Metabolism of methenolone acetate in a veal calf.

The use of anabolic steroids has been banned in the European Union since 1981. In this study, the metabolism of the anabolic steroid methenolone acetate, was investigated in a male veal calf. After daily oral administration of methenolone acetate, three main metabolites were detected in both urine and faeces samples. Among these metabolites, alpha-methenolone was apparently the main one, but 1-methyl-5alpha-androstan-3,17-diol and 3alpha-hydroxy-1-methyl-5alpha-androstan-17-one were also observed. The parent compound was still detectable in faeces. As a consequence, abuse of methenolone acetate as growth promoter can be monitored by analysing urine and faeces samples. A few days after the last treatment, however, no metabolites were observed. Alpha-methenolone was detectable in urine until 5 days after the last treatment, but in faeces no metabolites were detectable after 3 days.

New anabolic steroid illegally used in cattle-structure elucidation of 19-norchlorotestosterone acetate metabolites in bovine urine.

4-Chloro-estr-4-en-17-ol-3-one, trivially named 19-norclostebol acetate or 4-chloro-19-nortestosterone acetate (NClTA), has been identified on the European black market in the late 1990s for possible use in breeding animals. After oral and subcutaneous administration of NClTA to bovine, urine samples were collected over a period of three weeks, and chemical structure of main excreted urinary metabolites was determined. After oral administration, the most abundant metabolites were mainly reduced as 4-chloro-19-norandrostan-3xi-ol-17-one and 4-chloro-19-norandrostan-3xi,17xi-diol. They were identified until 1 week after administration. Following subcutaneous injection, 4-chloro-19-norandrostan-3xi-ol-17-one was again of major abundance, but so were 4-chloro-19-norandrost-4-ene-3xi,17xi-diol and 4-chloro-19-norandrost-4-en-3xi-ol-17-one. They were detected at least 3 weeks after administration. Whatever the route of administration, metabolites were found mainly glucurono-conjugated; the only exception was metabolite 4-chloro-19-norandrostan-3xi-ol-17-one which was identified both in the sulpho- and glucurono-fractions.

Androstadienetrione, a boldenone-like component, detected in cattle faeces with GC-MS(n) and LC-MS(n).

Boldenone (1,4-androstadiene-17-ol-3-one, Bol) has been the subject of a heated debate because of ongoing confusion about its endogenous or exogenous origin when detected in one of its forms in faecal or urine samples from cattle. An expert report was recently written on the presence and metabolism of Bol in various animal species. Androstadienedione (ADD) is a direct precursor of 17beta-boldenone (betaBol). It is a 3,17-dione; ssBol is a 17-ol-3-one. Not much is published on 1,4-androstadiene-3,17-diol, which is a 3,17-diol (ADL). If animals were exposed for a longer period to one of these analytes, a metabolic pathway would be initiated to eliminate these compounds. Similar to recent testosterone metabolism studies in the aquatic invertebrate Neomysis integer, ADD, ssBol and ADL could also be eliminated as hydroxymetabolites after exposure. The presence of 11-keto-steroids or 11-hydroxy-metabolites in faecal samples can interfere with a confirmation method by gas chromatography-negative chemical ionization mass spectrometry (GC-NCI-MS), after oxidation of corticosteroids with a double bond in the A-ring (e.g. prednisolone or its metabolite prednisone). The presence of androstadienetrione (ADT) in faecal samples of cattle has never been reported. The origin of its presence can be explained through different pathways, which are presented in this paper.

Differentiation between dexamethasone and betamethasone in a mixture using multiple mass spectrometry.

The objective of this study was to provide LC and GC-multiple mass spectrometry (MSn) data in positive and negative ion modes to prove the distinction between dexamethasone and betamethasone in a mixture of both components. Using GC-MS, the differentiation was based on a difference in the ratio of the ion traces of the two chromatographic peaks of the alpha and beta epimer with m/z 310 and 330. A minimum of 15% dexamethasone should be present in a mixture of both to detect it as present with a probability of 95%. In the same way betamethasone can be detected from 15% on. Because of the very similar structures of the dexamethasone and betamethasone epimers, no reversed-phase (RP) separations have been reported. Normal-phase separations have been reported in other studies. However because of the compatibility of RP mobile phases in the coupling with MS, the latter was the method of choice. In LC-MSn positive ion mode the product ion 355 was plotted against the sum of 337 and 319. With this combination dexamethasone and betamethasone could be discriminated in a mixture of 20 to 80% of each combination of analytes. In negative ion mode only two product ions were formed from the fragmentation of the acetate adduct, [M-H]- and [M-H-CH2O]-. The intensity of the fragment 391 ([M-H]-) was determined in the discrimination of the two epimers.

Determination of 16beta-hydroxystanozolol in urine and faeces by liquid chromatography-multiple mass spectrometry.

This paper describes the optimisation of the detection of stanozolol and its major metabolite 16beta-hydroxystanozolol in faeces and urine from cattle. Faeces are extracted directly with diisopropyl ether. Urine is first submitted to an enzymatic hydrolysis and then extracted over a modified diatomaceous earth column (Chem-Elut) with a mixture of diisopropyl ether-isooctane. In a final step an acidic back extraction is performed. For the LC-MS-MS detection two approaches are discussed. In a first approach the final extract is detected without derivatization, while the second approach makes use of a derivatization step for 16beta-hydroxystanozolol. While the MS-MS spectrum without derivatization exhibits extensive fragmentation, the spectrum of the derivative shows two abundant diagnostic ions with much more reproducible ion ratios. The derivatization method and the method without derivatization enable the detection of 16beta-hydroxystanozolol up to 0.03 microg l(-1) in urine and 0.07 microg kg(-1) in faeces. Until now there is no literature available for the detection of 16beta-hydroxystanozolol in faeces and urine at the ppt level.