Determination of β-19-nortestosterone and its metabolite α-19-nortestosterone in biological samples at the sub parts per billion level by high-performance liquid chromatography with on-line immunoaffinity sample pretreatment

Abstract

An immunoaffinity precolumn (immuno precolumn) packed with Sepharose-immobilized polyclonal antibodies against the anabolic hormone 17β-19-nortestosterone (β-19-NT) was used for the selective on-line pretreatment of raw extracts of urine, bile and tissue samples by high-performance liquid chromatography. Using UV detection (247 nm), β-19-NT and its metabolite 17α-19-nortes-tosterone (α-19-NT) can be determined in biological samples with a detection limit of 0.05 μg/kg. Owing to the high clean-up efficiency of the immuno precolumn and the large sample volumes used, confirmation by gas chromatography-mass spectrometry is possible at this level. In urine samples from a calf treated with 19-nortestosterone 17β-laurate, the maximum concentrations of β-19-NT (1.3 μg/l) and α-19-NT (3.1 μg/l) were found seven days after intramuscular administration. In a bile sample from this calf only α-19-NT (55 μg/l) was detected. In meat samples from three treated calves, the concentration of β-19-NT varied from 0.1 to 1.6 μg/kg and no α-19-NT could be detected. In liver samples from these calves, the concentrations of β-19-NT and α-19-NT were less than 0.05-0.1 and 0.5-0.9 μg/kg, respectively. In the corresponding kidney samples, the concentrations of β-19-NT and α-19-NT were 0.4-0.5 and 0.5-1.6 μg/kg, respectively. The application of the same immuno precolumn to the determination of 17β- and 17α-trenbolone, two structurally related steroids, is also demonstrated. © 1989.