Evaluation of Chromatographic and Immunological Methods for the Detection and Confirmation of Flumazenil in Urine

Flumazenil (FMZ, Flumazepil, Anexate, RO 15-1788) is a benzodiazepine antagonist useful in reversal of benzodiazepine sedation or intoxication. It reverses respiratory and CNS depression from benzodiazepine overdosage as well as the effects of short acting, more potent benzodiazepines such as midazolam used in surgery. This investigation evaluates standard chromatographic (TLC, GLC) and immunological (EMIT, FPIA) methods for the detection of FMZ in urine. FMZ was extracted and detected to a minimum concentration of 250 ng/ml using the TOXI-LAB TLC system. Acid hydrolysis (FMZ ≤ 100 μg/ml) did not produce aminobenzophenones which could be detected by TLC/UV visualization. Specimens containing ≤ 300 ng/ml FMZ exhibited no cross reactivity and concentrations of 5 to 100 μg/ml exhibited cross reactivity of less than 0.2% with the Abbott TDx Benzodiazepine Assay. Specimens containing up to 100 μg/ml FMZ did not produce a positive response with the Syva EMIT st Urine Benzodiazepine Assay. Confirmation of FMZ was made by GLC/FID. FMZ and flunitrazepam (internal standard) were extracted from basified urine (pH 9.2) with n-butyl chloride and separated using a glass, 6\u27x 2mm ID, 3% SP2250DA column at 250°C with He (flow rate 40 ml/min). FMZ eluted at 11.31 min with a retention index of 0.908 relative to flunitrazepam. Data indicate that FMZ can be detected at nanogram concentrations by TLC and GC. The lack of cross reactivity in EMIT and FPIA assays and the lack of production of aminobenzophenones with acid hydrolysis decrease the possibility of false positives and the need for confirmation of true negative specimens. Broad use of FMZ in chemical dependency treatment programs will generate an increased need for the detection of FMZ in biological fluids