Determination of benzodiazepines in human hair by on-line high-performance liquid chromatography using a restricted access extraction column.
A method is described for the identification of five frequently prescribed benzodiazepines (BZD) (clonazepam, diazepam, flunitrazepam, midazolam and oxazepam) in human hair samples by reversed phase HPLC, following on-line simple enrichment and clean-up on a restricted access extraction column. 50mg of powdered hair were incubated (2h at 45 degrees C) after sonication (1h) in 1 ml of the following solution (methanol:ammonia, 97.5/2.5, v/v). The aliquot was centrifuged and the methanolic phase transferred to a conical tube and evaporated under a gentle stream of nitrogen. The residue was reconstituted by adding 100 microl of a mixture of phosphate buffer (20mM, pH=2.2) and acetonitrile (94/6, v/v). A total of 80 microl were injected into the system with the column switching technique. The pre-column or clean-up column was washed with phosphate buffer pH=7.2. The drugs retained on the pre-column were then eluted in the back-flush mode and separated on a C(8) semi micro column, Lichrospher select B, 125 mm x 3 mm. The BZD were determined by a photodiode-array detector at 254 nm, using reference data (retention time and UV spectra) stored in a personal library. The method showed excellent linearity between 0.5 and 20 ng/mg of hair for clonazepam, flunitrazepam and midazolam and between 0.5 and 100 ng/mg of hair for diazepam and oxazepam. Finally, the present method has been applied to a number of forensic cases in our laboratory.
El Mahjoub
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Institut Universitaire de Médecine Légale, 9, avenue de Champel, 1211 Geneva 4, Switzerland.
Staub
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D000328:Adult; D014151:Anti-Anxiety Agents / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005445:Flunitrazepam / Q000032:analysis; D005554:Forensic Medicine / Q000379:methods*; D006197:Hair / Q000737:chemistry*; D006556:Heroin Dependence; D006801:Humans; D008297:Male; D008875:Middle Aged
D014151:Anti-Anxiety Agents; D005445:Flunitrazepam