title: Simultaneous quantification of diazepam, flunitrazepam and metabolites in reinforced clostridial medium by liquid chromatography-tandem mass spectrometry.
abstract: A novel liquid chromatography-tandem mass spectrometry method was validated for identification and quantification of diazepam, flunitrazepam and metabolites in reinforced clostridial medium (RCM), a complex matrix used to provide the nutrients required for bacterial growth. The method was designed for subsequent use in the investigation of gastrointestinal bacteria as a potential source of postmortem alteration of drugs of abuse and respective metabolite concentrations. A literature review yielded no experimental means or model for the extraction and analysis of samples from RCM or similar bacterial medium. Development and validation of a new experimental method were therefore critical. In future work, this method could be adapted and extended to similar organic compounds of interest. The calibration curves extended from 0.100 to 500 ng/mL. Analyte recoveries ranged from 95 to 119% and matrix effects from 97 to 119%. Bias was ≤±17.6%, within-run precision ≤12.2%, and between-run precision ≤11.7% across all concentration levels. The limits of detection and quantitation ranged from 0.100 to 1 ng/mL. Dilution integrity was maintained for 1:2 and 1:5 dilutions. Analytes were stable through two freeze-thaw cycles and processed samples for 48 h. Method robustness was evaluated by changes in buffer composition and column temperature as well as samples prepared by an alternate analyst.
authors.affiliation: Edgewood Chemical Biological Center, E3400 Ricketts Point Road, Aberdeen Proving Ground, Gunpowder, MD 21010, USA stephanie.m.martindale2.civ@mail.mil.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Martindale
authors.affiliation: Connecticut DPS Controlled Substances and Toxicology Laboratory, 278 Colony Street, Meriden, CT 06451, USA.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Powers
authors.affiliation: Department of Chemistry, West Virginia University, 1600 University Avenue, 208 Oglebay Hall, Morgantown, WV 26508, USA.
authors.forename: Suzanne C
authors.initials: SC
authors.lastname: Bell
mesh_terms: D002138:Calibration; D002853:Chromatography, Liquid; D003013:Clostridium; D003470:Culture Media / Q000737:chemistry*; D003975:Diazepam / Q000032:analysis*; D005445:Flunitrazepam / Q000032:analysis*; D041981:Gastrointestinal Tract / Q000187:drug effects / Q000382:microbiology; D057230:Limit of Detection; D015203:Reproducibility of Results; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry
chemical_list: D003470:Culture Media; D005445:Flunitrazepam; D003975:Diazepam