A sensitive GC-MS procedure for the analysis of flunitrazepam and its metabolites in urine

Analysis of urine specimens collected from individuals ingesting 2 and 4 mg flunitrazepam (FN) showed positive results by OnLine and OnTrak immunoassays for up to 60 h. Gas chromatographic-mass spectrometric (GC-MS) analysis of these specimens for FN, 3-OH-FN, 7-amino-FN, 7-amino-3-OH-FN, desmethyl-FN, and 3-OH-desmethyl-FN after glucuronidase treatment showed only low levels of 7-amino-FN with almost none of the other metabolites. These levels were far below the expected results based on the immunoassay data. This study reports on a GC-MS procedure for FN and the previously listed metabolites. The method is based on acid hydrolysis of the urine specimens, which converts FN and all its metabolites described previously to one of four amino-benzophenone derivatives (1-4) with oxazepam-d5 as the internal standard. Under the experimental conditions, the internal standard is converted to 2-amino-5-chloro-benzophenone-d5. The limit of detection for 7-amino-FN and 7-amino-desmethyl-FN and their 3-OH derivatives was less than 1 ng/mL. Analysis of urine specimens collected for 72-h postingestion of 1, 2, or 4 mg FN showed appreciable levels of benzophenone 3 (product of 7-amino-FN and 7-amino-3-OH-FN) and lower levels of benzophenone 4 (product of 7-amino-desmethyl-FN and 7-amino-3-OH-desmethyl-FN) with no detectable levels of benzophenones 1 and 2. The method makes it possible to confirm the presence of FN metabolites in urine at least 72-h postingestion of small doses of the drug.