How It’s Done
How the evidence is collected
Specimens sent for toxicology testing are usually collected by the forensic pathologist (who may also be an appointed “medical examiner” or “coroner” in some jurisdictions) or mortuary technician during an autopsy. Specimens must be properly identified, labelled and sealed as soon as practicable after collection. All specimens pertaining to a case must be collected and bagged separately in tamper-proof containers. Unique numbered seals are used to track all evidence for each case. Like any other evidence, the chain of custody must be preserved at all times, from the mortuary through the laboratory testing, reporting and storage, for court purposes. If the continuity of evidence is compromised, it can result in the case being dismissed in court.
Specimen collection
Table 1 (below) provides a snapshot of the kinds of samples commonly requested when investigating different manners of death, although there may be unique case needs that have to be addressed for some investigations. For example, exposure to volatile substances requires a sample of the fluid in the lung. Skeletal remains can be useful to determine prior exposure to drugs and other substances. In these cases hair can also be sampled.
Table 1. Recommended specimens collected in post-mortem cases.
Type of death case |
Recommended specimens |
Suicides, motor vehicle crashes, and industrial accidents |
Blood, urine, vitreous humour, liver |
Homicides and/or suspicious |
Blood, urine, vitreous humour, gastric contents, bile, liver, hair |
Drug-related |
Blood, urine, vitreous humour, gastric contents, bile, liver, hair |
Volatile substance abuse |
Blood, urine, vitreous humour, lung fluid or tied-off lung, liver |
Heavy metal poisoning and exposure to other poisons |
Blood, urine, vitreous humour, liver, hair, kidney |
Blood, Urine, Liver - Blood is often the specimen of choice for detecting, quantifying and interpreting drugs and other toxicant concentrations. Concentrations of drugs and other toxicants in blood may be useful for establishing recent drug ingestion and to determine the effect of a drug on the deceased at the time of death, or at the time the blood was taken. This can complicate the investigation when someone has been taking prescription medications for some time. For cases involving hospital treatment before death, blood samples taken soon after admission and immediately before death, should also be investigated particularly when poisoning is suspected before admission into hospital. Any treatment given can change the results of toxicology tests or be helpful in the investigation.
Post-mortem blood presents problems due to often variable condition and changes to concentrations from one place to another in the body after death. The degree of decomposition can also interfere with testing as these specimens can be difficult to analyse.
Urine is the most common sample used for drug testing in the workplace, but it is not always available for post-mortem testing. Urine testing results do not directly correlate to drug effects at the time of sample collection because of the time it takes the body to eliminate these drugs or their metabolites (the body’s breakdown products) in the urine. Its usefulness lies in the fact that the presence of a substance in the urine is a sign that the substance had been in the blood at an earlier time (usually within a few days) and had been somewhat processed (detecting these metabolites gives proof that the drug had been ingested). When urine is available, tests are also conducted for presence of drugs of abuse. Depending on the case and the results of initial testing other tissues may need to be analyzed, possibly including stomach contents, liver, etc., (see Table 1). Learn more about drug chemistry ▸
The liver is a primary solid tissue for use in post-mortem toxicology because it is where the body metabolizes most drugs and toxicants. Many drugs become concentrated in the liver and can be found even when there are no levels in the blood. In this latter situation, interpretation of findings is complex.
Vitreous Humour - is the clear, gel-like substance that fills the eye. It can be a useful fluid to screen for a range of drugs. Vitreous humour is commonly analyzed for blood alcohol concentrations. This is of particular interest in motor vehicle trauma, workplace accidents, suicides and homicides. Vitreous humour alcohol concentrations are a little higher than blood (about 20% on average), assuming there has been no degradation. Interpretation of other toxicological findings in vitreous humour is somewhat more complex.
