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We report on the development of solid phase microextraction probes for drug analysis, prepared with antibodies specific for benzodiazepines covalently immobilized to the surface. In the technique, immobilized antibody probes are exposed to a sample containing the drug for 30min. Extracted drugs are subsequently desorbed from the probes in 500μL of methanolic desorption solution, which is dried, reconstituted in a small volume of injection solution and analysed by LC-MS/MS. The antibodies were characterized both before and after immobilization, to facilitate the rational selection of antibodies for such analyses. Polyclonal and monoclonal antibodies were compared as was the impact of affinity purification of the polyclonal antibody to isolate the drug-specific fraction. The probes were evaluated for utility in analyzing 7-aminoflunitrazepam at sub ng/mL concentrations in urine, which is expected to be found …