Determination of beta-19-nortestosterone and its metabolite alpha-19-nortestosterone in biological samples at the sub parts per billion level by high-performance liquid chromatography with on-line immunoaffinity sample pretreatment.
An immunoaffinity precolumn (immuno precolumn) packed with Sepharose-immobilized polyclonal antibodies against the anabolic hormone 17 beta-19-nortestosterone (beta-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), beta-19-NT and its metabolite 17 alpha-19-nortestosterone (alpha-19-NT) can be determined in biological samples with a detection limit of 0.05 microgram/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 17 beta-laurate, the maximum concentrations of beta-19-NT (1.3 micrograms/l) and alpha-19-NT (3.1 micrograms/l) were found seven days after intramuscular administration. In a bile sample from this calf only alpha-19-NT (55 microgram/l) was detected. In meat samples from three treated calves, the concentration of beta-19-NT varied from 0.1 to 1.6 micrograms/kg and no alpha-19-NT could be detected. In liver samples from these calves, the concentrations of beta-19-NT and alpha-19-NT were less than 0.05-0.1 and 0.5-0.9 micrograms/kg, respectively. In the corresponding kidney samples, the concentrations of beta-19-NT and alpha-19-NT were 0.4-0.5 and 0.5-1.6 micrograms/kg, respectively. The application of the same immuno precolumn to the determination of 17 beta- and 17 alpha-trenbolone, two structurally related steroids, is also demonstrated.
Haasnoot
W
W
State Institute for Quality Control of Agricultural Products (RIKILT), Wageningen, The Netherlands.
Schilt
R
R
Hamers
A R
AR
Huf
F A
FA
Farjam
A
A
Frei
R W
RW
Brinkman
U A
UA
D000818:Animals; D001646:Bile / Q000032:analysis; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry; D006868:Hydrolysis; D007668:Kidney / Q000032:analysis; D008099:Liver / Q000032:analysis; D008460:Meat / Q000032:analysis; D009277:Nandrolone / Q000378:metabolism* / Q000652:urine; D014204:Trenbolone Acetate / Q000032:analysis
D009277:Nandrolone; D014204:Trenbolone Acetate