Other samples less commonly used for post-mortem toxicology
Stomach Contents - Because drugs and poisons can often be ingested, stomach contents can provide important investigative clues. In a case of potential overdose or acute poisoning, high concentrations of drugs or toxins may be detected, depending on how much time elapsed between ingestion and death. In many cases of acute poisoning, undissolved capsules or tablets may be discovered, allowing relatively simple drug or poison identification. The total amount of a drug or poison present in the stomach is more important than its concentration because it has not been processed by the body yet.
Bone and Bone Marrow - Bone, in particular bone marrow, can be used for testing when necessary, but the availability and condition of bones in skeletal remains may limit their usefulness. There are no data to suggest that bones from one part of the body are better than others for toxicology tests. However, it is always easier to extract samples from larger bones. Interpretation of these findings is often difficult when assisting in a death investigation, because the time that these toxins were deposited in the bones cannot be determined with reasonable certainty.
Hair and Nails - Hair specimens, usually taken from the back of the head, can be used to test for exposure to heavy metals and drugs over a period of weeks to months. Hair is predominantly used to test for drugs such as amphetamines, cocaine, marijuana (THC) and heroin, and more recently tests have been created to determine if the deceased was drinking heavily in the last few months before death. Drug analysis can also be done on finger- and toenails in order to provide an even longer potential window of exposure than hair. However, relatively little is known about how the nails process toxins, so interpretation of results is more difficult. Hair is subject to external contamination issues that can mitigate its value, so special sample preparations in the lab may be needed for a given case.
Testing
The testing of biological fluids and/or tissues for drugs and other substances is a complex process requiring sophisticated instrumentation and specially trained analysts. In the typical autopsy, fluids and tissue samples are collected specifically for toxicology testing. The types of samples collected from the body, and the testing targets in these biological fluids and tissues, are determined by circumstances of the case and the condition of the body.
Once at the laboratory, a series of tests will be conducted, usually on blood, for a large range of over-the-counter, prescription and illicit drugs as well as alcohol and other toxicants; e.g., metals, inhalants, environmentals (pesticides, insecticides), carbon monoxide, cyanide and many other possible toxins depending on investigative clues or specific tests requested. Table 3 and the “Systematic Toxicological Analysis” section (both provided below) spell out the many materials the medical examiner might harvest from a body during autopsy, and how they can be quickly screened in “truly unknown” cases. The biological evidence from autopsies are needed to support both “common” lab tests and help investigate some very specific potential causes of death.
The biological evidence from autopsies are needed to support both “common” lab tests and help investigate some very specific potential causes of death.
Routine testing, or testing without specific instructions to look for a particular substance, will generally include the drugs shown below in Table 2, but not all drugs. The list of the most common drugs detected is included in a standard attachment to all toxicology reports.
Table 2. Drugs included in routine post-mortem toxicology.
Substance(s) |
Examples |
Alcohol |
Chemically known as ethanol. Test also includes methanol and acetone1. |
Analgesics |
Paracetamol (acetaminophen), tramadol 9 (ConZip™, Ryzolt™, Ultracet, Ultram in the U.S.), Salicylates (aspirin) |
Antidepressants |
Tricyclics (e.g., imipramine, amitriptyline) ; SSRIs (fluoxetine [Prozac®], sertraline [Zoloft®]) |
Antihistamines (sedating) |
doxylamine, chlorpheniramine, diphenhydramine |
Antipsychotics |
old and newer generation including depot injections (subcutaneous or intramuscular injections of long-lasting medication); Haloperidol; Risperidone |
Benzodiazepines and “Z” drugs |
all available (diazepam [Valium®], alprazolam [Xanax®]; zolpidem (Ambien®, Ambien CR, Intermezzo®, Stilnox®, and Sublinox®), zopiclone (Imovane®, Zimovane), zaleplon (Sonata® and Starnoc) |
Cannabis |
tetrahydrocannabinol (THC) (Marijuana, pot) and its metabolites |
Cardiovascular drugs |
Diltiazem (calcium channelblockers), Disopyramide (names Norpace® and Rythmodan®), propranolol (beta blocker) |
Cocaine |
cocaine and its metabolites |
Narcotic analgesics |
codeine, methadone, pethidine, morphine, hydrocodone, oxycodone, fentanyl |
Stimulants |
Amphetamine, methamphetamine, MDMA (ecstasy), pseudoephedrine, fenfluramine, phentermine, caffeine |
Additional or specific requests to the toxicology laboratory can be made, depending on the circumstances of a case. The most common examples of specific drugs that analysts may be asked to look for are outlined in Table 3, below.
