Extraction and analysis of flunitrazepam/7-aminoflunitrazepam in blood and urine by LC–PDA and GC–MS using butyl SPE columns

The forensic toxicology community has recognized flunitrazepam and its metabolite (7-aminoflunitrazepam) as compounds of concern for several years. In this procedure, the analytes were extracted from whole blood and urine onto single mode solid phase cartridges (butyl) using nitrazepam as an internal standard. The columns were washed with distilled water and hexane. All three compounds were eluted from the sorbent using an ethyl acetate–methanol solvent mixture. After collection and evaporation of the solvent, the residue was dissolved in A, 0.1% (v/v) aqueous trifluoroacetic acid for HPLC–PDA analysis or B, ethyl acetate for derivatization with pentafluoropropionic anhydride (PFPA) for analysis by gas chromatography–mass spectrometry (selected ion monitoring, SIM). A limit of quantitation for this method using HPLC–PDA was found to be 5 and 1.0 ng mL−1 by SIM.