occupational history, if available. Decide the priorities for the analysis
1.3.1 Samples and sampling
In analytical toxicology, clinical chemistry, and related fields, the words ‘sample’ and ‘specimen’ are used to denote a portion of a body fluid, tissue, incubation medium, etc. obtained under defined conditions. The samples encountered may range from relatively pure solutions of a drug to a piece of putrefying tissue. Liquids, such as blood, oral fluid (principally saliva), urine, and cerebrospinal fluid (CSF), are generally easier to sample and to analyze than solids and semi-solids, which require homogenization or digestion prior to analysis.
Blood plasma or serum is used in clinical work if quantitative measurements are needed in order to assess dosage or monitor treatment as in TDM. Urine is commonly used in qualitative work such as substance misuse screening because collection is non-invasive and the concentrations of many drugs and their metabolites tend to be higher than in blood, thereby facilitating analyte detection. Further aspects related to samples and sampling are discussed in Chapter 2.
1.3.2 Choice of analytical method
In responding to a given analytical problem, many factors must be considered. It may seem self-evident that the method used should be appropriate for the intended analysis. In practice, the choice of method depends on several factors. These include (i) the circumstances under which an analysis is requested (i.e. the question being asked), (ii) the speed with which the result is required, (iii) the sample to be analyzed, (iv) the nature of the analyte (if known), (v) the expected concentration of any analyte(s), (vi) the time available for the analysis, (vii) the apparatus available, (viii) the existence of a validated method in the laboratory, and (ix) the training and experience of the analyst.
The nature of the sample and the expected concentration of any analyte(s) are obvious influences on the choice of method. It may be possible to measure the concentration of a known substance in a relatively pure solution directly using a simple technique such as UV spectrophotometry. However, if the sample is a piece of post-mortem tissue such as liver then a wholly different approach will be required. Typically, a representative portion of the tissue will have to be homogenized and the analyte obtained in a relatively pure form by LLE, for example, of the homogenate at an appropriate pH. Further purification or extract concentration steps may be needed prior to instrumental analysis. In the case of organic poisons, this will usually be by a chromatographic method such as GC or LC because both qualitative and quantitative information can be obtained during the course of the analysis. The choice of instrument may influence the choice of sample preparation procedure, although this is not always the case.
For optically active (chiral) drugs (Table 1.4), the desired clinical activity resides predominantly in one enantiomer, the eutomer. The other enantiomer (the distomer) may be either pharmacologically inactive, or have different properties from its enantiomer, so administration of a racemate (a 50:50 mixture of enantiomers, Table 1.5) is the same as giving different compounds as far as the body is concerned. The supply of optically active compounds as pure enantiomers is sometimes indicated by the name used (dexamfetamine, dextropropoxyphene, escitalopram, levorphanol), but this does not always apply (hyoscine, morphine, physostigmine). Moreover, it is thought that some 50 % of currently used drugs are chiral, of which some 88 % are supplied as racemates, usually without any indication of the fact (Nguyen et al., 2006). Atropine is the approved name for (±)-hyoscyamine, for example.
With the exception of amfetamine (Section 22.4.21.1) and other misused drugs where the enantiomers have different actions such as dextromethorphan/levomethorphan (levorphanol) and perhaps escitalopram (Section 22.4.3), there are few clear indications for providing chiral methodology for routine analytical toxicology at present. In part this is because chiral analysis at the sensitivity required for the analysis of biological samples is difficult. It should be noted that not only is MS achiral, but also that positional isomers cannot be differentiated by MS unless either resolved chromatographically, or by differing fragmentation patterns. Nevertheless, chromatographic methods have made a major contribution to the development of pharmacology and therapeutics by providing methods to separate enantiomers on a preparative scale and on occasions in biological samples (Fortuna et al., 2014).
1.3.3 Method validation and implementation
Whatever analytical method is used it must be validated, i.e. it must be shown to be ‘fit for purpose’. Method validation is important not only when developing a method (Peters et al., 2007), but also when implementing a method for routine use (Wille et al., 2017). A number of terms important to understanding method validation are given in Table 1.6. A fundamental starting point in any assay is obtaining certified pure reference material, or at least the best approximation to such material that can be sourced. When preparing primary standards, particular attention should be paid to the Mr of salts and their degree of hydration (water of crystallization). Analytical results are normally reported in terms of free acid or base and not of a salt (Section 3.1.1).
A method must possess adequate sensitivity for the task in hand. The limit of sensitivity is a term often used to describe the limit of accurate measurement, but this is better defined as the lower limit of quantification (LLoQ). The limit of detection (detection limit) is a better term for limit of sensitivity. Whatever terminology is used, assessing the presence or absence of a compound at the limit of assay sensitivity is always difficult and ideally reporting a positive finding in such circumstances requires corroboration from other evidence.
Table 1.4 Summary of chiral nomenclature
| Number of chiral centres | If n = number of optical centres there will be 2nisomers. Molecules with two optical centres can exist as four molecules: two diasteromers (diastereoisomers), each consisting of two enantiomers, i.e. there are two pairs of enantiomers. The exception to this is if two molecules have a plane of symmetry (a plane that divides a molecule into two parts, each a mirror image of the other) and therefore cancel out their net optical rotation. In such cases they are known as meso forms |
| Nomenclature | Enantiomers possess a unique property in that they rotate plane-polarized light to the same extent, but in opposite directions. This is the basis of the (+)/(–) or d/l notation, the former being preferred as it avoids confusion with D/L, however it does not unequivocally distinguish between enantiomers because some molecules may change rotation on forming salts. The notation tells nothing about the absolute configuration, i.e. the spatial arrangement, of the atoms |
| Rotation of plane polarized light |
Rotates to the right – dextrorotatory (+) Rotates to the left – levorotatory
|
