Background image

USDTL Research

Highly sensitive micro-plate enzyme immunoassay screening and NCI-GC-MS confirmation of flunitrazepam and its major metabolite 7-aminoflunitrazepam in hair

J Anal Toxicol. 1999 Oct;23(6):429-35.
Negrusz A, Moore C, Deitermann D, Lewis D, Kaleciak K, Kronstrand R, Feeley B, Niedbala RS.
Department of Pharmaceutics and Pharmacodynamics, College of Pharmacy, University of Illinois at Chicago, 60612, USA. DOI: 10.1093/jat/23.6.429

Flunitrazepam (Rohypnol) is a benzodiazepine used in the treatment of insomnia as a sedative hypnotic and as preanesthetic medication in European countries and Mexico. Although it has no medicinal purpose in the United States, the occurrence of its abuse is increasing. Sexual abuse of both men and women while under the influence of so-called “date-rape” drugs has been the focus of many investigations. Reported date-rape drugs include flunitrazepam (FN), clonazepam, diazepam, oxazepam, gamma-hydroxybutyrate, and many others. FN has been banned in the United States because of its alleged use in such situations. Unfortunately, the detection of FN or its metabolites 7-aminoflunitrazepam (7-AFN) and desmethylflunitrazepam in a single specimen such as urine or blood is difficult in criminal situations because of the likelihood of single-dose ingestion and the length of time since the alleged incident. Hair provides a solution to the second of these problems in that drugs tend to incorporate into hair and remain there for longer periods of time than either urine or blood. There are various techniques for the detection of FN in plasma, blood, and urine, but little work has been done with hair. Hair collection is a virtually noninvasive procedure that can supply information on drug use for several months preceding collection. The objective of this paper was to determine if a commercially available micro-plate enzyme immunoassay system was sufficiently sensitive for the routine screening of 7-AFN in hair by the development of extraction procedures and optimization of the immunoassay kit. Further, this study used the same solid-phase extraction to isolate FN and its major metabolite, 7-AFN, and gas chromatography-mass spectrometry with negative ion chemical ionization for confirmation. Two seven-point standard curves were established ranging from 0.5 pg/mg to 100 pg/mg for 7-AFN and 2.5 pg/mg to 200 pg/mg for FN with respective deuterated internal standards. A replicate analysis of controls was performed to establish inter- and intraday variabilities. Two suicide cases along with one alleged date-rape case and one case of an emergency room patient whose blood screened positive for benzodiazepines were analyzed. All the hair specimens screened positive for benzodiazepines using micro-plate enzyme immunoassay. Two cases, including the date-rape case, were negative for FN and 7-AFN, and two postmortem hair samples were confirmed positive for FN and its metabolite.

Published by: United States Drug Testing Laboratories on

Contact USDTL


Client Services

By Phone: 1.800.235.2367
Business Hours (CST)
Monday....................7am-7pm Tuesday....................7am-7pm Wednesday.............7am-7pm Thursday..................7am-7pm Friday........................7am-7pm Saturday...................8am-5pm

Contact Client Services

Newsletters, Posters, and Catalogs

Our print materials will keep you up to date on the latest news in drug and alcohol testing.

Request Literature

Request Your Collection Supplies

For your convenience, USDTL provides test collection supplies at no additional charge.

Order Supplies