Immunoaffinity precolumn for selective sample pretreatment in column liquid chromatography: Immunoselective desorption

Summary

An automated liquid chromatographic system is described using immunoaffinity precolumns for sample pretreatment. The system consists of a column-switching unit allowing preconcentration from a large volume of sample (e.g. 15 ml of urine) onto an immunoaffinity precolumn (containing polyclonal antibodies immobilized on Sepharose). After sorption, the analytes are desorbed by a mixture of two cross-reacting solutes, followed by reconcentration on a C-18-bonded silica precolumn, and then separation on a C-18-bonded silica analytical column. Using oestrogen steroids as model compounds and UV absorbance detection, the minimum detectable concentration is ca. 200 ng/l with a repeatability of 6–8%. The total analysis time is 45 min which allows the unattended analysis of 30 samples per day. The features of the sample pretreatment method, especially of the immunoselective desorption, are evaluated and the general applicability of the system is discussed.