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Abstract
A high-throughput ultra-performance liquid chromatography (UPLC) method with UV detection was elaborated and validated for the simultaneous quantification of flunitrazepam and its major metabolite, 7-aminoflunitrazepam, in human plasma. After a simple liquid-liquid extraction with ethyl acetate, chromatographic separation was performed on a BEH C18 chromatographic column with a binary mixture of acetonitrile/10 mM potassium monohydrogen phosphate (pH = 8.5) as the mobile phase, under gradient elution (flow rate = 0.275 mL/min). The total run time of the chromatographic analysis was 4.2 min. UV detection was performed at 314 nm for flunitrazepam and at 242 nm for 7-aminoflunitrazepam. To our knowledge, this is the first reported UPLC-PDA method for the quantification of flunitrazepam and 7-aminoflunitrazepam in human plasma. The assay was applied successfully for the analysis of the selected two analytes in real human plasma samples obtained from a healthy volunteer, after ingestion of a single dose of flunitrazepam.