Direct quantitative analysis of benzodiazepines, metabolites, and analogs in diluted human urine by rapid resolution liquid chromatography–tandem mass spectrometry

Rapid resolution liquid chromatography (RRLC) coupled with triple quadrupole mass spectrometry (QqQ-MS) was developed for direct quantitative analysis of benzodiazepines (BDZs) and their metabolites, as well as BDZ analogs (zopiclone, zolpidem, and zaleplon) in diluted human urine. A perfluorophenyl column packed with sub-2-μm particles and a C8 precolumn were applied to accomplish RRLC analysis with symmetric peak shapes. Urine samples were diluted 10-fold with Milli-Q water prior to autoinjection for direct quantification of 34 target analytes with negligible matrix effect. Gradient elution and dynamic multiple reaction monitoring were used for simultaneous quantitation of 34 BDZs, metabolites, and BDZ analogs. Good recovery was obtained in the range of 80.2–98.5% and the limit of detection ranged from 0.01 ng/mL to 0.5 ng/mL for all 34 target analytes in spiked urine. Moreover, good precision and accuracy were obtained for quantitative determination in diluted urine samples by the proposed RRLC/QqQ-MS method for intra-day/inter-day assays in the ranges of 0.1–8.8%/0.1–8.9% and 91.2–106.1%/89.6–104.6%, respectively. The applicability of this newly developed RRLC/QqQ-MS method was demonstrated by quantitative determination of BDZs, metabolites, and BDZ analogs in various clinical urine samples.