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 simultaneous determination of five frequently prescribed benzodiazepines: clonazepam, diazepam, flunitrazepam, midazolam and oxazepam was developed. A 50-microl plasma sample was directly injected into a BioTrap 500 MS (hydrophobic polymer) column. After a washing step with a mixture of phosphate buffer and acetonitrile, the retained benzodiazepines were back-flushed into a reversed-phase (LiChrospher Select B C8) column with a mobile phase of acetonitrile-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 the 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 described is relatively simple and rapid because no off-line manipulation of the sample is required: the total analysis time is approximately 30 min.
El Mahjoub
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Institut Universitaire de Médecine Légale, Geneva 4, Switzerland.
Staub
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D001569:Benzodiazepines / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D001569:Benzodiazepines