Systematic Evaluation and Validation of Benzodiazepines Confirmation Assay Using LC-MS-MS

Benzodiazepines is a large drug group used extensively to treat sleep disorders and anxiety; these drugs are frequently associated with various crimes such as murder and drug-facilitated sexual assault. With growing use and misuse of these compounds, confirmatory assays are increasingly required in clinical laboratories. In this study, 4 β-glucuronidase enzymes were systematically evaluated for their hydrolysis efficiencies. Additionally, the matrix effects and extraction recoveries of three protein precipitation plates were systematically evaluated. The recombinant IMCSzyme β-glucuronidase enzyme showed higher hydrolysis efficiency than did β-glucuronidase from abalone, Patella vulgata and Helix pomatia. Lower ion suppression and more favorable overall recovery were observed in the Supelco protein precipitation plate than in the two other plates. Analytes were separated on a superficially porous particle column (ultra biphenyl) within 4.5 min and characterized through electrospray ionization-tandem mass spectrometry in the positive ion multiple-reaction monitoring mode. The accuracy, carryover, extraction recovery, detection limit, matrix effect, quantification limit and precision of the method were validated. Sixty opioid-positive urine samples were analyzed; a high incidence of benzodiazepines was detected in these samples. The proposed technique is robust and suitable for efficiently monitoring the numerous benzodiazepines that occur in urine samples.