Simultaneous determination of benzodiazepines in whole blood or serum by HPLC/DAD with a semi-micro column.

ABSTRACT

A simple and sensitive high performance liquid chromatographic (HPLC) method using a semi-micro column, C8 reversed-phase column (3 mm i.d.) and a low flow rate 0.3 ml/min was developed and validated for the determination of five frequently prescribed benzodiazepines clonazepam, diazepam, flunitrazepam, midazolam and oxazepam. Quantification was performed at 220 nm with methylclonazepam as internal standard. The method involved a simple extraction from alkalinized blood (1 ml) into 1-chlorobutane and provided excellent sensitivity, recovery, accuracy and reproducibility for benzodiazepines in therapeutic or toxic concentrations.