A Sensitive GC-MS Procedure for the Analysis of Flunitrazepam and its Metabolites in Urine

Abstract

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.

Analytical developments in toxicological investigation of drug-facilitated sexual assault

“…In the past few years, considerable information about socalled date-rape drugs and drug-facilitated sexual assault has accumulated; at the same time an increase in the number of scientific publications on the subject has been observed [1,2,3,4,5,6]. In a typical scenario, a sexual predator surreptitiously spikes the drink of an unsuspecting person with a sedative drug for the purpose of "drugging" and subsequently sexually assaulting the victim while under the influence of this substance.…”

“…Flunitrazepam is readily (80-90%) absorbed through the GI tract and metabolized almost completely by the liver. Its metabolism includes reduction to 7-aminoflunitrazepam and then to the N-glucuronide, demethylation to the N-demethyl metabolite, hydrolysis to the 3-OH metabolite and then to the O-glucuronide [2,11,12]. Approximately 90% of its metabolites are excreted through the urine and 10% in the feces [2,10].…”

Laboratory Confirmation of Flunitrazepam in Alleged Cases of Date Rape

“…All specimens underwent a three-step analytical process. 2 The first step involved the Abuscreen OnTrak immunoassay (Roche Diagnostic Systems, Indianapolis, IN), which qualitatively detects benzodiazepines (including flunitrazepam). The second step involved pretreatment of the urine with ␤-glucuronidase followed by analysis using an automated Abuscreen OnLine (Roche Diagnostic Systems) immunoassay system.…”

“…Pretreatment with ␤-glucuronidase increases the sensitivity of the immunoassay for flunitrazepam. 2 Finally, each specimen underwent gas chromatographymass spectrometry (GC-MS) to confirm the qualitative results obtained in the first two steps and to distinguish specific benzodiazepines.…”

“…The limits of detection for flunitrazepam metabolites are 100 ng/mL (OnTrak), 50 ng/ mL (OnLine), and 1 ng/mL (GC-MS). 2 …”