Simultaneous HPLC analysis of the hypnotic benzodiazepines nitrazepam, estazolam, flunitrazepam, and triazolam in plasma

A new analytical methodology was developed for simultaneous high-performance liquid chromatographic quantitation of nitrazepam, estazolam, flunitrazepam, and triazolam in plasma. After addition of the internal standard nor-prazepam, biological samples and calibration standards were extracted at basic pH into diethyl ether-methylene chloride (2:1, v/v). The reconstituted extract was separated on a reversed-phase Nova Pak C18 column with acidic buffer (6mM)-acetonitrile-methanol (64:23:13, v/v) as mobile phase. The detection was performed at 242 nm. Within-run and day-to-day precision values were about 2-8%. The method is thus sensitive and reproducible for use in clinical and experimental toxicokinetic studies.