Abstract: An efficient method of SEP-PKA solid-phase extraction and reversed-phase, isocratic high-performance liquid chromatographic method for the simultaneous quantitation of nitrazepam, clonazepam and flunitrazepam in human urine is presented. The recoveries with SEP-PAK C18 cartridgeswere excellent for all these drugs. The drugs were separated on a reversed-phase YMC A-303 C18 co-lumn with acetonitrile-0.01 mol/L ammonium acetate (45:55, v/v) as the mobile phase. The detectionwas performed at 254 nm. Within-day and day-to-day precision were about 1~5%. The method is sensitive and reproducible and can be used in clinical and experimental toxicokinetic studies.

Key words: SEP-PAK C18 cartridge, Benzodiazepine, HPLC