Degradation and transformation of 17α-trenbolone in aerobic water-sediment systems.

Synovex® ONE is an extended-release implant containing the active ingredients estradiol benzoate and trenbolone acetate for use in beef steers and heifers. Trenbolone acetate is rapidly hydrolyzed in cattle to form 17ß-trenbolone and its isomer, 17α-trenbolone, which are further transformed to a secondary metabolite, trendione. As part of the environmental assessment for the use of Synovex ONE, data were generated to characterize the fate of 17α-trenbolone, which is the principal metabolite found in cattle excreta, in the environment. A study was conducted to determine the degradation and transformation of [14 C]-17α-trenbolone in 2 representative water-sediment systems under aerobic conditions. The same transformation products, 17ß-trenbolone and trendione, were formed, principally in the sediment phase, in both systems. From the production of these transformation products, the 50% disappearance time (DT50) values of 17ß-trenbolone and trendione were determined, along with the DT50 values of the parent compound and the total drug (17α-trenbolone + 17ß-trenbolone + trendione). The DT50 values for the total system (aqueous and sediment phase) and for the total residues (17α-trenbolone + 17ß-trenbolone + trendione) in the 2 systems were 34.7 d and 53.3 d, respectively. Environ Toxicol Chem 2017;36:630-635. © 2016 SETAC.

Degradation and transformation of 17α-estradiol in water-sediment systems under controlled aerobic and anaerobic conditions.

One of the principal metabolites in cattle excreta following the administration of Synovex® ONE, which contains estradiol benzoate and trenbolone acetate, is 17α-estradiol. As part of the environmental assessment of the use of Synovex ONE, data were generated to characterize the fate of 17α-estradiol in the environment. Studies were conducted to determine the degradation and transformation of 17α-[14 C]-estradiol in 2 representative water-sediment systems each under aerobic and anaerobic conditions. The same transformation products-estriol, 17ß-estradiol, and estrone-were formed, principally in the sediment phase, under both conditions in both systems. From the production of these transformation products, the 50% disappearance time (DT50) values of estrone and 17ß-estradiol were determined, along with the DT50 values of 17α-estradiol and the total drug (17α-estradiol + 17ß-estradiol + estrone). The results indicate that 17 α-[14 C]-estradiol was more persistent under anaerobic conditions than under aerobic conditions and that 17 α-[14 C]-estradiol was less persistent than its transformation products. The DT50 values for the total system (aqueous and sediment phases) and for the total residues (17α-estradiol, 17ß-estradiol, and estrone) were selected for use in modeling the environmental fate of estradiol benzoate. For aerobic degradation in the water-sediment system, the DT50 was 31.1 d, and it was 107.8 d for the anaerobic system. Environ Toxicol Chem 2017;36:621-629. © 2016 SETAC.

Effects of the antimicrobial contaminant triclocarban, and co-exposure with the androgen 17ß-trenbolone, on reproductive function and ovarian transcriptome of the fathead minnow (Pimephales promelas).

Triclocarban (TCC) is an antimicrobial agent routinely detected in surface waters that has been hypothesized to interact with the vertebrate endocrine system. The present study examined the effects of TCC alone and in combination with the model endocrine disruptor 17ß-trenbolone (TRB) on fish reproductive function. Adult Pimephales promelas were continuously exposed to either 1 µg TCC/L or 5 µg TCC/L, to 0.5 µg TRB/L, or to a mixture (MIX) of 5 µg TCC/L and 0.5 µg TRB/L for 22 d, and a variety of reproductive and endocrine-related endpoints were examined. Cumulative fecundity was significantly reduced in fathead minnows exposed to TRB, MIX, or 5 µg TCC/L. Exposure to 1 µg TCC/L had no effect on reproduction. In general, both TRB and MIX treatments caused similar physiological effects, evoking significant reductions in female plasma vitellogenin, estradiol, and testosterone, and significant increases in male plasma estradiol. Based on analysis of the ovarian transcriptome, there were potential pathway impacts that were common to both TRB- and TCC-containing treatment groups. In most cases, however, those pathways were more plausibly linked to differences in reproductive status than to androgen-specific functions. Overall, TCC was reproductively toxic to fish at concentrations at or near those that have been measured in surface water. There was little evidence that TCC elicits reproductive toxicity through a specific mode of endocrine or reproductive action, nor that it could augment the androgenic effects of TRB. Nonetheless, the relatively small margin of safety between some measured environmental concentrations and effect concentrations suggests that concern is warranted. Environ Toxicol Chem 2017;36:231-242. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Optimisation and validation of a new analytical method for the determination of four natural and synthetic hormones using LC-ESI-MS/MS.

