Dr James Watterson, Associate Professor, Department of Forensic Science at Laurentian University in Ontario, told SelectScience® about his forensic toxicology research and the technologies he utilizes in his work. Dr Watterson’s primary interest is the characterization of drug and metabolite disposition in skeletal remains and he has employed a variety of chromatography and mass spectrometry based analyses.
Dr Watterson has made significant findings in the toxicological analysis of bone. Although this area is less commonly used in forensic casework, Dr Watterson’s team have been able to detect a range of drugs in skeletal remains. Very little reference data are available to facilitate meaningful interpretation from measurements of drugs from bone. Using experimental animal models, the team found a great deal of variation in drug and metabolite levels across different bones in the skeleton. With such large variation (approximately 20-50 fold) across different bones, toxicological interpretation of drug levels are not possible, beyond confirmation of exposure to the drug at some point. However, the ratio levels of a drug and its primary metabolite were found to be less variable and more discriminatory between different exposure patterns (e.g. acute vs. repeated doses), than the individual analyte levels themselves. These findings suggest that the relationship between drug and metabolite levels may be more informative and enable discrimination between different patterns of drug exposure. This is now being further investigated by the team.
Now that we have switched to UPLC-qTOF-MS as our primary analytical approach, the advantages are absolute: substantially reduced sample preparation requirements, vastly improved sensitivity and selectivity, and a much greater of analytes that may be assayed.
Dr. James Watterson
Detection of drugs and metabolites
Dr Watterson’s laboratory uses standard analytical toxicology techniques to detect drugs and metabolites. They have demonstrated the utility of screening by immunoassay, as well as using more comprehensive extraction and analytical procedures to isolate the drugs from the bone matrix, including microwave-assisted extraction and solid-phase extraction, followed by gas chromatography mass spectrometry (GC/MS), ultra performance liquid chromatography with photo diode array (UPLC-PDA) or ultra performance liquid chromatography with time-of-flight mass spectrometry (UPLC-ToF MS).
The team is now investigating the probative power of quantitative drug-metabolite relationships in bone using LC-MS techniques, which offer greater sensitivity and selectivity for analyses of a number of different metabolites. As Dr Watterson explains, “Now that we have switched to UPLC-qTOF-MS as our primary analytical approach, the advantages are absolute: substantially reduced sample preparation requirements, vastly improved sensitivity and selectivity, and a much greater of analytes that may be assayed.” The choice of LC column for such analyses is also of critical importance, the correct column chemistry is essential for efficient separation. For example, the team used Raptor Biphenyl columns, with long column geometry and small particle/fused-core stationary phase, to maximize resolution of polar metabolites of phenothiazine drugs, using UPLC-PDA. This approach was critical in the characterization of these metabolites and of the phenothiazine oxidation products, which are produced during sample preparation. Dr Watterson comments, “the biphenyl column has been very helpful in resolving those compounds too, as we applied this method to UPLC-qTOF-MS”. Dr Watterson advises choosing reagents and columns carefully, as background impurities are much more visible using current technologies.
Dr Watterson’s future research goals include expanding skeletal tissue analysis to include chemometric approaches, similar to those used in metabolomics, with the hypothesis that such analyses may better enable patterns associated with drug exposure variations to be identified. These more quantitative analyses involve analysis of more metabolites and require highly sensitive and selective LC-MS techniques.
The team is also working on characterization of the forensic utility of dried blood spot (DBS) analysis. This technique has been applied to clinical toxicology and drug development studies, and the team is now investigating the utility of DBS for cases of drug impaired driving, again using highly sensitive and selective LC-MS techniques.
Dr Watterson remarks that forensic toxicology is now struggling to keep up with the appearance of a wide range of novel psychoactive substances (NPSs) that are synthetic variants of existing substances, such as the synthetic cathinones, or “bath salts.” This is because the human pharmacology of these compounds is poorly understood and they are usually in use long before certified reference materials are available to forensic laboratories. Dr Watterson comments that high resolution mass spectrometry techniques will be key for toxicological investigation of these substances in the future.