Sample analysis
General methodology the forensic toxicologist should remember Orfila’s maxim ‘The presence of a poison must be proved in the blood and organs before it can be considered as a cause of death’. There are typically four main steps in any toxicological examination:
1. Detection– to detect any drugs or poisons in the samples submitted by means of screening procedures.
2. Identification– to identify conclusively any drugs, metabolites or poisons present by means of specific relevant physicochemical tests.
3. Quantification– to quantify accurately those drugs, metabolites or poisons present.
4. Interpretation– to interpret the analytical findings in (2) and (3) in the context of the case, the information given and the questions asked by the investigating officer. Note the distinction above between detection and identification. In forensic toxicology, as with many other areas of chemistry, there is a clear difference between these two aspects. Colour tests, thin-layer chromatography, immunoassays and other screening tests are commonly applied in toxicology. These tests rely on detecting a particular interaction with a functionality of the compound being tested for. For colour tests this interaction is generally via a reaction with a functional group present in the chemical structure of a substance or group of substances. However, other substances that may be present may have this same functional group and hence give the same or a very similar interaction. Thin-layer chromatography (TLC) (Chapter 13) relies on the molecular interactions of analytes with a solvent or mixture of solvents and an inert medium called the stationary phase to enable separation from other substances present in the sample. However, some of the other substances present may have similar molecular interactions and hence may behave similarly to drugs and poisons such that complete separation is not achieved. If techniques such as GC and/or HPLC are not available and the analyst has to rely on TLC, at least two, and preferably three, non-correlating TLC systems should be employed in order to improve the discrimination of the analysis. The specificity of an immunoassay is only as good as the specificity of the antigen–antibody interaction. As we will see in the chapter on immunoassays, for some drug assays an antibody with broad specificity for the drug class under test is often deliberately employed to minimise the number of tests that need to be carried out to detect members of a drug group. Thus, in the case of opiates, immunoassays may give a positive result in the presence of diamorphine (heroin), morphine or codeine. The reporting of a positive result for such an immunoassay without being certain which of these substances is present could have serious implications for the interpret ation of cases in a court of law. Toxicologists typically refer to the results of these initial tests as presumptive, i.e. there is a strong indication that a particular substance or class of substances may be present but further tests are required to confirm the identity of the particular substance.
The detection of the drug or poison is the most difficult part, as the nature of the poison may not be known. Hence toxicologists employ screening tests for a wide range of drugs or poisons. General screening methods are usually more flexible than special methods and can therefore be applied to a wide variety of materials. They are essential for the investigation of unknown poisonings, and have some advantages even when the toxic agent is known or suspected. Once a toxic agent has been detected, specific analytical procedures can be used to identify it conclusively. Most analytical procedures in toxicology rely on a combination of chromatography to separate out the substances in the sample and some form of spectroscopy to detect and/or identify the separated substances. The most commonly employed chromatographic techniques include TLC, GC (Chapter 18), HPLC and capillary electrophoresis (CE) (Chapter 20). Spectroscopic techniques used include ultraviolet–visible and fluorescence UV-visible, infrared (IR) and near infrared (NIR), Raman and MS.
In toxicological analyses where a body fluid or tissue is being analysed, maximum efficiency is gained by coupling a chromatographic technique with a suitable detection technique (so called hyphenation) so that separation and detection can be carried out in-line, typically with automation of sample introduction to the analytical instrument and automated data collection. This also allows analysis to be carried out on a 24 h basis. Hyphenated techniques most commonly used are GC-MS, HPLC-UV or HPLC-fluorescence and HPLC-MS (generally abbreviated to LC-MS). As we will see in Chapter 18, GC can be combined with several other types of detector but the powerful combination offered by GC-MS analysis has made it the work horse instrument in most modern analytical toxicology laboratories. LC-MS offers several advantages over GC-MS and is finding more and more uses in toxicology laboratories. CE-MS is still a developing technique and has yet to find routine use in many toxicology laboratories. However, the advantages that it offers in terms of relatively simple sample preparation and the simplicity of analysing certain types of analytes that are more difficult to analys by GC or HPLC mean that its use is likely to increase in the future as the technology matures. Nuclear magnetic resonance (NMR) spectroscopy is the workhorse technique of most organic chemistry laboratories, enabling identification of a compound from an NMR spectrum. However, the relative insensitivity of NMR and the difficulty of interpreting a spectrum of a complex mixture has meant that NMR is not a technique used in most toxicology laboratories. LC-NMR instruments have been developed. At the present time these instruments are costly relative to GC MS and LC-MS and considerably more complex to use, and thus they are not in routine use in most toxicology laboratories. As a result, NMR is not covered in this textbook. IR, NIR and Raman spectroscopies are covered because, although they are not used in combination with chromatographic techniques in most toxicology laboratories, they are used to identify bulk drugs.
