Elsevier Pathology

Clinical Pathology: Clinical Chemistry

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255) A 28-year-old woman who does not take any medications or drugs awoke in her hotel room a few hours after eating a cookie that was offered to her by a gentleman she met earlier that day. Although she felt fine, her belongings were stolen. After a police investigation, she was taken to the emergency department, where a urine sample was collected approximately 12 hours after the patient ate the cookie. Urine screening for benzodiazepines (with a cutoff 300 μg/L) was negative. Because of a suspected “roofie” intoxication, the sample was treated with β-glucuronidase and reanalyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS); the following concentrations were found: flunitrazepam less than 0.25 ng/mL, 7-aminoflunitrazepam 46 ng/mL, 7-aminodesmethylflunitrazepam of 30 ng/mL (see Figure 1). Based on these results, the best conclusion is that the patient was drugged with which one of the following?
Mean metabolite excretion profiles after a 2.0-mg single dose of flunitrazepam, corrected for creatinine concentration (n = 8). (From Forsman M, Nystrom I, Roman M, et al. Urinary detection times and excretion patterns of flunitrazepam and its metabolites after a single oral dose. J Anal Toxicol 2009;33:491-501.)

• Flunitrazepam (Rohypnol), also known as a “roofie,” is often used for drug-facilitated sexual assault or robbery. It is added to drinks (i.e., by “drink spiking”), but it can also be eaten in cookies laced with flunitrazepam. Police investigations are often made difficult because of the anterograde amnesia in the victims caused by the drug.

• Flunitrazepam is absorbed quickly with a Tmax of 20 to 30 minutes and a half-life of 13 to 19 hours. The sedative effect varies between 4 and 8 hours. The drug is metabolized extensively by the liver, and many metabolites have been identified in plasma and urine. The primary urinary metabolites are 7-aminoflunitrazepam and 7-aminodesmethylaminoflunitrazepam.

• Because of cross-reactivity, immunoassays for benzodiazepines can also detect flunitrazepam and its metabolites in the urine, especially after deglucuronidation. However, different immunoassays have different cross-sensitivities toward flunitrazepam and its metabolites. Therefore, some assays yield positive results for a particular sample, whereas other assays will be negative. Higher doses of the drug (e.g., 2 mg) are more likely to yield a positive result compared with lower doses of the drug (e.g., 0.5 mg).

• After intake of the drug, there is a time course for flunitrazepam and its metabolites in urine, with maximal values of 7-aminoflunitrazepam at approximately 24 hours after intake and an increasing ratio of 7-amonidesmethylflunitrazepam to 7-aminoflunitrazepam over time (see Figure 2). The ratio of the two, which can be determined by LC-MS/MS, can be used to determine the approximate time of intake.

• This is a forensic case, and care must be taken regarding maintaining a careful chain of evidence.

Forsman M, Nystrom I, Roman M, et al: Urinary detection times and excretion patterns of flunitrazepam and its metabolites after a single oral dose. J Anal Toxicol 2009;33:491–501.

Elliott SP, Burgess V: Clinical urinalysis of drugs and alcohol in instances of suspected surreptitious administration (“spiked drinks”). Sci Justice 2005;45(3):129–134.

Daderman AM, Strindlund H, Wiklund N, et al: The importance of a urine sample in persons intoxicated with flunitrazepam-legal issues in a forensic psychiatric case study of a serial murderer. Forensic Sci Int 2003;137(1):21–27.

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