹-progesterone was not detected, and 20α-dihydro-Δ¹-progesterone was detected in both pre- and post-administration samples (only one ≥ LLOQ of 50 pg/mL at 132 pg/mL). A regulatory urine sample associated with altered feed consumption resulted in detection of boldenone and 20α-dihydro-Δ¹-progesterone at 43 ng/mL and 898 pg/mL, respectively. Therefore, a similar biomarker approach to that previously published for geldings (Δ¹-progesterone > 50 pg/mL or 20α-dihydro-Δ¹-progesterone > 100 pg/mL indicates a microbial origin) could be applied to colt urine samples with boldenone concentrations exceeding the International Threshold. This approach can be used to give confidence that reported findings in colts are caused by steroid administrations rather than microbial activity.