Extraction and analysis of clonazepam and 7-aminoclonazepam in whole blood using a dual internal standard methodology.
In this paper, a simple and robust method for the determination of clonazepam and its primary metabolite (7-aminoclonazepam) in whole blood is described. Clonazepam (klonopin) is a popular prescription drug that has been implicated in the field of drug facilitated sexual assaults (DFSA). Clonazepam, 7-aminoclonazepam and the internal standards (deuterated analogues for GC-MS analysis and nitrazepam for analysis by LC-PDA/GC-MS) were spiked into blood samples. The samples were buffered with a pH 6-phosphate solution (5 mL) and extracted from phenyl spe columns. The columns were washed with 5% acetonitrile in pH 6-phosphate buffer (3 mL) and eluted with ethyl acetate (2 x 3 mL). The eluents were evaporated for further chromatographic analysis. For GC-MS, the samples were derivatized prior to analysis. When performed with LC-PDA the samples were reconstituted in distilled water. From this method LOQs of 5 ng/mL of sample is easily achievable by either chromatographic system. By using GC-MS in EI mode, 1 ng/mL of sample can be detected. Data is presented here to show the simplicity and efficiency of the extraction scheme. By employing the properties of GC-MS and LC-PDA, this extraction and analysis procedure further extends the number of tools open to the forensic toxicologist for the analysis of this drug.
Hackett
Jeffery
J
Forensic Toxicology Laboratory, Center for Forensic Sciences, 100 Elizabeth Blackwell Street, Syracuse, NY 13210, USA. hljhack@ongov.net
Elian
Albert A
AA
D000328:Adult; D000927:Anticonvulsants / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D002998:Clonazepam / Q000031:analogs & derivatives* / Q000097:blood*; D005260:Female; D053593:Forensic Toxicology / Q000379:methods*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008297:Male; D009567:Nitrazepam / Q000097:blood; D052616:Solid Phase Extraction
D000927:Anticonvulsants; C020860:7-aminoclonazepam; D002998:Clonazepam; D009567:Nitrazepam