A rapid liquid chromatographic-tandem mass spectrometric method was developed for the simultaneous determination of four natural and synthetic hormone residues (progesterone, testosterone, trenbolone acetate and zeranol) in animal tissue samples. Sample preparation was optimised to minimise time and solvent consumption. Meat samples were mechanically homogenised and digested in a procedure that gave similar recoveries to those enzymatically hydrolysed by Helix pomatia. Efficient extraction was achieved using acidified acetonitrile (1% acetic acid). Chromatographic conditions were optimised to minimise matrix effects. Analytes were separated using a C18 column with gradient elution using ammonium formate solution in methanol (MeOH)/water (1:9) and MeOH mobile phases. Finally, residues were qualitatively and quantitatively determined by electrospray ionisation tandem mass spectrometry in multiple reaction monitoring mode. Different parameters for LC-MS/MS (e.g., declustering potential and collision energy) were optimised using API 6500QT; all analytes were measured using positive-mode electrospray ionisation (ESI+) except zeranol which was measured in negative mode (ESI-). Due to LC-MS/MS signal enhancement/suppression, the determination of hormones was based on matrix-matched standard calculations. The method was validated for the four hormones on meat samples at different fortification levels and showed accepted performance criteria according to European Commission Decision 2002/657/EC. Decision limits and detection capabilities were estimated for all analytes.

Sorption, uptake, and biotransformation of 17ß-estradiol, 17α-ethinylestradiol, zeranol, and trenbolone acetate by hybrid poplar.

Hormonally active compounds may move with agricultural runoff from fields with applied manure and biosolids into surface waters where they pose a threat to human and environmental health. Riparian zone plants could remove hormonally active compounds from agricultural runoff. Therefore, sorption to roots, uptake, translocation, and transformation of 3 estrogens (17ß-estradiol, 17α-ethinylestradiol, and zeranol) and 1 androgen (trenbolone acetate) commonly found in animal manure or biosolids were assessed by hydroponically grown hybrid poplar, Populus deltoides x nigra, DN-34, widely used in riparian buffer strips. Results clearly showed that these hormones were rapidly removed from 2 mg L(-1) hydroponic solutions by more than 97% after 10 d of exposure to full poplar plants or live excised poplars (cut-stem, no leaves). Removals by sorption to dead poplar roots that had been autoclaved were significantly less, 71% to 84%. Major transformation products (estrone and estriol for estradiol; zearalanone for zeranol; and 17ß-trenbolone from trenbolone acetate) were detected in the root tissues of all 3 poplar treatments. Root concentrations of metabolites peaked after 1 d to 5 d and then decreased in full and live excised poplars by further transformation. Metabolite concentrations were less in dead poplar treatments and only slowly increased without further transformation. Taken together, these findings show that poplars may be effective in controlling the movement of hormonally active compounds from agricultural fields and avoiding runoff to streams.

Trenbolone acetate metabolite transport in rangelands and irrigated pasture: observations and conceptual approaches for agro-ecosystems.

