The analysis of drugs and solvents in forensic toxicology by combined GC- and LC-MS
The work described in this thesis was in the field of Forensic Toxicology, which is the study and practice of toxicology for legal purposes. Three different application areas were considered: (a) the analysis of drugs in blood by gas chromatography-mass spectrometry (GC-MS) following isolation from the biological matrix by solid phase extraction procedures, (b) the analysis of paint solvents in blood by gas chromatography (GC) and GC-MS following dynamic headspace elution from blood, and (c) an evaluation of Thermospray/Plasmaspray (TSP/PSP LC-MS) liquid chromatography-mass spectrometry as the basis of drug screening techniques in forensic toxicology. (a) In the first of these areas, a sensitive, specific and reliable method was developed for the analysis of basic drugs in blood, using morphine and buprenorphine as model compounds which are also drugs commonly abused in the Glasgow area. A novel extraction procedure was used, based on a commercially available chemically-modified silica containing surface-bound benzenesulphonylpropyl groups, which served as a cation exchange resin. Several methods for the initial treatment of the biological matrix wre examined and the one selected involved absorption of the blood sample on diatomaceous earth and solvent elution of the crude extract containing any drugs present in the sample. This extract was then purified using the cation exchange resin: conditions suitable for the efficient retention of basic drugs and subsequent elution were also examined and developed. The extraction efficiencies for morphine were 925% and 954% at concentrations of 35 and 560ng/ml blood, respectively, for buprenorphine 836% and 875% , at concentration of 0.5 and 8ng/ml respectively, and for other drugs were better than 85% . The end-step analytical technique chosen for this method was GC-MS, because of analytical and legal requirements with respect to sensitivity and specificity. The polar nature of the model compounds, and of many other drugs likely to be encountered in this field, required chemical modification of the substances prior to gas chromatography. A comparison was made of three silyl ether derivatives - the trimethylsily (TMS), ethyldimethylsilyl (EMS) and tert-butyldimethylsilyl (MTBS) ethers. The conditions required for derivative formation were examined, including the choice of silyl donor reagent, solvents, temperature of reaction and time to completion. The EDMS donor, diethyltetramethyldisilazane, was selected on the basis of the midl reaction conditions required for the test compounds and also because this reagent caused less interference during GC-MS analysis. EDMS ethers gave satisfactory gas chromatographic behaviour and the presence of prominent ions at high mass was shown to be advantageous for specific and sensitive detection by SIR-MS with little background interference. The developed method was considered to be useful for both clinical and post-mortem blood samples containing morphine and buprenorphine down to the low picogram/millilitre level, and therefore adequate for the intended purpose. The method was subsequently applied to 13 samples submitted for analysis to the Department of Forensic Medicine and Science and found to be satisfactory. (b) In the second area of application, dynamic head space (DHS) elution was used for the analysis of paint solvents in blood. Volatilised solvents were trapped on a Tenax-GC cartridge and were subsequently analysed by GC-MS. The extraction efficiency of this method for C18-C12 n-alkane hydrocarbons, which were present in the paint materials, was better than 90% and sensitivity down to pg/ml levels was obtained. Solvent extraction procedures for these hydrocarbons were evaluated using high purity solvent. The extraction efficiency was better than 85% . Analysis of extracts by GC or GC-MS suffered from interference from the solvent front which reduced the sensitivity. The DHS method was applied to a pilot study for occupational monitoring of a group of painters to assess the presence of paint solvents in their blood. Two venous blood samples were collected at the beginning and at the end of a working week from each subject. They showed the presence of several solvents similar to those present in paint material and the levels in the second series of samples were higher than those of the first series. The differences between the levels in the two series were statistically significant for n-nonane, n-undecane and alkylbenzenes. The levels in the first sample indicated incomplete clearance of these solvents from the body during the weekend, and the second samples indicated solvent uptake during the working week. Solvent contamination in the extraction system was tackled by several approaches but still hindered the accurate estimation of solvent levels in blood. (c) In the third area of application, the operating parameters which control the sensitivity of the mass spectrometer using the TSP/PSP LC-MS interface were evaluated. These included the effects of the probe temperature and discharge voltage on sensitivity and mass spectral fragmentation pattern and the effects of the mobile phase constituents on sensitivity and mass spectral peak stability. Solvent systems containing ammonium acetate buffer and an organic modifier such as acetonitrile produced the best results in plasmaspray LC-MS. Three model HPLC-MS analyses were developed for mixtures of basic drugs, barbiturates and opiates using both the plasmaspray positive and negative ion modes. During the development of the mobile phases, the optimization of chromatography by organic modifiers was assessed. The quality of chromatography obtained was not always as good as expected in conventional HPLC, but the combination of chromatographic and mass spectral data could be used for identification and quantification purposes. A compilation of PSP mass spectra of drugs commonly encountered in forensic toxicology was produced. These mass spectra provided mostly molecular weight information with little structural information.