Published: Journal of Analytical Toxicology, ISSN 0146-4760, Volume 26, Number 7, October 2002, pp. 471-478

Deposition of 7-Aminoclonazepam and Clonazepam in Hair Following a Single Dose of Klonopin™
Adam Negrusz[1], Andrew M. Bowen[1], Christine M. Moore[2], Sheila M. Dowd[3], Mary Jane Strong[3], and Philip G. Janicak[3]
[1]Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612; and
[2]United States Drug Testing Laboratories, Inc., 1700 South Mount Prospect Road, Des Plaines, Illinois 60018; and
[3]Department of Psychiatry, College of Medicine, University of Illinois at Chicago, 1601 West Taylor Street, Chicago, Illinois 60612

The objective of this paper was to determine whether benzodiazepine clonazepam (CLO) and its major metabolite 7-aminoclonazepam (7-ACLO) could be detected in hair collected from healthy volunteers after receiving a single 3-mg dose of Klonopin (clonazepam). Such data would be of great importance to law enforcement agencies trying to determine the best time interval for hair collection from a victim of drug-facilitated sexual assault (DFSA) in order to reveal drug use. Ten healthy volunteers (6 women and 4 men, 23–49 years old) participated in the study. The following hair samples were collected from each volunteer: one before CLO administration, and 1, 3, 5, 14, 21, and 28 days after. All hair samples were pulverized and 50-mg aliquots were sonicated in methanol and digested with 0.1N HCl at 55°C for 18–24 h. Internal standard, diazepam-d5 (DIAZ-d5) was used. Both extracts were combined and extracted using HCX solid-phase extraction columns. After derivatization with HFBA all extracts were analyzed using highly sensitive negative chemical ionization gas chrometography–mass spectrometry. Standard curves for CLO (20–100 pg/mg) and 7-ACLO (1–20 pg/mg) were prepared by spiking aliquots (50 mg) of negative hair and had correlation coefficients of 0.985 and 0.989, respectively. In addition, two levels of control hair were prepared for CLO and 7-ACLO. All method validation parameters were within acceptable limits. 7-ACLO was detected in hair of 6 out of 10 volunteers. In two cases 7-ACLO appeared in hair three days after CLO intake and remained detectable for the entire 28-day study period (3.6–8.4 pg/mg and 2.7–3.0 pg/mg), and in two subjects it was detectable 21 days later (4.9 and 2.7 pg/mg and 1.2 and 23 pg/mg ). In two volunteers 7-ACLO was detected only on day 28 (1.8 and 3.3 pg/mg). CLO was not detected in any of the samples.

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