Determination of benzodiazepines in human urine and plasma with solvent modified solid phase micro extraction and gas chromatography; rationalisationof method development using experimental design strategies

Solid phase micro extraction (SPME) and gas chromatographic analysis was us ed for the analysis of several benzodiazepines (oxazepam, diazepam, nordiaz epam, flunitrazepam and alprazolam) in human urine and plasma. Several fact ors likely to affect the analyte recovery were screened in a fractional fac torial design in order to examine their effect on the extraction recovery. Parameters found significant in the screening were further investigated wit h the use of response surface methodology. The final conditions for extract ion of benzodiazepines were as follows: Octanol was immobilised on a polyac rylate fibre for 4 min. The fibre was placed in the sample and extraction t ook place at pH 6.0 for 15 min. Urine samples were added to 0.3 g ml(-1) so dium chloride. In plasma, the extraction recovery was less than in urine an d releasing the benzodiazepines from plasma proteins followed by protein pr ecipitation was found necessary prior to sampling. The method was validated and found linear over the range of samples. The limits of detection in uri ne were determined to be in the range 0.01-0.45 mu mol l(-1). The correspon ding limits of detection in plasma were in the range 0.01-0.48 mu mol l(-1) . Finally, the method developed was applied to determine some benzodiazepin es after administration of a single dose. This method offer sufficient enri chment for bioanalysis after a single dose of high dose benzodiazepines as diazepam, but for low dose benzodiazepines as flunitrazepam, further sensit ivity is needed. (C) 1998 Elsevier Science B.V. All rights reserved.