Semiautomated High-Performance Liquid Chromatographic Method for the Determination of Benzodiazepines in Whole Blood

A semiautomated method for the determination of five frequently prescribed benzodiazepines (BZD) (clonazepam, diazepam, flunitrazepam, midazolam, and oxazepam) in whole blood samples by reversed-phase high-performance liquid chromatography following simple online enrichment and clean-up on a short precolumn is described. After precipitation of protein and red cells with a mixture of organic solvents (methanol/acetonitrile, 50:50), the aliquot is centrifuged and the organic upper phase evaporated under a gentle stream of nitrogen. The residue is reconstituted by adding 500 microL of a mixture of phosphate buffer (20mM, pH 2.2) and acetonitrile (70:30, v/v). The sample is then directly introduced into the column-switching column. The precolumn is first washed with phosphate buffer at pH 7.2. Compounds retained on the precolumn are then eluted in the back-flush mode and separated on a C8 semi-microcolumn (Lichrospher select B, 125 x 3 mm). The BZD studied are determined by a diode-array detector at 254 nm. The method shows excellent linearity between 25 and 1,000 ng/mL for clonazepam, flunitrazepam, and midazolam and between 25 and 5,000 ng/mL for diazepam and oxazepam. The recoveries are around 80% for clonazepam and oxazepam and around 90% for the three others. Coefficients of variation for between-day and within-day assays are < 15% for low concentrations close to the limit of quantitation and < 5% for high concentrations.