Flunitrazepam excretion patterns using the Abuscreen OnTrak and OnLine immunoassays: comparison with GC-MS

Area of Science:

• Forensic Toxicology
• Analytical Chemistry
• Pharmacology

Background:

• Immunoassays are widely used for drug screening, but their specificity and quantitative accuracy can be limited.
• Flunitrazepam (FNP) is a potent benzodiazepine with potential for abuse, necessitating reliable detection methods in biological samples.

Purpose of the Study:

• To compare the performance of two immunoassays (OnLine and OnTrak) against gas chromatographic-mass spectrometric (GC-MS) analysis for detecting flunitrazepam and its metabolites in human urine.
• To evaluate the impact of beta-glucuronidase treatment on immunoassay results.

Main Methods:

• Urine samples were collected from individuals following a single oral dose of flunitrazepam (1mg or 4mg).
• Immunoassay analysis was performed using OnTrak (100 ng/mL nordiazepam cutoff) and OnLine (with and without beta-glucuronidase).
• GC-MS analysis was conducted using standards for FNP and its major metabolites, with beta-glucuronidase treatment.

Main Results:

• Flunitrazepam doses of 1mg did not yield positive immunoassay results; 4mg doses did, with detection windows ranging from 4-12 hours initially to 18-60 hours finally.
• Beta-glucuronidase treatment increased OnLine immunoassay values by 20-60% but did not extend detection times.
• GC-MS confirmed 7-amino-flunitrazepam (7-amino-FNP) as the primary detectable metabolite, with peak concentrations correlating to immunoassay peaks, but accounting for only 15-20% of the immunoassay-detected cross-reactive metabolites.

Conclusions:

• Both immunoassays can detect flunitrazepam metabolites, but the 4mg dose is required for reliable detection within the studied timeframe.
• 7-amino-FNP is the main metabolite detected by GC-MS, and immunoassays may overestimate the total drug burden due to cross-reactivity with other metabolites.