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

Abstract

Solid phase micro extraction (SPME) and gas chromatographic analysis was used for the analysis of several benzodiazepines (oxazepam, diazepam, nordiazepam, flunitrazepam and alprazolam) in human urine and plasma. Several factors likely to affect the analyte recovery were screened in a fractional factorial design in order to examine their effect on the extraction recovery. Parameters found significant in the screening were further investigated with the use of response surface methodology. The final conditions for extraction of benzodiazepines were as follows: Octanol was immobilised on a polyacrylate fibre for 4 min. The fibre was placed in the sample and extraction took place at pH 6.0 for 15 min. Urine samples were added to 0.3 g ml −1 sodium chloride. In plasma, the extraction recovery was less than in urine and releasing the benzodiazepines from plasma proteins followed by protein precipitation was found necessary prior to sampling. The method was validated and found linear over the range of samples. The limits of detection in urine were determined to be in the range 0.01–0.45 μmol l −1. The corresponding limits of detection in plasma were in the range 0.01–0.48 μmol l −1. Finally, the method developed was applied to determine some benzodiazepines after administration of a single dose. This method offers sufficient enrichment for bioanalysis after a single dose of high dose benzodiazepines as diazepam, but for low dose benzodiazepines as flunitrazepam, further sensitivity is needed.