Fundamentals of Analytical Toxicology. Robin Whelpton

Figure 1.1 The three key steps in systematic toxicological analysis

      The aim of the differentiation/detection step is to identify all relevant compounds in the minimum amount of time. This requires a combination of relatively non-specific (‘universal’) assays and highly selective methods. Immunoassays, particularly if the antibody has wide cross-reactivity, are useful for identifying classes of drugs. TLC has the advantage that all the non-volatile materials in the extract remain on the plate, whereas with GC and LC there is always the possibility that compounds have not been eluted from the analytical column. Obviously, one analytical technique cannot separate and identify all the possible compounds of interest. Only a finite number of compounds can be resolved on a single TLC plate, for example.

      The greater the number and range of techniques that are available, the greater the probability that unknown substance(s) will be identified correctly. Investigation of the responses of various analytes to different detectors can provide valuable information about the nature of a compound. LC with diode array detection (LC-DAD) can provide spectral information to aid peak assignment, but is limited by relatively poor sensitivity and selectivity (Section 10.3.1). In contrast, hyphenated techniques such as GC-MS can provide robust analyte identification, particularly when combined with computerized libraries of electron ionization (EI) fragmentation data that can be searched rapidly to confirm compound identity (Grapp et al., 2016). In addition, positive ion chemical ionization (PCI) MS can be used to give an indication of the molar mass of a substance (Section 13.3.1.2).

Analytes may be chemically modified to improve their chromatographic properties or ‘detectability’, but derivatization can also give useful qualitative information. One classic example is the so called ‘acetone-shift’ (reaction of acetone with a primary amine to give the corresponding Schiff's base). Amfetamine, for example, reacts with acetone to form N-(1-methyl-2-phenylethyl)propanimine (Figure 1.2). If the product does not chromatograph as expected, then the unknown substance was not amfetamine.

Figure 1.2 Reaction of amfetamine with acetone

      The third step in STA is to compare the observed data with validated database information. Clearly, databases used in compound identification need to be regularly updated, and must include information on not only parent compounds, but also metabolites, common interferences, and possible contaminants. It is important that the analytical techniques used in establishing such databases are reproducible, both within and between laboratories.

       1.2.3 Ethanol and other volatile substances

      Enzymatic methods for plasma ethanol using alcohol dehydrogenase with spectrophotometric measurement of a coenzyme, for example, are available in kit form for clinical chemistry analyzers. GC analysis of ethanol either by direct injection of blood or urine diluted with deionized water, or by static headspace sampling (HS-GC), is also widely used, particularly in forensic work. GC-FID is advantageous because methanol, 2-propanol, and acetone may be separated and measured simultaneously. Methanol poisoning from ingestion of synthetic alcoholic drinks is one of the few causes of acute poisoning ‘epidemics’ and measurement of blood methanol is important in confirming the diagnosis and in monitoring treatment (Section 22.4.1.2). Detection of high concentrations of acetone, itself a metabolite of 2-propanol, and vice versa, may aid the diagnosis of ketoacidosis (Belsey & Flanagan, 2016).

      Many more volatile compounds may be encountered in acute poisoning arising, for example, from deliberate inhalation of vapour in order to become intoxicated [volatile substance misuse, ‘glue sniffing’, solvent abuse, inhalant abuse, volatile substance abuse (VSA)]. Some of these volatile compounds have metabolites, which may be measured in urine in order to assess exposure, notably hippuric and methylhippuric (toluric) acids (from toluene and the xylenes, respectively) and trichloroacetic acid (from trichloroethylene). However, most volatile substances are excreted unchanged in exhaled air and therefore whole blood is the best sample for detecting and identifying these compounds (Section 22.4.23).

       1.2.4 Trace elements and toxic metals

In order to help diagnose either deficiencies or chronic poisoning with these substances, where alterations from target ranges of only a few μg L^–1 (parts per billion, ppb, i.e. parts per thousand million) of blood or serum can be important, good accuracy and reproducibility are essential (Wang et al., 2014). Sample contamination during collection (for example from sample tubes, or even from syringe needles in the case of chromium and manganese) and within the laboratory itself can be a serious source of error. This applies especially to widespread elements such as lead and aluminium.

Modern methods for measuring toxic metals in biological materials (Table 1.2) vary enormously in terms of complexity, cost, accuracy, and sensitivity. In the case of serum iron, however, reliable kits based on the formation of a coloured complex remain widely used in clinical chemistry. Some techniques (isotope dilution mass spectrometry, neutron activation analysis), on the other hand, are in reality reference methods. Atomic absorption spectrophotometry (AAS) with either flame or electrothermal atomization using a graphite furnace has been employed widely, but is being superseded by inductively coupled plasma-mass spectrometry (ICP-MS).

Table 1.2 Methods for the analysis of toxic metals in biological materials

Technique	Mode	Variant
Electrochemical	PotentiometricCoulometric	Ion selective electrodes(Differential pulse) polarographyAnodic/cathodic stripping voltammetry (A/CSV)a
Spectrophotometric	Optical Emission (OES)	Flame emission photometry (FEP)b Direct-current plasma Inductively coupled plasma (ICP)
	Atomic Absorption (AAS)	FlameHydride generationElectrothermalCold vapour
	X-Ray	Fluorescence
	Nuclear	Neutron activationProton activation
Mass spectrometric		Inductively coupled plasma (ICP-MS)

      ^aAlso known as potentiometric stripping analysis (PSA)

      ^bNormally refers to the use of filters to select the emission wavelength – used mainly for potassium, lithium, and sodium assay

      ICP-MS is a multi-element technique that can detect and measure isotopes with detection limits of μg L^–1 to ng L^–1. Different isotopes of an element can also be measured. For some elements, the relative abundance of the isotopes depends upon the source of the metal. Therefore, by measuring the isotope ratios of an element such as lead in a sample from a chronically poisoned patient with those found in material present in the patient's immediate environment it may be possible to localize