Determination of Chronic Flunitrazepam Abuse by Hair Analysis using GC-MS-NCl

A 38-year-old male was found comatose at home with an empty package of Rohypnol (2-mg tablets, 10 per package) near the body. Many other drugs, which had recently been stolen from a pharmacy, were also found. The judge in charge of the investigation of the stolen pharmaceuticals asked our laboratory to determine if the subject was a chronic user of flunitrazepam, leading our laboratory to develop a procedure for the detection of this benzodiazepine in human hair. The method involved decontamination of hair samples with dichloromethane, incubation in Sorenson buffer (pH 7.6) in the presence of diazepam-d5 used as internal standard, direct liquid-liquid extraction with diethylether-chloroform (80:20, v/v), derivatization with heptafluorobutyric anhydride and analysis by gas chromatography coupled to mass spectrometry in the negative chemical ionization mode of detection. Flunitrazepam and its major metabolite, 7-amino-flunitrazepam, were both detected. The concentrations determined in the proximal hair segment were 89.5 and 24.0 pg/mg for flunitrazepam and 7-amino-flunitrazepam, respectively.