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 alpha- and beta-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-dichloromethanehexane, 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 alpha- and beta-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 degrees C for over 25 weeks did not identify any significant loss of residues.
MacNeil
James D
JD
Centre for Veterinary Drug Residues, Saskatoon Laboratory, Canadian Food Inspection Agency, 116 Veterinary Rd, Saskatoon, Saskatchewan, Canada S7N 2R3. jmacneil@inspection.gc.ca
Reid
JoAnn
J
Neiser
Constance D
CD
Fesser
Adrian C E
AC
D000097:Acetonitriles; D045930:Anabolic Agents / Q000032:analysis*; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D004350:Drug Residues / Q000032:analysis; D004355:Drug Stability; D005615:Freezing; D007753:Laboratories; D008099:Liver / Q000737:chemistry*; D018482:Muscle, Skeletal / Q000737:chemistry*; D011786:Quality Control; D012044:Regression Analysis; D015203:Reproducibility of Results; D019346:Sodium Acetate; D014204:Trenbolone Acetate / Q000032:analysis*
D000097:Acetonitriles; D045930:Anabolic Agents; D019346:Sodium Acetate; D014204:Trenbolone Acetate; C032159:acetonitrile