To assess the relative ecological risks of trenbolone acetate (TBA) use in agro-ecosystems, we evaluated the spatiotemporal dynamics of TBA metabolite transport during irrigation and rainfall events. Within a pasture, TBA-implanted heifers (40 mg TBA, 8 mg estradiol) were briefly penned (24 h) at high stocking densities (500 animal units (AU)/ha), prior to irrigation. Irrigation runoff concentrations of 17α-trenbolone (17α-TBOH) 0.3 m downslope were 11 ng/L in the wetting front, but quickly decreased to ∼0.5 ng/L, suggesting mass transfer limitations to transport. At 3 and 30 m downslope, efficient attenuation of 17α-TBOH concentrations is best explained by infiltration and surface partitioning. At plot scales, transport through vegetated filter strips resulted in <0.5-7 ng/L 17α-TBOH concentrations in rainfall-induced runoff with partial subsequent attenuation. Thus, even under intense grazing scenarios, TBA-metabolite transport potential is expected to be low in rangelands, with ecological risks primarily arising from uncontrolled animal access to receiving waters. However, 17α-TBOH concentrations in initial runoff were predicted to exceed threshold levels (i.e., no observed adverse effect levels [NOAELs]) for manure concentrations exceeding 2.0 ng/g-dw, which occurs throughout most of the implant life. For comparison, estrone and 17ß-estradiol were modeled and are likely capable of exceeding NOAELs by a factor of ∼2-5 in irrigation runoff, suggesting that both endogenous and exogenous steroids contribute to endocrine disruption potential in agro-ecosystems.

Bioavailability and fate of sediment-associated trenbolone and estradiol in aquatic systems.

Endocrine disrupting effects in aquatic organisms have been observed in systems influenced by steroid hormones. Associating endocrine disruption with aqueous concentrations of steroids alone may overlook the influence of source-sink dynamics in sediments on steroid hormone bioavailability. The objective of this study was to determine the fate of 17ß-estradiol and 17ß-trenbolone in two field sediments and to evaluate the corresponding bioavailability of the compounds to the fathead minnow (Pimephales promelas). Steroid fate was evaluated using analytical chemistry and verified by assessing the biological activity using yeast based in vitro assays. Effective bioavailability of the steroids was inferred from changes in hepatic vitellogenin expression (increased expression in males exposed to 17ß-estradiol, and reduced expression in females exposed to 17ß-trenbolone). In experiments conducted with 17ß-estradiol, no induction of hepatic vitellogenin mRNA expression was observed in male fish exposed to sediment-associated 17ß-estradiol. In contrast, female minnows exposed to sediment-associated 17ß-trenbolone experienced significant reductions in hepatic vitellogenin compared to negative controls. In both systems, the parent compounds were shown to degrade rapidly to the more persistent metabolites, estrone and trendione, both of which were found predominantly associated with the sediments. Results from the yeast estrogen screen indicate a reduction in biological activity as biotransformation of 17ß-estradiol occurs; results from the yeast anti-estrogen screen were inconclusive and unable to substantiate 17ß-trenbolone fate in aquatic systems. Collectively, these data support the contention that steroid hormones associated with the sediment can become bioavailable to fish, and that sediment characteristics influence the observed bioavailability of these compounds.

Analysis of trenbolone acetate metabolites and melengestrol in environmental matrices using gas chromatography-tandem mass spectrometry.

Studies demonstrate that exposure to steroid hormones in receiving waters can adversely impact reproduction of aquatic organisms. In particular, exogenous steroid hormones widely used as growth promoters in animal agriculture are of high concern, yet no gas chromatography-tandem mass spectrometry (GC/MS/MS) analytical methods for the detection of these compounds in complex environmental matrices is described in the literature. This study utilizes analytical methods based upon N-methyl-N-(trimethylsilyl)trifluoro-acetamide-iodine (MSTFA-I(2)) derivatization for the analysis of metabolites of trenbolone acetate (TBA), including 17α-trenbolone, 17ß-trenbolone, and trendione, and melengestrol acetate in receiving waters and surface soils associated with animal agriculture. Results suggest method detection levels of 0.5-1 ng/L for the trenbolone metabolites, while detection of melengestrol is qualitative only. Isotope dilution methods employing d3-17ß-trenbolone were used to improve steroid quantification. Method recoveries in spiked samples collected from a variety of representative receiving waters generally ranged from 80-120% with consistent and low standard deviation (generally<10%) for replicate analysis. Analysis of a storm water runoff sample from a commercial confined animal feeding operation (CAFO) that used TBA implants detected 17ß-trenbolone and trendione at concentrations of 31 and 52 ng/L, respectively. Analysis of surface soils at a commercial CAFO using TBA implants detected 17α-trenbolone at concentrations between 4-6 ng/g dry weight. Method development efforts suggested that the concentration of I(2) in MSTFA, the removal of I(2) from sample extracts after derivatization, and the use of Florisil clean-up to reduce organic matter matrix were vital aspects of steroid hormone quantification at low (<30ng/L) concentrations in complex environmental matrices.

