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

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.