A rapid and sensitive method is presented for the simultaneous determination of flunitrazepam, norflunitrazepam, 7-aminoflunitrazepam, and 7-acetamidoflunitrazepam in serum, plasma, and urine. The compounds were extracted by a mixed-mode solid-phase procedure following analysis by high-performance liquid chromatography and ultraviolet detection and using methylclonazepam as the internal standard. The method revealed high recoveries and showed good precision and linearity for all compounds. The limit of detection was at least 1 ng/ml serum (plasma) for all compounds.

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A rapid and selective cleanup procedure based on immunoadsorption is described for the simultaneous extraction of diazepam and its free or glucuronidated metabolites nordiazepam, temazepam, and oxazepam from urine. The method can also be used for the extraction of lorazepam and lorazepam-glucuronide. Because the samples do not have to be hydrolyzed before extraction, valuable information is preserved. With the exception of lorazepam-glucuronide, recoveries between 86 and 100% were obtained at spiking levels up to 200 ng of benzodiazepine or glucuronide per milliliter of urine. Using methanol/water (90:10, v/v) as an eluent, the immunoadsorber could be used at least 20 times. High-performance liquid chromatograms of urine samples from patients receiving low therapeutic dosages of diazepam or lorazepam are shown to demonstrate the high purity of the extracts.

A sensitive, simple, and rapid method for the determination of flunitrazepam and its major metabolites (7-aminoflunitrazepam, 7-acetamidoflunitrazepam, and norflunitrazepam) in serum and plasma is presented. The on-line procedure uses an immobilized, highly reusable antibody against benzodiazepines for selective extraction from serum followed by analysis by high-performance liquid chromatography with ultraviolet detection. This reliable method provides a limit of detection of 1 ng/mL serum, and results are obtained in less than 40 min.