Fate and occurrence of steroids in swine and dairy cattle farms with different farming scales and wastes disposal systems.

Fate and occurrence of fourteen androgens, four estrogens, five glucocorticoids and five progestagens were investigated in three swine farms and three dairy cattle farms with different farming scales and wastes disposal systems in China. Twenty-one, 22, and 12 of total 28 steroids were detected in feces samples with concentrations ranging from below method limit of quantitation (

Estrogens and synthetic androgens in manure slurry from trenbolone acetate/estradiol implanted cattle and in waste-receiving lagoons used for irrigation.

The increasing size of concentrated animal feeding operations has led to a concomitant increase in the land-application of manure, which has spawned research on the concentrations and environmental risk assessment of natural and synthetic hormones in animal manures. 17ß-Trenbolone acetate (TBA) is widely used in the United States for improving daily gains in beef cattle and is often administered in combination with 17ß-estradiol (17ß-E2). Trenbolone (TB) and E2 isomers and their metabolites were quantified in manure collection pits and lagoon effluent from beef cattle implanted with the commercial anabolic preparation Ravoler-S (containing 140 mg 17ß-trenbolone acetate and 28 mg 17ß-E2). Manure pit and lagoon effluent samples were collected weekly for 9 weeks post implanting and analyzed using reverse-phase liquid chromatography tandem mass spectrometry. 17α-TB was the most abundant androgen with the highest concentration observed 2 weeks post implant. 17ß-TB and trendione peaked at the end of week 2 and 4, respectively. For the estrogens, the highest concentrations for estrone (E1), estriol (E3), and 17α-E2 were observed after week 4, 6, and 8, respectively. 17ß-E2 concentrations were the lowest of the estrogens and erratic over time. In lagoon water, which is used for irrigation, 17α-TB and E1 had the highest detected hormone concentrations (1.53 and 1.72 µg L(-1), respectively). Assuming a 1-2 order dilution during transport to surface water, these hormone levels could lead to concentrations in receiving waters that exceed some of the lowest observable effect levels (LOELs) reported for hormones (e.g., 0.01-0.03 µg L(-1)).

Hormone discharges from a midwest tile-drained agroecosystem receiving animal wastes.

Manure is increasingly being viewed as a threat to aquatic ecosystems due to the introduction of natural and synthetic hormones from land application to agricultural fields. In the Midwestern United States, where most agricultural fields are tile-drained, there is little known about hormone release from fields receiving animal wastes. To this end, seven sampling stations (four in subsurface tile drains and three in the receiving ditch network) were installed at a Midwest farm where various types of animal wastes (beef, dairy, and poultry lagoon effluent, dairy solids, and subsurface injection of swine manure) are applied to agricultural fields. Water flow was continuously monitored and samples were collected for hormone analysis during storm events and baseline flow for a 15 month study period. The compounds analyzed included the natural hormones 17α- and 17ß-estradiol, estrone, estriol, testosterone, and androstenedione and the synthetic androgens 17α- and 17ß-trenbolone and trendione. Hormones were detected in at least 64% of the samples collected at each station, with estrone being detected the most frequently and estriol the least. Testosterone and androstendione were detected more frequently than synthetic androgens, which were detected in fewer than 15% of samples. Hormone concentrations in subsurface tile drains increased during effluent irrigation and storm events. Hormones also appeared to persist over the winter, with increased concentrations coinciding with early thaws and snowmelt from fields amended with manure solids. The highest concentration of synthetic androgens (168 ng/L) observed coincided with a snowmelt. The highest concentrations of hormones in the ditch waters (87 ng/L for total estrogens and 52 ng/L for natural androgens) were observed in June, which coincides with the early life stage development period of many aquatic species in the Midwest.

