Detection and determination of common benzodiazepines and their metabolites in blood samples of forensic science interest. Microcolumn cleanup and high-performance liquid chromatography with reductive electrochemical detection at a pendent mercury drop electrode.

Benzodiazepines in the blood samples typical of forensic science work are recovered from 100-250 microliters amounts of blood (diluted with aqueous sodium octyl sulphate to suppress protein binding) onto microcolumns of Porapak-T, and finally eluted into 60-microliters volumes of aqueous acetonitrile. The eluates may be taken directly for analysis by high-performance liquid chromatography (HPLC) with reductive amperometric detection at a pendent mercury drop electrode held at potentials down to -1.2 V vs. Ag/AgCl. For high sensitivity work the electrode is preceded by a coulometric detector fitted with porous carbon electrodes held at 0 V (proprietary reference electrode). The technique detects all of the commonly encountered benzodiazepines and others except clobazam, which contains no azomethine group. The detection limits generally are in the range 1-5 ng/ml (40-200 pg HPLC-injected) in hemolyzed human blood, with recovery values of 84-95%, depending on the actual benzodiazepine, over the range examined (less than or equal to 2.14 micrograms/ml). The respective values for the metabolites of nitrazepam are 8-12 ng/ml and 75-84%. The technique is very much less susceptible to the interferences afflicting other commonly applied techniques, and facilitates considerably the analysis of degraded samples.