Validated method for the therapeutic drug monitoring of flunitrazepam in human serum using liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry with an ion trap detector

An HPLC–MS–MS method for the quantitative analysis of flunitrazepam in human serum was established. The method features a very simple liquid–liquid extraction, the use of a standard 4-mm HPLC column, isocratic elution using a buffer-free solvent, short retention times in connection with good peak resolution and the sensitivity and selectivity of an ion trap MS–MS detector. The procedure enables unambiguous identification of analytes by their product ion spectra, as well as sensitive quantitation (limit of quantitation for flunitrazepam=0.5 ng/ml). This feature was used for the confirmation of HPLC–UV results for nitrazepam.