Immunoaffinity pre-column for selective on-line sample pre-treatment in high-performance liquid chromatography determination of 19-nortestosterone.
A liquid chromatographic column-switching system for automated sample pretreatment and determination of the anabolic hormone beta-19-nortestosterone (beta-19-NT) and its metabolite alpha-19-nortestosterone (19-norepitestosterone) in calf urine is described. The system consists of an immunoaffinity pre-column (immuno pre-column) packed with Sepharose-immobilized polyclonal antibodies against beta-19-NT, a second pre-column packed with C18 bonded silica and an analytical C18 column. Urine (25 ml) is directly loaded on the immuno pre-column, where the analytes of interest are trapped by the immobilized antibodies. Next the analytes are desorbed selectively with a solution containing an excess of the cross-reacting steroid hormone norgestrel and transferred, via the second pre-column, to the analytical column. The recovery of beta-19-NT in spiked urine samples was over 95%. The detection limit was 50 ng/l for a 25-ml urine injection. The system showed no loss of analytical performance over a 6-month period, during which about 100 samples were analysed with the same immuno pre-column. The general applicability of this sample pretreatment method is discussed.
Farjam
A
A
Department of Analytical Chemistry, Free University, Amsterdam, The Netherlands.
de Jong
G J
GJ
Frei
R W
RW
Brinkman
U A
UA
Haasnoot
W
W
Hamers
A R
AR
Schilt
R
R
Huf
F A
FA
D000818:Animals; D001322:Autoanalysis; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D007120:Immunochemistry; D007163:Immunosorbent Techniques; D009277:Nandrolone / Q000276:immunology / Q000652:urine*
D009277:Nandrolone