Quantitation of clonazepam and its major metabolite 7-aminoclonazepam in hair

J Anal Toxicol. 2000 Oct;24(7):614-20.
Negrusz A, Moore CM, Kern JL, Janicak PG, Strong MJ, Levy NA.
Department of Pharmaceutics and Pharmacodynamics, College of Pharmacy, University of Illinois at Chicago, 60612, USA. DOI: 10.1093/jat/24.7.614

Clonazepam (CLO) is an anticonvulsant benzodiazepine approved by the Food and Drug Administration for use in the treatment of seizures. It produces pharmacological effects (depression, amnesia) similar to other compounds from the same therapeutic class, and in combination with alcohol, its CNS-depressant action can be significantly potentiated. As with some other benzodiazepines, CLO is a drug possibly used in “date-rape” situations. A method using solid-phase extraction followed by a highly sensitive negative chemical ionization gas chromatography-mass spectrometry for the simultaneous quantitation of CLO and its major metabolite 7-aminoclonazepam (7-ACLO) in hair was developed and validated. The method has potential application to alleged drug-facilitated rape cases. To determine the feasibility of detecting 7-ACLO and CLO in hair, specimens were collected from 10 psychiatric patients treated with CLO, divided into 2-cm segments, and analyzed. Standard curves for 7-ACLO (1-1000 pg/mg) and CLO (10-400 pg/mg) had correlation coefficients of 0.998. All precision and accuracy values were within acceptable limits. 7-ACLO was present in measurable quantities (1.37-1267 pg/mg) in 9 out of 10 patient samples. CLO concentrations in hair were much lower (10.7-180 pg/mg). In 4 out of 10 cases, CLO was not detected in hair. Two patients who had never been treated with CLO before received a single 2-mg dose of the drug. Approximately three weeks later, hair samples were collected, and measurable quantities of 7-ACLO (4.8 pg/mg) were detected in the first segment (proximal) of one of those samples, and traces of the drug were present in the other sample. We concluded that the 7-ACLO is being deposited in hair in much higher quantities than the parent drug and remains there for extended periods of time. Our study also indicates that it is possible to detect 7-ACLO after a single dose of CLO as in the typical date-rape scenarios.