A highly sensitive method for the determination of 7-aminonitrazepam, a metabolite of nitrazepam, in human urine using high-performance electrospray liquid chromatography tandem mass spectrometry.

A highly sensitive method has been developed for the determination of urinary 7-aminonitrazepam (7-ANZP), the major metabolite of nitrazepam, using high-performance electrospray liquid chromatography tandem mass spectrometry. The samples were prepared for analysis by adding 7-aminoclonazepam (7-ACZP, internal standard), hydrolysis with beta-glucuronidase and liquid-liquid extraction. Mass spectral acquisition was achieved by selectively monitoring the reaction between the two diagnostic transition reactions. Qualitative analysis was based on the retention time, and the quantitation was carried out by comparison with the internal standard. The recoveries of different concentrations of 7-ANZP from spiked blank samples was 89.0-95.2%, and the relative standard deviation was below 6.4%. The limit of determination in urine was 0.07 ng/mL, and the limit of quantitation was 0.5 ng/mL in the linear range of 1-50 ng/mL. This method possesses the merits of convenient operation, high sensitivity and good repeatability, making it a practical method for analysis of 7-ANZP in urine.