Determination of 7-aminoflunitrazepam in urine by dispersive liquid–liquid microextraction with liquid chromatography–electrospray-tandem mass spectrometry

Dispersive liquid–liquid microextraction (DLLME) and liquid chromatography–electrospray-tandem mass spectrometry (LC–ES-MS/MS) procedure was presented for the extraction and determination of 7-aminoflunitrazepam (7-aminoFM2), a biomarker of the hypnotic flunitrazepam (FM2) in urine sample. The method was based on the formation of tiny droplets of an organic extractant in the sample solution using water-immiscible organic solvent [dichloromethane (DCM), an extractant] dissolved in water-miscible organic dispersive solvent [isopropyl alcohol (IPA)]. First, 7-aminoFM2 from basified urine sample was extracted into the dispersed DCM droplets. The extracting organic phase was separated by centrifuging and the sedimented phase was transferred into a 300 μl vial insert and evaporated to dryness. The residue was reconstituted in 30 μl mobile phase (20:80, acetonitrile:water). An aliquot of 20 μl as injected into LC–ES-MS/MS. Various parameters affecting the extraction efficiency (type and volume of extraction and dispersive solvent, effect of alkali and salt) were evaluated. Under optimum conditions, precision, linearity (correlation coefficient, r2 = 0.988 over the concentration range of 0.05–2.5 ng/ml), detection limit (0.025 ng/ml) and enrichment factor (20) had been obtained. To our knowledge, DLLME was applied to urine sample for the first time.