Fundamentals of Analytical Toxicology. Robin Whelpton

and for laboratory analyses to monitor occupational exposure to certain chemicals has increased. There has also been increased demand for ‘on-site’ testing, be it either in the clinic, or at the roadside.

      Despite analytical advances, it remains impossible to look for all poisons in all samples at the sensitivity required to diagnose poisoning. It is vital therefore that the reason for any analysis is kept clearly in mind. Although the underlying principles remain the same in the different branches of analytical toxicology, the nature and the amount of specimen available can vary widely, as may the time scale over which the result is required and the purpose for which the result is to be used. All these factors may in turn influence the choice of method(s) for a particular analysis. Over the last 10 years the dramatic expansion in the range of compounds that may be misused to achieve intoxication has further complicated the role of the analytical toxicologist (Pasin et al., 2017), as has the use of such unusual substances as ²¹⁰Po and the nerve agent ‘Novichok’ as murder weapons (Harrison et al., 2017; Vale et al., 2018).

      

       1.2.1 Analytical methods

Immunoassays have found wide application in analytical toxicology. A range of techniques, for example enzyme-multiplied immunoassay technique (EMIT) and cloned enzyme donor immunoassay (CEDIA), are available and are often highly sensitive. Enzyme-based assays, such as that for paracetamol (acetaminophen), have also been described. However, all of these assays have the disadvantage that antibodies, enzymes, or specific binding proteins have to be prepared for each analyte or group of analytes before an analysis is possible. On the other hand, these and similar assays may often be used directly in small volumes of aqueous media (‘homogenous assay’), in contrast to chromatographic methods, which often require some form of sample preparation procedure, for example liquid–liquid extraction (LLE), otherwise known as solvent extraction, prior to the analysis. Moreover, although immunoassays can be very sensitive, some may be poorly selective, the antibody recognizing several structurally similar molecules. Sometimes this cross-reactivity can be exploited, as in screening for classes of misused drugs such as benzodiazepines and opiates (Box 1.1).

      There are practical considerations. Immunoassay is a batch process and therefore readily amenable to automation, whilst GC and LC involve sequential assays on the same instrument. Whilst automated sample preparation and shorter columns/faster analysis times can help, this remains a limiting factor. However, the infinitely greater selectivity of chromatographic methods even without MS detection means that such methods are often a prerequisite if the results are to withstand scrutiny in a court of law.

      LC has achieved wide application in analytical toxicology since the 1970s. Gases and very volatile solvents excepted, many analytes are amenable to analysis by LC or a variant of the basic procedure. GC, on the other hand, is restricted to the analysis of either compounds or derivatives that are both stable and volatile at temperatures up to approximately 350 °C. This being said, GC with modern bonded-phase capillary columns, temperature programming, nitrogen–phosphorus detection (NPD), and MS detection either when used alone, or linked in series (MS/MS), has tremendous sensitivity and selectivity especially when combined with a selective sample preparation procedure.

      The use of LC in the qualitative analysis of drugs and other poisons has been limited by the lack of a sensitive universal detector analogous to the flame ionization detector (FID) in GC and the poor performance of gradient elution systems. However, a range of sensitive MS detectors is now available for use with LC and together with modern narrow-bore packed columns and eluent gradient programming, major advances have been made. The advent of accurate mass detection systems, i.e. detectors that can measure mass-to-charge ratio (m/z) to four decimal places, together with other parameters that can be measured to increase the certainty of peak assignment, has been yet another milestone (Grapp et al., 2018). However, the high purchase and maintenance costs of such systems remains a major barrier to their widespread use.

Box 1.1 Opiates, opioids, and opium

       Opium is the dried residue obtained from the white, viscous fluid that exudes from the unripe seed head of the opium poppy (Papaver somniferum) when it has been cut

       The opium poppy is the major source of the drug morphine, but also contains the closely related alkaloids codeine and thebaine. Opium also contains meconin, noscapine, papaverine, and reticuline, amongst other compounds, which are not structurally related to morphine and are not analgesic

       Purified morphine and thebaine are used as starting materials for semi-synthetic drugs such as dihydrocodeine and oxycodone

       Acetylating morphine produces diamorphine, which was introduced in Germany in 1895 as an antitussive under the name Heroin. The addictive properties of diamorphine quickly became apparent and the importation, production, and use of diamorphine was banned in the US in 1924, for example

       The term ‘heroin’ is now generally used to refer to the impure diamorphine obtained by treating semi-purified opium with an acetylating agent, usually acetic anhydride. Acetylcodeine is a common contaminant of heroin

       Strictly, the term ‘opiates’ refers to substances obtained from opium, not all of which have morphine-like properties, whereas ‘opioids’ are materials with morphine-like properties

       The term ‘opioid’ can thus apply to not only naturally occurring and semi-synthetic compounds such as morphine and oxycodone, but also synthetic drugs such as methadone and dextropropoxyphene, as well as naturally occurring transmitters, for example met-enkephalin and leu-enkephalin

       The term ‘narcotic’ originally referred to any psychoactive compound with sleep-inducing properties. In the US it has since become associated with opiates and opioids, commonly morphine and heroin, as well as stimulants such as cocaine, but is clearly non-specific and is best avoided

      An important consideration is that the MS detector is a reaction detector, i.e. the signal obtained is dependent on chemical reaction(s) occurring in the ionization source. Therefore, the signal obtained is dependent not only on the analyte, but also on the possible presence of co-eluting compounds that may affect the ionization of the analyte. This is not normally a problem in GC-MS because the carrier-gas (usually helium) has little influence and the selectivity of the system is such that the analyte is usually fairly pure when it arrives at the ionization source. Not so with LC-MS, where either the suppression, or enhancement of the analyte signal due to the presence of co-eluting compounds can be significant.

       1.2.2 Systematic toxicological analysis

The problem in systematic toxicological analysis (STA, poisons screening, drug screening, unknown screening) is to detect reliably as wide a range of compounds as possible in as little sample (plasma/serum/whole blood, urine, vitreous humour, stomach contents or vomit, or tissues) as possible at high sensitivity, but with no false positives (Maurer, 1999). Ideally some sample should be left to permit confirmation of the results using another technique and if indicated quantitation of any poison(s) present to aid clinical interpretation of the results.

In poisons screening it is important to adopt a systematic approach in order to eliminate possible contenders and to ‘home in’ on any compound(s) of interest present. STA can be divided into three key stages (Figure 1.1). The aim of the sample preparation step is to retain all the toxicologically important substances whilst removing potentially interfering sample matrix components. Thus, a wide range of analytes of interest, including lipophilic and polar, acidic, basic, and neutral species, should be isolated. To increase the yield of analyte(s) when analyzing urine, for example, the sample may be treated with β-glucuronidase/arylsulfatase to hydrolyze conjugated metabolites, but this may not be necessary if the conjugates themselves can