Quantification of benzodiazepines in whole blood and serum

A high-performance liquid chromatography method for the determination of benzodiazepines and their metabolites in whole blood and serum using mass spectrometry (MS) and photodiode array (PDA) detection is presented. The combination of both detection types can complement each other and provides extensive case relevant data. The limits of quantification (LOQ) with the MS detection lie between 2 and 3 μg/l for the following benzodiazepines or metabolites: 7-amino-flunitrazepam, alprazolam, desalkyl-flurazepam, desmethyl-flunitrazepam, diazepam, flunitrazepam, flurazepam, α-hydroxy-midazolam, lorazepam, midazolam, nitrazepam, nordazepam and oxazepam, respectively 5 μg/l for lormetazepam and 6 μg/l for bromazepam. The LOQ of clobazam determined with the PDA detector is 10 μg/l. A convenient approach for determining the measurement uncertainty of the presented method—applicable also for other methods in an accreditation process—is presented. At low concentrations (<10 μg/l), measurement uncertainty was estimated to be about 50%, and at concentrations >180 μg/l, it was estimated to be about 15%. One hundred and twenty-eight case data acquired over 1 year are summarised.