Simultaneous determination of nitrazepam and its metabolites in urine by micellar electrokinetic capillary...

We applied micellar electrokinetic capillary chromatography to simultaneous separation and determination of nitrazepam and its major metabolites, 7-aminonitrazepam and 7-acetamidonitrazepam, in spiked urine. Prior to electrophoresis, the three compounds were successfully extracted from the spiked urine with commercial disposable solid-phase cartridges. The optimum running buffer for the separation was prepared by combining 85 parts of 60 mM sodium dodecyl sulphate-6 mM phosphate-borate, adjusted to pH 8.5, with 15 parts of methanol. The separation order, completed within 25 min, was 7-aminonitrazepam > 7-acetamidonitrazepam > nitrazepam, at an applied potential of 20 kV. We obtained reproducible electropherograms in successive repetitions, and few other peaks or interferences appeared in the electropherogram. The detection limits of the three compounds were 50-100 pg (0.1-0.2 microgram/ml of analyte in spiked urine), and the recoveries were 78.9-100.8% for 1 microgram/ml and 84.1-100.3% for 5 micrograms/ml. The application of this method to forensic or clinical samples is demonstrated.