Systematic drug identification
One of the most difficult tasks the forensic toxicologist can face is to investigate a suspicious death when there is no information to guide them as to what substances may be present in samples submitted for analysis. The toxicologist is trying to find an answer to the question ‘Is there any drug or poison present that may have contributed to death?’ This is the proverbial ‘needle-in-a-haystack’ situation. A systematic approach must then be taken. Systematic toxicological analysis takes advantage of the separation of an unknown substance in standard TLC systems (or other chromato graphic systems) to establish the probable identity of the substance by reference to a database of candidate compounds using a statistical comparison approach, such as the mean list method. Suitable chromatographic techniques for systematic toxicological analysis must meet the following criteria:
1. The drugs must exhibit acceptable chromato graphic properties in the separation system.
2. The RF values for the drugs must be distributed evenly over the full RF range.
3. The RF values are standardised in such a way that good interlaboratory reproducibility is obtained.
4. When more than one separation system is used, there must be a low correlation of RF values in the selected systems. TLC systems that meet these requirements are described below.
Since pH-dependent extractions are custom arily used in drug extraction and work-up procedures, different TLC systems are used to separate acidic and basic drugs, with neutral drugs likely to occur in both fractions. The Committee for Systematic Toxicological Analysis of The International Association of Forensic Toxicologists (TIAFT) recommended 11 separation systems for drug identification (Table 13.9). Four systems (1 to 4a) are to separate neutral and acidic drugs and seven systems (4b to 10) are to separate neutral and basic drugs. Reference data are available for about 1600 toxicologically relevant substances. For general drug screens, the use of two separation systems with a low correlation is recommended: systems 2 and 4(a) for neutral and acidic drugs and systems 5 and 8 for neutral and basic drugs (systems 7 and 8 are nearly as good). Combining colour reactions with the TLC data improves the certainty of identification significantly. Four colour reactions are carried out on the same plate in sequence. After each step the colour is noted and encoded by means of a colour chart (1, yellow; 2, orange; 3, brown; 4, red; 5, purple; 6, black; 7, blue; 8, green; 0, no spot observed). The sequence consists of formaldehyde vapour and Mandelin’s reagent, water, fluorescence under 366 nm irradiation and modified Dragendorff’s reagent. Other sequential colour reactions can be encoded and utilised in the same way. The Merck Tox Screening System (MTSS) contains the TIAFT TLC database and several other useful tools for searches using other chromatographic and spectroscopic databases and user-created databases. Hard-copy lists of RF values are also available (de Zeeuw, 1992) Romano et al. (1994) presented data for 443 drugs in four TLC systems using high performance silica gel TLC plates (Table 13.10). These systems use slight modifications of the mobile-phase compositions recommended by TIAFT. The UniTox system uses three TLC systems (Table 13.11). System 1 is designed to separate neutral and acidic drugs and systems 2 and 3 to separate basic, amphoteric and quater nary drugs. Two of the separation systems are based on reversed-phase separations designed to complement the more familiar silica gel separations. The database contains over 375 drugs of general toxicological interest, including a large number of amfetamines. The Toxi-Lab system is a TLC kit for toxicological drug screening; it contains equipment for extraction, development, detection and identification. Separations are performed on unsupported, particle-embedded glass-fibre sheets with holes punched in them to receive samples and standards as extraction or reference disks. A combination of silica gel and reversed-phase separations together with sequential colour reactions is used for identification and confirmation purposes. The database is designed for computer search es with results enter ed in a standard format. Pesticides are a further class of toxic substances of interest to systematic toxicological analysis because of their general availability, toxicity and potential confusion with drugs. Erdmann et al. (1990) developed a database for 170 commonly used pesticides separated in three standardised TLC systems (Table 13.12). The systems in Table 13.12 supplement those in Table 13.9, in which many common pesticides migrate with the solvent front. Systems 1 and 2 are recommended for general screening and system 3 for the identification of special compounds not distinguished in the first two systems.
