An immunoaffinity precolumn containing immobilized antibodies raised against the synthetic steroid hormone beta-19-nortestosterone, has been used for the automated sample pretreatment of urine samples containing beta-19-nortestosterone or the related steroids norethindrone and norgestrel. The sample pretreatment system was coupled on-line to a capillary GC. The on-line connection between the immunoaffinity precolumn and the capillary GC was realized with an interface that consisted of a 10 mm X 2 mm i.d. reversed-phase pre-column and a diphenyltetramethyldisilazane-deactivated GC retention gap. After preconcentration on the immunoaffinity precolumn the analytes were eluted and reconcentrated on the reversed-phase precolumn. Subsequently, this precolumn was desorbed with 75 microL of ethyl acetate, which was directly introduced into the retention gap by using partially concurrent solvent evaporation. The system allows the automated pretreatment and GC analysis of 5-25-mL urine samples for the ppt-level determination of 19-norsteroids. The general applicability and potential of on-line immunoaffinity-capillary GC systems are discussed.
An immunoaffinity precolumn (immuno-precolumn) containing an immobilized antibody directed against estrogen steroids, was used as a model system for the evaluation of different desorbing techniques, suitable for on-line coupling to column liquid chromatography (LC). Desorption of estrogen analytes from the immuno-precolumn proved to be impossible with the buffers and chaotropic solutions frequently used in affinity desorption. Micellar solutions are effective in obstructing the antibody-antigen reaction, but their use as desorbing solutions was not found to be practical because of the large interferences introduced into the chromatograms. Desorption with aqueous solutions at elevated temperature, created by microwave action or conventional heating, although effective is not practical in this instance, because the agarose used in this study as the stationary phase for the immuno-precolumn is prone to heat decomposition. The most effective and practical approach is desorption with a methanol-water mixture (95 + 5 v/v). On-line dilution of the eluate allows the concentration of the desorbed analytes using a reversed-phase LC system with subsequent separation and ultraviolet detection. The performance of the system with spiked urine and plasma samples, which were introduced directly into the system, was evaluated and the results were compared with immunoselective desorption.
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