Micellar electrokinetic capillary chromatography of benzodiazepines in human urine

The determination of the major urinary compounds of eight common benzodiazepines, flunitrazepam, diazepam, midazolam, clonazepam, bromazepam, temazepam, oxazepam, and lorazepam, by micellar electrokinetic capillary chromatography (MECC) is shown to be a simple and attractive approach for confirmation testing of these drugs in human urine. After enzymatic hydrolysis and extraction using mixed-mode solid-phase cartridges and a two-step elution protocol, fractions were analyzed in a phosphate/borate buffer (pH 9.3) containing 75 mM sodium dodecyl sulfate and small amounts of isopropanol, methanol and/or acetonitrile using an instrument with on- column multi-wavelength detection. The presence of these compounds could unambiguously be confirmed in patient urines which tested positive for benzodiazepines using a commercial enzyme multiplied immunoassay screening technique (EMIT). The sensitivity of the MECC assay is demonstrated to be better than that of EMIT. MECC analysis of one patient urine which tested negatively employing EMIT revealed the presence of lorazepam, this demonstrating that false-negative results from the initial immunological screening process can be recognized using MECC. For one example, 7-aminoflunitrazepam, the MECC data are shown to agree well with those obtained by gas chromatography/mass spectrometry.