Evaluation of boldenone formation and related steroids transformations in veal faeces by liquid chromatography/tandem mass spectrometry

Abstract

It is established that bovine urine can result positive for boldenone and androstadienedione in consequence of faecal contamination. The simple transfer of steroids to urine is one minor aspect of faecal contamination. A high de novo production of steroids in faeces after deposition and in faeces-contaminated urine is almost certainly due to microbial activity, although the precursor compounds and transformations leading to the presence of these illegal steroids are unclear. We developed a simple in vitro method - incubation of faecal matter suspended in 0.9% saline - to induce steroid transformations in faeces, and analyzed the products by liquid chromatography/tandem mass spectrometry, without the need for prior extraction. Norethandrolone was the internal standard. The linearity (R(2): 0.987-0.999), sensitivity (LODs: 0.3 to 1.0 ng/mL; LOQs: 1.0 to 3.0 ng/mL), precision (intra-day CVs: 2.6-8.2; inter-day CVs: 4.5-11.5) and accuracy (percentage recovery: 89-120%) were calculated for the studied steroids. Androstenedione, androstadienedione, alpha- and beta-boldenone, testosterone and epitestosterone transformations were investigated. Mutual interconversion of steroids was observed, although 17beta-hydroxy steroids had low stability compared with 17alpha-hydroxy and 17-keto steroids. The results suggest that this simple in vitro system may be an effective way of studying hormone transformations in faeces and, after analogue studies, in faeces-contaminated urine.

Occurrence and fate of androgens, estrogens, glucocorticoids and progestagens in two different types of municipal wastewater treatment plants

“…31 Transformation has been reported for some steroids in the environmental media. 30,[32][33][34]42 Testosterone was found to be transformed to androstenedione (AED) in soil. 32 Both trenbolone isomers (17a and 17b) could be transformed to trendione in soil under aerobic conditions.…”

“…30 Transformation of cortisol and cortisone into prednisolone and prednisone, respectively, and 4-androstene-3,17-dione (AED) to androsta-1,4-diene-3,17-dione (ADD) was observed in bovine faeces. 34,42 Progesterone could be transformed biologically to 17a-hydroxyprogesterone, and subsequently into androstenedione, and other androgens. 33 The present and previous studies suggest that aerobic, anoxic and anaerobic biological processes could lead to degradation or transformation of these steroids in WWTPs.…”

Prednisolone and prednisone neo-formation in bovine urine after sampling

“…Moreover, there is a close resemblance in the A ring of prednisolone, prednisone (pregna-1,4-diene-17a, 21-diol-3,11,20-trione), androstadienedione (androsta-1,4-diene-3,17-dione; ADD) and its precursors/metabolites a-and b-boldenone (androsta-1,4-diene-17a-ol-3-one and androsta-1,4-diene-17b-ol-3-one) on the one hand, and of cortisol, cortisone (pregn-4-ene-17a, 21-diol-3,11,20-trione), androstenedione (androst-4-ene-3,17-dione; AED) and its precursors/metabolites epitestosterone and testosterone (androst-4-ene-17a-ol-3-one and androst-4-ene-17b-ol-3-one) on the other, as shown in Figure 1. In a previous study, we described the dehydrogenation of the androgenic steroids testosterone, epitestosterone and AED: saline-suspended faeces spiked with the examined anabolic steroids were analysed by HPLC-MS/MS and the conclusions were that neo-formation of dehydrogenated steroids can occur, 'AED possibly being the pivotal compound giving rise to boldenone in contaminated urine' through its transformation into ADD (Arioli et al, 2008). Similarly, when we checked the dehydrogenation of cortisol and cortisone in faecal suspensions, we observed their transformation into prednisolone and prednisone, respectively (Arioli et al, 2010).…”

Mouldy feed: A possible explanation for the excretion of anabolic‐androgenic steroids in horses

“…[32] L-cysteine (0.5 g/L) (SAFCSupply Solutions, St Louis, MO, USA) was added to improve anaerobicity. [33] Hemin solution was prepared by adding 2 mL concentrated NH 4 OH solution (25%, Merck, Darmstadt, Germany) and 0.1 g hemin to 500 mL sterile ultrapure water and sterilized afterwards (121°C, 15 min, 1 atm). After filtration over a 0.45 μm filter (Merck, Darmstadt, Germany), pH was adjusted to 7.0 and the medium was sterilized again.…”