Modification of mRNA expression after treatment with anabolic agents and the usefulness for gene expression-biomarkers.

With this feasibility study a first step towards a new monitoring system for hormonal treatments was done. Screening of regulation and function of anabolic sex steroids via modified gene expression of mRNA in various tissues could be a new approach to trace treatments with unknown drugs or newly combined cocktails. In the study, uterus, liver and muscle tissue from 24 cycling heifers were taken after the animals were treated either with Melengestrol Acetate (MGA), Finaplix-H (200 mg Trenbolone Acetate) or Ralgro (36 mg Zeranol) for 56 days. In every treatment group always two heifers were given 1-fold, 3-fold and 10-fold doses of the standard preparation, the control group without any treatment consisted of two animals. The different tissue gene expression profiles were investigated via the candidate gene approach. Totally 57 candidate genes were selected according to their functionality by screening the actual literature and composed to functional groups: angiogenesis, apoptosis, cell cycle, endocrine factors, energy metabolism, inflammatory factors, muscle function, oncogenes, protein metabolism and transcription factors. Gene expression was measured using quantitative real-time RT-PCR (qRT-PCR) technology. From 24 tested candidate genes in the liver, 17 showed a significant regulation. Eight genes were influenced by MGA, 9 by Finaplix-H, and 4 by Ralgro. For the muscle tissue 19 genes were tested with the result that in the neck muscle 11 genes were regulated and in the hind limb muscle 8 genes. In the neck 5 genes were affected by MGA, 6 by Finaplix-H and 3 by Ralgro. Only 2 genes were influenced by MGA in the hind limb muscle. Finaplix-H affected 6 and Ralgro 4 genes. In the uterus 29 target genes were tested and 13 were significantly influenced by the anabolic sex steroids. Under Finaplix-H treatment eight target genes were regulated and Ralgro and MGA showed a significant regulation in four target genes. The highest gene expression changes under anabolic treatment were observed in the uterus. The analyzed genes showed significant regulations but further studies, testing different animal husbandry conditions will be needed to identify meaningful expression patterns for the different tissues. With the investigation of the regulation and possible function of anabolic sex steroids via gene expression, a preparatory work for the development of an expression pattern for drug screening was made.

Analysis of synthetic 19-norsteroids trenbolone, tetrahydrogestrinone and gestrinone by gas chromatography-mass spectrometry.

Tetrahydrogestrinone, gestrinone and trenbolone are synthetic 19-norsteroids with androgenic properties sharing a labile conjugated ketotrienyl motif. Their LC-MS analyses tend to overcome classical derivatization problems, a shortcoming to the use of GC-MS. Therefore, alternative derivatization procedures were evaluated. The procedure with methoxylamine: pyridine followed by TMSImid: MSTFA gave the best results. This is attributed to the stability of the MO-TMS derivatives hindering the formation of artifacts and tautomerism. A full method is presented including SPE, hydrolysis and liquid-liquid extraction. It was possible to confirm the analytes below 2 ng/mL in urine, being the method robust and cost effective also for screening proposes.

Residues from anabolic preparations after good veterinary practice.

