Simple high-performance liquid chromatographic column-switching technique for the on-line immunoaffinity extraction and analysis of flunitrazepam and its main metabolites in urine

A sensitive, simple and rapid method without sample pretreatment is presented for the simultaneous determination of flunitrazepam and its main metabolites (norflunitrazepam, 7-amino- and 7-acetamidoflunitrazepam) in urine. The single-step procedure is based on a column-switching technique which uses an immobilized antibody in an extraction column following concentration on a precolumn and separation on an analytical column. UV detection was performed at 254 nm. The reusability of the antibody exceeds 88 runs and a complete analysis was performed in less than 40 min. The method shows coefficients of variation below 9.9% and rates of recovery greater than 92% tested at the level of 50 ng/ml urine. The limit of detection was below 2 ng/ml urine for the four compounds.