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Benzodiazepines: Laboratory Detection Challenges

Posted December 1, 2011

Cassie L. Boggs, MD

Benzodiazepines are a class of drug that has a wide variety of medical uses, including treatment for insomnia, alcohol withdrawal, anxiety disorders, seizure disorders, and muscle spasms. They are also used as induction agents for general anesthesia. Additionally, benzodiazepines are used recreationally to enhance the effects of heroin and cocaine, to lessen the symptoms of withdrawal of various drugs, and to render a victim unconscious prior to sexual assault. The symptoms of benzodiazepine ingestion include dizziness, disorientation, lack of coordination, and slurred speech. In addition to the apparent sedative, hypnotic, and disinhibitory effects, many benzodiazepines also have amnesic effects at therapeutic doses. At supratheraputic levels, patients can develop muscular hypotonia, hypotension, bradycardia, coma, and death. The effects of benzodiazepines can be difficult to differentiate from those of ethyl alcohol, so confirmation of the presence of benzodiazepines often requires a urine drug screen.

The half-lives of the various benzodiazepines range greatly. For example, midazolam (Versed) has a half-life of only 1.5–2.5 hours, but flurazepam (Dalmane) has a half-life ranging from 40–200 hours when one takes active metabolites into account.

In the clinical laboratory, immunoassays are the most widely used screening technique for drugs such as benzodiazepines. Immunoassays have multiple formats with commonly used acronyms such as EMIT (enzyme-mediated [or multiplied] immunologic technique), FPIA (fluorescence polarization immunoassay), and ELISA (enzyme-linked immunosorbent assay). These methods are inexpensive and fast, but they have important limitations and a negative result by these methods does not exclude the presence of the drug.

All immunoassay technologies are based on a competitive binding process between antibody and antigen, which in this instance is a drug. The antibodies used are most sensitive and specific for the drug used to generate the antibodies, which for benzodiazepine immunoassays is commonly oxazepam; however, many of the benzodiazepines do not form oxazepam during metabolism and, therefore, have the potential for being missed on screening if only this method of detection is used. In addition, the levels of the drugs that are present in the patient’s blood or urine at the time of specimen collection can be quite low and often can be below the level of detection for most immunoassay formats. As a result, a negative screen does not exclude the possibility that the patient has benzodiazepines in his or her blood or urine. Given that the therapeutic blood concentrations for benzodiazepines ranges from 2–1000 ng/mL depending on the specific benzodiazepine, blood concentrations below the level of detection are not uncommon.

The various benzodiazepines have a wide range of cut-off values for detection by immunoassay platforms (See Chart 1). These values range greatly because of the amounts of each metabolite that is formed from the parent compound and the level of cross-reactivity between the antibody or antibodies used. For this reason, it is important to know what methods a laboratory uses and what the strengths and weaknesses of those methods are. This information can be found on the package insert of the immunoassay used by the laboratory. If the laboratory result does not coincide with the clinical picture of the patient, discussion with the toxicology laboratory personnel or laboratory director may help to explain the discrepancy. Additionally, if the clinical suspicion of a particular drug, such as lorazepam (Ativan), use is communicated to the laboratory, the laboratory can use a testing method that is sensitive for that drug in the initial screen.

The strengths and weaknesses of the various platforms for benzodiazepine detection have been documented. One of the known problems with detection of benzodiazepines on screening tests is detection of lorazepam, which is used as an induction agent and to treat violent or psychotic behavior. As shown in Chart 1, the cut-off for level of detection for lorazepam with the EMIT and FPIA platforms is very high. In order to minimize the risk of a false negative result, some forensic toxicology laboratories and pain management monitoring laboratories now use a separate assay for detection of lorazepam in addition to a general benzodiazepine screen. The positive results are confirmed by a higher order method such as gas chromatography-mass spectroscopy; but if the screening test is negative and the physician provides no clinical impression or data, the higher order method will not be employed.

Chart 1: Threshold of detection (ng/mL)
AnalyteEMITFPIA
desalkylflurazepam100400
noridiazepam60200
oxazepam200300
lorazepam1000750
temazepam70300
OH-ethylflurazepam60300
OH-alprazolam60200
OH-triazolam100220

Suggested Reading

  • Drummer OH. Benzodiazepines—Effects on Human Performance and Behavior. Forensic Science Review. 2002;14:2–10.
  • Tenore PL. Advanced urine toxicology testing. J Addictive Dis. 2010;29:436–448.
  • Frasier AD. Psychotropic Agents: The Benzodiazepines. In: Shaw L, ed. The Clinical Toxicology Laboratory: Contemporary Practice of Poisoning Evaluation. Washington, DC: AACC Press; 2001:211–221.

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NewsPath® Editor: C. Leilani Valdes, MD, FCAP
This newsletter is produced in cooperation with the College of American Pathologists Public Affairs Committee and the NewsPath Editorial Board and may be reproduced in whole or in part as a service to the medical community. Copyright © 2011 by the College of American Pathologists.
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