Microemulsion electrokinetic chromatography versus capillary electrochromatography-UV-mass spectrometry for the analysis of flunitrazepam and its major metabolites

Benzodiazepines, namely flunitrazepam and its three major metabolites, were successfully separated by microemulsion electrokinetic chromatography. Separation was achieved using an untreated fused-silica capillary (48 cm (effective length 40 cm) x 50 num) at 25 kV; detection was performed by UV at 220 nm. The microemulsion system consisted of 70 mM octane, 800 mM 1-butanol, 80 mM sodium dodecyl sulfate (SDS) and 10 mM borate buffer, pH 9. Very high efficiencies (up to 400 000 plates) and resolution better than 3 were achieved. Since this technique is not compatible with mass spectrometry (MS) detection, a capillary electrochromatographic (CEC) method was developed to separate flunitrazepam and its metabolites. The effects of mobile phase composition and pH as well as voltage and temperature were systematically investigated. The optimized CEC method allowed the baseline separation of the investigated compounds. For the on-line coupling of CEC with electrospray ionization-mass spectrometry, the column was connected to a void fused-silica capillary using a Teflon connection. This configuration was found efficient and suitable for hyphenation of commercial CEC and MS instrumentation using commercially available CEC columns.