Analysis of biofluids for flunitrazepam and metabolites by electrospray liquid chromatography/mass spectrometry

A rapid and sensitive liquid chromatography/electrospray ionization mass sp ectrometry (LC/MS) procedure has been developed for the analysis of bioflui ds containing flunitrazepam and its metabolites. Specimens were spiked with deuterated analogs of the analytes. Urine specimens were enzymatically hyd rolyzed and blood specimens were untreated. Extractions were carried out us ing CleanScreen DAU SPE cartridges. The drugs were separated on a C18 colum n using a methanol-water-ammonium hydroxide (60:40: 0.03 v/v) mobile phase. After determination of base peaks using full scan mass spectrometry, the m ass spectrometry method was optimized to operate in selected-ion monitoring (SIM) mode for the base peak of each analyte. Positive findings were confi rmed by LC/MS/MS using the same mobile phase and column. This analytical pr ocedure allows for the detection of low levels of flunitrazepam and metabol ites in biofluids. It is useful for ascertaining the role of flunitrazepam in cases of drug-facilitated sexual assault.