The purpose of this study was to determine the endogenous concentrations of estrogens, particularly estradiol-17beta (E2beta, in edible tissues of beef cattle (females and intact and neutered males) and the concentrations of E2beta, and trenbolone beta and alpha (betaTb, alphaTb) after an E2beta and/or trenbolone acetate (TA) ear implant. Radioimmunoassays were validated for quantitation of E2beta (active isomer), E2alpha, estrone (E1), betaTb and alphaTb for bovine muscle, liver, kidney and fat tissues. The criteria of accuracy, precision, specificity and sensitivity were applied according to the standards of the U.S. Food & Drug Administration. In steer tissues, endogenous E2beta was <15 ppt, as was heifer muscle; but heifer liver and kidney were 3-fold greater. An E2beta implant in steers had no effect on muscle E2beta concentration, but increased E2beta in liver and fat 4- and 3-fold, respectively, but by 24 h post-implant removal, E2beta had fallen by half. Tissue E1 concentrations in cyclic females were similar to E2beta, but rose many fold greater than did E2beta during gestation; E2beta rose 3-fold during gestation. After E2beta/TA implant, steer tissues had E2beta concentrations equal to (for muscle and fat) and one-half (for liver) the E2beta measured in E2beta implant only steers; betaTb was in a low range (250-380 ppt) in muscle, liver and fat and alphaTb was even lower, except in liver (800-1500 ppt). An implant of TA only (no E2beta) resulted in betaTb and alphaTb concentrations 2-3-fold greater in liver, kidney and fat, but no greater in muscle than betaTb in tissues of E2beta/TA implant steers. In conclusion, anabolic implants in steers resulted in tissue E2beta concentrations less than the FDA allowable increment and betaTb in the lowest quartile (0.25) of a part per billion 30 days after implant.

Hormone contents in peripheral tissues after correct and off-label use of growth promoting hormones in cattle: effect of the implant preparations Filaplix-H, Raglo, Synovex-H and Synovex Plus.

Certain hormonal growth promoters are licensed in several beef producing countries outside the European Union (EU). Use in compliance with Good Veterinary Practice is mandatory. As risk assessment of hormone residues in animal tissues up to now has neglected potential off-label use, the present study dealt with two topics: 1) multiple treatment with the implant preparations Finaplix-H (200 mg trenbolone acetate), Ralgro (36 mg zeranol) and Synovex-H (200 mg testosterone propionate plus 20 mg estradiol benzoate) in heifers (1-fold, 3-fold and 10-fold dose), and 2) non-approved treatment of female veal calves (1-fold dose of Synovex-H or Synovex Plus with 200 mg trenbolone acetate plus 28 mg estradiol benzoate). Residues of estradiol-17beta, estradiol-17alpha, estrone and testosterone, trenbolone-17beta, trenbolone-17alpha and trendione or zeranol, respectively, were measured in loin, liver, kidney and peri-renal fat by high performance liquid chromatography/enzyme immunoassay (HPLC/EIA) after liquid-liquid extraction and solid-phase clean-up. The hormone residues in the multiple-dose experiments were dose-dependent and partially exceeded the threshold values: in the liver in one animal after 3-fold dose and in two animals after 10-fold dose of Finaplix-H, and in the liver and kidney after 3-fold and 10-fold dose of Synovex-H. Mean hormone residues in calves were mainly below those of heifers and did not infringe threshold values.

Detection of anabolic residues in misplaced implantation sites in cattle.

Eight weeks before slaughter, 26 heifers, 2 calves, and 1 steer were implanted with licensed anabolic preparations at off-label injection sites. After slaughter, 24 of 31 implantation sites (77%) were detected. Residual pellets of Revalor H contained a mean of 42.9 mg trenbolone acetate (range 19.8-57.7 mg) and 4.6 mg (1.96-6.45 mg) estradiol, corresponding to 30% (19.8-57.7%) and 32.7% (14.0-46.6%) of the originally applied dose, respectively. In the tissue areas containing residual Revalor H pellets, total residues ranged from 14.8 microg to 12.6 mg trenbolone acetate, 41.7 microg to 1.45 mg trenbolone, and 11.1 microg to 3.39 mg estradiol. The outer tissue areas of the injection sites contained <2 microg hormones. The preparations Synovex H, Finaplix H, Implus S, and Component EC behaved similarly to Revalor H. Residues of Synovex Plus were low, whereas the Compudose silicone rubber contained 58.8% of the implanted dose, but left no significant tissue residues. If implantation sites are processed in meat manufacturing, international threshold levels of the respective substances will be exceeded in tons of meat products.

