Types of Toxicology Analytical Methods

Once samples in a forensic toxicology case have been collected, they must be handled, secured, and stored as any other evidence, with chain of custody documentation maintained from the time of collection until delivery to the analytical laboratory (that will have their own internal protocols to maintain chain of custody) (Murphy, 2012; Gwaltney-Brant, 2016). Analytical testing of samples can be expensive, especially if the identity of the toxicant has not been determined and multiple tests are run without a rational plan. Therefore, the information obtained from the history, clinical signs, response to therapy, diagnostic testing, and necropsy findings should be evaluated to determine the toxicants that are the most likely candidates. Consultation with a veterinary toxicologist can be very helpful in whittling down the list of potential toxicants; the toxicologist should also be able to provide guidance as to which analytical methods are likely to be the most fruitful.

Unlike life in television land, where one machine is able to identify multiple poisons from a single sample in less than the hour it takes the television show to run, the real world of toxicology must utilize a variety of techniques in order to identify and quantitate toxicants in samples. There is no such thing as a "tox screen” that will pick up any poison—the screening test offered by diagnostic laboratories (e.g., heavy metal screen, convulsant screen) are actually just tests for each individual substance that are bundled together as a convenience to the person requesting the analysis. Each analysis that is performed ultimately destroys some of the collected sample, and running multiple tests can rapidly deplete the sample (as well as the client’s wallet). The turnaround time for an analysis can be as short as a few hours or as long as several days or even weeks depending on the type of analysis performed. Samples being analyzed must be processed to isolate the fraction of the sample that contains the suspected toxicant. Tissue digestion, incubation, and the analysis itself can take hours or days, all of which add to the turnaround time.

Analysis of toxicology samples can be as simple as visualization of, say, a sample of feed for toxic plants or as elaborate as combining several sophisticated techniques as mass spectrometry and liquid chromatography (Levine, 2010). Physical inspection includes both visual and olfactory

Table 14.2 Sample collection for toxicology

Sources: Dinis-Olivera et al. (2010); Galey and Hall (1990); Millo et al. (2008).

Veterinary Forensic Toxicology Investigations evaluation of a sample, requiring little more than a pair of eyes +/— magnifying equipment. Visualization is used to identify toxic plants, toxic mushrooms, toxic algae, and gross contaminants in gastrointestinal contents, feeds, or other samples. Visualization is also used to determine some physical characteristics of samples or suspect materials, such as pH, dissolution in solvent, and density determination. Using olfaction to detect telltale odors from certain toxicants (e.g., the bitter almond odor to cyanide or the rotten fish odor to zinc phosphide) is another component of physical evaluation. Bioassays utilize living animals to aid in diagnosing poisoning cases, generally by exposing a normal animal to the suspect poison. Since they do not directly identify the poison, bioassays have little use in forensic toxicology, and they are rarely performed anymore except to detect the presence of botulinum toxin. Chemical reactions may be used as qualitative tests to indicate the presence of certain compounds, for example, the formation of Prussian blue indicates the presence of cyanide. However, these tests do not tell us how much of the toxicant is present, and further quantitative testing is required to determine whether toxic levels are present. Spectroscopy and spectrophotometry are used to detect compounds in liquid samples based on characteristic wavelength signatures. Immunoassays utilize antibodies to identify and quantitate compounds by means of a color change on a test strip or test well. Because they are relatively inexpensive, immunoassays are frequently used as screening tests, but any results would need to be confirmed by more robust techniques as they are prone to false positives. Mass spectrometry ionizes a compound down to its component atoms then separates the ions based on their mass-to-charge ratios. The mass spectra output gives the mass and relative amounts of the different ions, allowing determination of its chemical formula but not its chemical structure. Mass spectrometry is a rapid method that can be used alone to detect metals such as lead and arsenic, but it is when mass spectrometry is combined with chromatography techniques that its real power shines. Chromatography is commonly used to detect drugs, pesticides, and other organic compounds. Chromatography separates compounds based on their chemical properties, and the separated compounds can then be analyzed for their unique chemical properties. Chromatography may be performed on chemicals in liquids (high-performance liquid chromatography [HPLC]), solids (thin-layer chromatography [TLC]), and gas matrices (gas chromatography [GC]). GC followed by mass spectrometry is currently considered to be the gold standard of chemical analysis (Levine, 2010).

Interpretation of analytical results is an essential component of veterinary forensic toxicology (Ensley, 1995). With modern analytical equipment able to detect compounds down to the parts per trillion (ppt) level, it is now possible to detect the presence of compounds well below levels that are expected to be associated with toxicosis (Gwaltney-Brant, 2016). For instance, anticoagulant rodenticides such as brodifacoum are stored in the liver, so if a dog’s liver is tested and shown to have detectable concentrations of brodifacoum, that does not necessarily mean that the dog died of brodifacoum toxicosis. It does tell us the dog was exposed to brodifacoum at some point before the sample was taken, but we would have to compare the amount found in the dog’s liver with the amount that would be expected in a toxicosis. We would also have to determine if the clinical syndrome that the dog experienced was consistent with brodifacoum toxicosis, which causes uncontrollable bleeding. So, just because a poison is found in a tissue does not necessarily mean that a toxicosis occurred. Most veterinary diagnostic laboratories have developed nomograms of normal and toxic concentrations for common veterinary toxicants; assistance with interpretation of diagnostic laboratory results is another area where consultation with a veterinary toxicologist can be beneficial.