Determination of residual levels of naloxone, yohimbine, thiophanate, and altrenogest in porcine muscle using QuEChERS with liquid chromatography and triple quadrupole mass spectrometry.

A quick, easy, cheap, effective, rugged, and safe QuEChERS (method) was used for the simultaneous detection of four veterinary drug residues, namely naloxone, yohimbine, thiophanate, and altrenogest, in porcine muscle, using liquid chromatography with electrospray ionization triple quadrupole tandem mass spectrometry. Because of the unavailability of a suitable internal standard, matrix-matched calibrations were used for quantification, with determination coefficients ≥ 0.9542. The accuracy (expressed as recovery %) ranged from 60.53 to 83.25%, and the intra- and interday precisions (expressed as relative standard deviations) were <12%. The limits of quantification were 5, 0.5, 2, and 5 ng/g for naloxone, yohimbine, thiophanate, and altrenogest, respectively. Samples purchased from local markets in Seoul, Republic of Korea, revealed no traces of the target analytes. The developed method described herein is sensitive and reliable and can be applied to quantify the tested veterinary drugs in animal tissues.

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.

Liquid chromatography-tandem mass spectrometry analysis of 17α-trenbolone, 17ß-trenbolone and trendione in airborne particulate matter.

Trenbolone acetate (TbA) is a potent synthetic anabolic steroid that was approved by the FDA as a growth promoter in beef cattle in 1987. Given the endocrine-modulating activity of TbA and its metabolites in all vertebrates, a sensitive and reliable analytical method is needed to detect TbA and related residues in environmental matrices. We have developed a method that incorporates solid phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of the three major TbA metabolites (trendione, 17ß-trenbolone, 17α-trenbolone) in total suspended particulate matter (TSP) samples. Sample preparation involved pressurized liquid extraction followed by cleanup on solid-phase extraction cartridges. The procedure was optimized to obtain maximum recovery and minimum signal suppression/enhancement from matrix effects. Analytes were separated with a Phenomenex Gemini-NX C18 analytical column (150 mm × 2.0 mm, 3 µm particle size) using an aqueous methanol gradient at a flow rate of 0.2 mL/min. Column effluent underwent positive electrospray ionization (ESI). Two or more diagnostic product ions were acquired from analyte specific precursor ions for unambiguous confirmation and quantification. The method detection limit was 3.27-4.87 ng/g of particulate matter (PM). Method accuracy, determined with analyte recoveries, ranged between 68% and 117%, and method precision, expressed as relative standard deviation, was below 15% at spiked levels of 6.67, 33.3, and 167 ng/g PM. Analysis of TSP samples demonstrated the presence of the target species associated with PM in the vicinity of beef cattle feeding operations.

Stereoselective sorption by agricultural soils and liquid-liquid partitioning of trenbolone (17alpha and 17beta) and trendione.

Trenbolone acetate (TBA) is a synthetic anabolic hormone used for growth promotion in beef cattle, which excrete primarily 17alpha-trenbolone along with small amounts of 17beta-trenbolone and trendione. To aid in predicting transport of manure-borne TBA metabolites, multiconcentration sorption isotherms for 17alpha- and 17beta-trenbolone and trendione were generated with five autoclaved-sterilized soils that represented a range in soil properties. Hormone concentrations were measured independently in solution and soil phases, and quantified using liquid chromatography with electrospray mass spectrometry. In addition, partition coefficients between apolar hexane and water (K(hw)) and bipolar octanol and water (K(ow)) were measured for the three androgens to better ascertain the mechanisms that may be responsible for the sorption differences observed between isomers. In all five soils, trendione sorbed the most, and 17alpha- and 17beta-trenbolone isomers exhibited different sorption magnitudes. 17beta- trenbolone consistently sorbed a factor of 2 more than 17alpha-trenbolone. For all three androgens, sorption is proportional to the soil organic carbon (OC) content with average log OC-normalized distribution coefficients (log K(oc), L/kg OC) of 2.77 +/- 0.12 for 17alpha-trenbolone, 3.08 +/- 0.1 for 17beta-trenbolone and 3.38 +/- 0.19 for trendione, which suggests the dominance of hydrophobic partitioning. However, differences in K(hw) values between 17alpha- and 17beta-trenbolone were small indicating differences are not simply due to differences in aqueous activity. In contrast, similarly different K(ow) and K(oc) values for the two isomers indicate the likely contribution of H-bonding to stereoselective sorption.

A fast immunoassay for the screening of alfa-trenbolone in bovine urine

Brazil is one of the many countries that forbids the use of anabolic compounds, which generates difficulties on monitoring its use, once it has one of the biggest cattle herd. Therefore, several anabolic compounds are used, including trenbolone acetate. With the agreement of “Ministério da Agricultura, Pecuária e Abastecimento", an ELISA based test was done with the production of polyclonal antibodies in rabbits and testing in steers urine which received trenbolone acetate implants. The test showed to be cheap, easy and reliable to use in bovine urine to determine alfa- trenbolone, trenbolone acetate major metabolite, released in the animals urine until 60 days after implant was been injected. The results in comparison to the ones obtained by the commercial kit used by the Brazilian "Ministério da Agricultura, Pecuária e Abastecimento" were similar, with no significant differences.

O Brasil está entre os países que mantêm o uso de anabolizantes proibido, o que gera dificuldades muito grandes no monitoramento destas substâncias, uma vez que possui o maior rebanho bovino. Contudo, diversas substâncias são largamente utilizadas, entre as quais o acetato de trembolona. Com o consentimento do Ministério da Agricultura, Pecuária e Abastecimento, foram produzidos anticorpos policlonais para um teste baseado em ELISA ("enzyme linked immunosorbent assay") capazes de detectar acetato de trembolona na urina de bovinos tratados com trembolona. O teste apresentou baixo custo e de fácil execução para a detecção de alfa-trembolone, o principal metabólito na absorção do acetato de trembolona, liberado na urina dos animais. Os resultados foram similares aos obtidos com o "kit" comercial usado pelo Ministério da Agricultura, Pecuária e Abastecimento do Brasil.

Melanin as an adsorbent for drug residues.

The binding of seven veterinary drugs (clenbuterol, chlorpromazine, diethylstilbestrol, 19-nortestosterone, salbutamol, salicylic acid and trenbolone) to melanin from Sepia officinalis was investigated. Basic and hydrophobic drugs were the most strongly bound. Desorption by ethanol was complete for neutral drugs but only partial for the basic drugs, which suggests that binding of the latter involves an ionic component. A method of synthesizing melanin in an immobilized form (melanin-PS) on the surface of porous silica was developed. When the drug binding properties of melanin-PS were investigated, its capacity to bind the basic drug clenbuterol was found to be higher (5.9 nmol mg-1) than that for the neutral hydrophobic drug 19-nortestosterone (0.56 nmol mg-1); for both drugs the attainment of binding equilibrium with melanin-PS was relatively rapid (< 5 min). By virtue of its binding kinetics, high capacity and mechanical robustness, melanin-PS offers potential for use in chromatography or solid-phase extraction and may additionally enable modelling of drug-melanin interactions.

[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.