Determination of 19-nortestosterone, testosterone and trenbolone by gas chromatography-negative-ion mass spectrometry after formation of the pentafluorobenzylcarboxymethoxime-trimethylsilyl derivatives.

The known reaction of 3-ketosteroids with carboxymethoxylamine (to form the corresponding carboxymethoximes), followed by esterification of the carboxyl group with pentafluorobenzyl bromide, has been used to obtain derivatives of 19-nortestosterone, testosterone and trenbolone suitable for high-sensitivity detection with gas chromatography-negative-ion chemical ionization mass spectrometry. These derivatives, after further silylation of the alcoholic groups of the steroids, showed excellent chromatographic and spectrometric characteristics and were detectable in the low picogram range. The derivatization gave rise to the formation of two isomers which were distinguishable by gas chromatography. The existence of the two isomers was also confirmed by high-performance liquid chromatography. Examples of the usefulness of this derivatization procedure are given for the analysis of 19-nortestosterone, testosterone and trenbolone in meat and urine samples. By the use of immunoaffinity extraction and addition of deuterated internal standards (synthesized by isotopic exchange), the new derivatization procedure allowed a correct identification and quantitation of the steroids and reached very low detection limits [0.02 ppb (10(9] for 19-nortestosterone and testosterone, 0.06 ppb for trenbolone].

[Synthetic anabolic androgens in the fecal material of cattle: radioimmunological determination after purification by high performance liquid chromatography]. / Androgènes anabolisants de synthèse dans les matières fécales des bovins: dosage radio-immunologique après purification par chromatographie liquide à haute performance.

In answer to the mandatory control of the illegal use of anabolizing agents in meat-producing animals imposed by the EEC in farms, a method of analysis of faeces involving high performance liquid chromatography (HPLC) and radioimmunoassay (RIA) has been described. Four HPLC fractions were collected and submitted to corresponding RIA: 17 beta- and 17 alpha-trenbolone, 17 beta-nortestosterone, 17 alpha-nortestosterone and 17 alpha-methyltestosterone. The mean extraction and purification yield was estimated at 44 +/- 7% using tritiated 17 alpha-methyltestosterone as internal standard. Detection limits of the 3 hormones were estimated at 0.2-0.3 ng/g of faeces. About 50 samples can be analysed per week by this method.

Identification and quantitation of trenbolone in bovine tissue by gas chromatography-mass spectrometry.

Identification and quantitation of trace amounts of trenbolone in bovine tissue by capillary gas chromatography-mass spectrometry-selected-ion monitoring (GC-MS-SIM) has been developed. Three-phase liquid-liquid extraction using a mixture of water-acetonitrile-dichloromethane-hexane was utilized for the sample extraction from tissue. Target compounds were extracted from the tissue into the acetonitrile layer. The residue from this extraction was then subjected to solid-phase extraction by C18 and silica gel disposable cartridges using methanol-water and benzene-acetone as eluents. To overcome extensive matrix interferences, preparative reversed-phase high-performance liquid chromatographic separation was used with an octadecyl-bonded column using methanol-water as mobile phase for sample clean-up prior to GC-MS analysis. A structural analogue of trenbolone, 19-nortestosterone, was chosen as the internal standard for quantitation by GC-MS. The sample was co-injected with N,O-bis (trimethylsilyl) trifluoroacetamide-1-(trimethylsilyl) imidazole (95:5, v/v) for flash heater derivation. Identification and quantitation were simultaneously carried out by SIM of characteristic ions of the trimethylsilyl derivatives of trenbolone and 19-nortestosterone. The limit of detection for trenbolone and epitrenbolone was 0.5 ppb in muscle and liver tissue. A comparison of sensitivity and specificity between GC-MS under electron ionization in addition to positive- and negative-ion chemical ionization conditions using methane reagent gas is also discussed.