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Single-Laboratory Validation of a Modified Liquid Chromatographic Method with UV Detection for Determination of Trenbolone Residues in Bovine Liver and Muscle

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Trenbolone acetate is a synthetic testosterone analog registered for use in a number of countries as a growth-promoting hormone, applied as an implant in the ears of feedlot cattle. The method is intended for the detection and quantitation of trace amounts of α- and β-trenbolone in bovine tissues (muscle, liver) by liquid chromatography (LC) with UV detection and eliminates the use of the structural analog, 19-nortestosterone, as an internal standard. Trenbolone residues are extracted from tissues that have been homogenized in sodium acetate with a 3-phase liquid–liquid extraction by adding a mixture of water–acetonitrile–dichloromethane– hexane, with trenbolone residues preferentially partitioned into the middle acetonitrile layer. The extract is passed through solid-phase extraction cartridges (both C18 and silica gel) using, respectively, methanol–water and acetone–toluene as eluents. Reversed-phase high-performance LC separation is performed, an octadecyl-bonded column with methanol–acetonitrile–water used as mobile phase for sample analysis. The limit of detection is 0.2 ng/g in muscle tissue and 0.6 ng/g in liver tissue, with coefficients of variation of 3.5–12.1% for α- and β-trenbolone at concentrations from 0.2 to 4.0 ng/g fortified in muscle and 3.3–26.0% from liver fortified at 0.6–10.0 ng/g. Absolute recoveries of 40–130% were observed, but the use of fortified matrix curves eliminated recovery correction. Critical control points were identified in a pH adjustment step and an evaporation step during method validation, which included ruggedness testing. Analysis of incurred tissues (bovine liver and muscle) stored at –20°C for over 25 weeks did not identify any significant loss of residues.
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Document Type: Research Article

Affiliations: Centre for Veterinary Drug Residues, Saskatoon Laboratory, Canadian Food Inspection Agency, 116 Veterinary Rd, Saskatoon, Saskatchewan, Canada S7N 2R3

Publication date: 2003-09-01

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  • The Journal of AOAC INTERNATIONAL publishes refereed papers and reviews in the fields of chemical, biological and toxicological analytical chemistry for the purpose of showcasing the most precise, accurate and sensitive methods for analysis of foods, food additives, supplements and contaminants, cosmetics, drugs, toxins, hazardous substances, pesticides, feeds, fertilizers and the environment available at that point in time. The scope of the Journal includes unpublished original research describing new analytical methods, techniques and applications; improved approaches to sampling, both in the field and the laboratory; better methods of preparing samples for analysis; collaborative studies substantiating the performance of a given method; statistical techniques for evaluating data. The Journal will also publish other articles of general interest to its audience, e.g., technical communications; cautionary notes; comments on techniques, apparatus, and reagents.
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