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. 50 mg of powdered hair were incubated (2 h at 45 degreesC) after sonication (1 h) 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 mul of a mixture of phosphate buffer (20 mM, pH = 2.2) and acetonitrile (94/6, v/v). A total of 80 mul 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 precolumn 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. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.