Simultaneous Determination and Quantification of 12 Benzodiazepines in Serum or Whole Blood Using UPLC/MS/MS

The benzodiazepines are a large, commonly prescribed family of psychoactive drugs. We describe a method permitting the simultaneous detection and quantification of 12 benzodiazepines in serum using ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS). Analytes included alprazolam, temazepam, oxazepam, nordiazepam, clonazepam, lorazepam, diazepam, chlordiazepoxide, midazolam, flunitrazepam, 7-aminoclonazepam, and 7-aminoflunitrazepam. Sample pretreatment is simple consisting of protein precipitation using cold acetonitrile (ACN) mixed with the deuterated internal standards. Samples were capped and vortexed for 5 min to ensure maximum precipitation. Following a 5-min centrifugation period, 400 �L of the supernatant was transferred to a clean tube and evaporated down under nitrogen. Samples were reconstituted in 200 �L of a deionized water:ACN (80:20) mixture and transferred to appropriate vials for analysis. Chromatographic run time was 7.5 min, and the 12 analytes were quantified using multiple reaction monitoring (MRM) and 6-point calibration curves constructed for each analyte at concentrations covering a clinically significant range.