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Liquid Chromatography – Tandem Mass Spectrometry Techniques for Drugs of Abuse as Applied to Bacterial and Forensic Toxicology Stephanie Marie Martindale The ability of bacterial species to transform complex chemical substances has been well documented. Some of these species are native to the human gastrointestinal tract and play an active role in the postmortem decomposition process. These species have potential to cause biotransformations that affect compound-to-metabolite ratios within the human body, especially after death. Postmortem changes such as these have rarely been evaluated or taken into consideration but they have potential to supply valuable information, especially concerning compound identification and confirmation. The purpose of this research was to investigate the effects of Escherichia coli, Bacteroides fragilis, and Clostridium perfringens on drugs of abuse, and to compare these metabolites to those produced during normal human metabolism. To analyze and quantify these effects, a novel liquid chromatography triple quadrupole tandem mass spectrometry method was developed for diazepam, flunitrazepam, and metabolites in Reinforced Clostridial Medium. Reinforced Clostridial Medium is a complex matrix designed to provide the nutrients necessary to promote growth of bacterial species, particularly Clostridia, other anaerobes, and specimens in clinical samples. To date, similar methods of extraction, quantitation, and analysis of drugs of abuse in a nutrient medium matrix have not been published. After validation, the method was applied to assess the specific effects of human gastrointestinal species on the targeted drugs of abuse. Experiments explored the effects of individual bacterial species as well as a mixed culture on the benzodiazepine compounds under anaerobic conditions. Diazepam and flunitrazepam were selected for biotransformation studies because of their potential for abuse and prevalence in analysis of both clinical and forensic specimens. Additionally, the human metabolites for these compounds had been previously identified and reference standards were commercially available. A series of bacterial blanks were prepared in Reinforced Clostridial Medium and incubated under experimental conditions. None of the bacterial specimens produced interferences with the target analytes that satisfied peak shape, retention time, ion ratio, and limit of quantitation requirements. Analyte blanks were also prepared for analysis. Investigation of analyte blanks and the mass balance for the reaction suggested that diazepam performed a partial, unassisted degradation when incubated under experimental conditions. However, neither nordiazepam, oxazepam, nor temazepam registered an increase between preto post-incubation concentrations. The degradation product was therefore unable to be identified under current experimental conditions. In biotransformation studies, diazepam showed a decrease in concentration between preand post-incubation for the Escherichia coli and Bacteroides fragilis series of samples, while changes were minimal for Clostridium perfringens and the mixed culture. Findings suggested that Escherichia coli and Bacteroides fragilis were acting on diazepam; however there was no increase in concentration for the human metabolites monitored. The discrepancy in mass balance was potentially indicative of a unique bacterial metabolite not produced during normal human metabolism. Analysis of the flunitrazepam drug blanks likewise exhibited a decrease in concentration. Unlike what was observed in the diazepam blank experiments however, there was a corresponding increase in concentration for the 7-aminoflunitrazepam metabolite in post-incubation samples. This accounted for some of the flunitrazepam conversion. In biotransformation studies, samples incubated with Escherichia coli demonstrated a greater decrease in flunitrazepam concentration than what was seen in analyte blank samples, while Bacteroides fragilis, Clostridium perfringens, and the mixed culture resulted in nearly complete conversion of flunitrazepam. Increased 7aminoflunitrazepam concentrations accounted for the majority of the conversion; however data suggested production of a minor metabolite that was not monitored in the current analysis. These experiments served as a pilot study and proof of concept. Unlike previous research, animal models were not required for experimental purposes, which eliminated the ethical considerations and guidelines required for animal research. Frozen stock cultures of bacterial samples provided a theoretically endless supply of specimens for transformation studies, reducing cost. The streamlined methodology allowed for quantities of experimental variations to be performed in unison, conserving time. Extraction procedures, a narrow bore column, and low flow rate for instrumental analysis minimized solvent consumption and hazardous waste production, making analysis both cost-effective and environmentally friendly. The presented experimental methodology serves as a template and can be adapted and applied to a realm of possibilities including investigation of natural products, biodegradation of agricultural and environmental contaminants, isolation and repurpose of native bacterial enzymes, as well as further studies on pharmaceutical compounds and drugs of abuse. An additional method was developed and validated for quantitation of cocaine, fentanyl, and metabolites in Reinforced Clostridial Medium, which will be applied in similar fashion. The applications can easily be expanded to include alternative bacterial species as well. Ultimately, this methodology would be ideal to study compounds that are too toxic or lethal for animal and human metabolic investigations. This would be particularly useful in military explorations of exposure to incapacitation and chemical warfare agents.

Liquid Chromatography -- Tandem Mass Spectrometry Techniques for Drugs of Abuse as Applied to Bacterial and Forensic Toxicology