High-performance liquid chromatographic method for the determination of benzodiazepines in plasma or serum using the column-switching technique

A column-switching high-performance liquid chromatographic method for the s imultaneous determination of five frequently prescribed benzodiazepines: cl onazepam, diazepam, flunitrazepam, midazolam and oxazepam was developed. A 50-mu l plasma sample was directly injected into a BioTrap 500 MS (hydropho bic polymer) column. After a washing step with a mixture of phosphate buffe t and acetonitrile, the retained benzodiazepines were back-flushed into a r eversed-phase (LiChrospher Select B C-8) column with a mobile phase of acet onitrile-phosphate buffer. The method showed excellent linearity from 50 to 1000 ng/ml for clonazepam, flunitrazepam and midazolam and from 50 to 5000 ng/ml for diazepam and oxazepam. The recoveries were around 98% for all th e benzodiazepines studied. The relative standard deviation for between and within-day assay was <20% for low concentrations close to the values of the limit of quantification and <4% for high concentrations. The procedure des cribed is relatively simple and rapid because no off-line manipulation of t he sample is required: the total analysis time is approximately 30 min. (C) 2000 Elsevier Science B.V. All rights reserved.