Determination of flunitrazepam and 7-aminoflunitrazepam in human urine by on-line solid phase extraction liquid chromatography–electrospray-tandem mass spectrometry

A method using an on-line solid phase extraction (SPE) and liquid chromatography with electrospray-tandem mass spectrometry (LC–ES-MS/MS) for the determination of flunitrazepam (FM2) and 7-aminoflunitrazepam (7-aminoFM2) in urine was developed. A mixed mode Oasis HLB SPE cartridge column was utilized for on-line extraction. A reversed phase C18 LC column was employed for LC separation and MS/MS was used for detection. Sample extraction, clean-up and elution were performed automatically and controlled by a six-port valve. Recoveries ranging from 94.8 to 101.3% were measured. For both 7-aminoFM2 and FM2, dual linear ranges were determined from 20 to 200 and 200–2000 ng/ml, respectively. The detection limit for each analyte based on a signal-to-noise ratio of 3 ranged from 1 to 3 ng/ml. The intra-day and inter-day precision showed coefficients of variance (CV) ranging from 4.6 to 8.5 and 2.6–9.2%, respectively. The applicability of this newly developed method was examined by analyzing several urine samples.