Sweeping technique combined with micellar electrokinetic chromatography for the simultaneous determination of flunitrazepam and its major metabolites

A sweeping technique, in conjunction with micellar electrokinetic chromatography, for the simultaneous determination of flunitrazepam and its major metabolites, 7-aminoflunitrazepam and N-desmethylflunitrazepam, is described. The optimized conditions for the sweeping and separation were a pH 9.5 buffer, 25 mM borate, 50 mM cetyltrimethylammonium bromide, 30% MeOH (v/v), and a 151-mm injection length. The calibration functions were all linear with the coefficient of determination (r²) exceeding 0.996 for the three target compounds. Using the sweeping procedure, the limits of detection were determined to be 13.4, 5.6, and 12.0 ng/mL for flunitrazepam, 7-aminoflunitrazepam, and N-desmethylflunitrazepam, respectively, and the sensitivity enhancement for each compound was within the range of 110-200 fold. The RSDs for the retention time and the peak area were less than 4.10%. The optimized sweeping method was also used to examine a spiked urine sample. We conclude that sweeping with micellar electrokinetic chromatography has considerable potential use in clinical and forensic analyses of flunitrazepam and its metabolites.