Table 3. Drugs not routinely included in death investigation toxicology screens.
Substance(s) |
Examples |
Anti-convulsants |
(some only) lamotrigine (Lamictal®), valproate, phenytoin |
Barbiturates |
Phenobarbital, butalbital, many other prescribed (or otherwise obtained) Barbs |
Designer drugs |
synthetic cathinones and synthetic cannabinoids (“bath salts”, “plant food”, “spice”, etc.) |
Digoxin |
used to treat arrhythmias and heart failure |
GHB |
gamma-hydroxybutyrate(“Juice”, “Liquid Ecstasy”, Fantasy) |
LSD |
hallucinogen rarely seen in coroners cases |
Environmental Toxicants |
carbon monoxide; cyanide; pesticides; herbicides; metals |
Poison
Death by poison can happen in a variety of ways, for example through recreational exposure by inhaling solvents such as butane lighter fluid or fuels, ingesting plant-derived substances like Angel’s Trumpet, accidental exposure to a substance used in the workplace or even accidently produced in the home (like carbon monoxide), or suicidal ingestion of a poison such as strychnine, pesticides, cyanide, etc. These all require specialized tests and the laboratory is alerted to their possible usage or involvement in the death when requests for toxicology testing are submitted.
Who conducts the analysis?
Scientists/toxicologists analyze the specimens submitted and then provide interpretation of the significance of the results based on information received about the case. These analysts may work in crime laboratories, medical examiners’ offices, government health laboratories or even independent providers, depending on the criminal justice structure in a given community.
Certification for individuals in the United States and Canada as a forensic toxicology specialist or diplomate is available through the American Board of Forensic Toxicology (ABFT). Certification is based upon the candidate’s personal and professional record of education and training, experience, achievement and a formal examination. In Europe, individuals may become part of the European Register of Toxicologists through EuroTox, through a similar process. In Japan, the Japanese Society of Toxicology (JSOT) has a diplomate process. International discussion and participation in toxicology is widespread.
How and Where the Analysis is Performed
Forensic toxicologists employ a large number of analytical techniques to determine the drugs or poisons relevant to a case investigation; the capacity of a laboratory to conduct routine toxicological analysis will vary dependent upon equipment, technical capability and analyst experience. When needed, there are specialty toxicology labs that can test for virtually any potential toxin or metabolite in almost every kind of post-mortem sample.
A laboratory should be accredited to perform the analytical work and must be subject to regular inspections by approved accreditation personnel. Certification for forensic toxicology laboratories in the United States and Canada is also available through ABFT, and is voluntary and additional to accreditation. All laboratory tests must be validated, fully documented and fit for purpose. This will ensure that laboratory can reproduce accurate and reliable results for medicolegal investigations. All laboratory tests should conform to standard operating procedures, results are confirmed to meet standards, and reported results are peer reviewed by a second toxicologist before being released. In fact, the American toxicology community recently completed a two-year effort to improve these standards, under the name of “Scientific Working Group on Toxicology” (SWGTOX), in order to meet more stringent legal and scientific challenges.
Immunoassays - Most commonly used drug screening tests involve immunoassay techniques. Immunoassays are laboratory tests that use antibodies to detect a reaction with specific substances. Immunoassay screening tests are designed to detect whether a sample is positive or negative for the targeted drug. Four interpretations of a screening drug test are possible:
- a true-positive result occurs when the test correctly detects the presence of a drug
- a false-positive result is one where the test incorrectly detects the presence of a drug where no drug is present
- a true-negative result occurs when the test correctly confirms the absence of a drug
- a false-negative result is one where the test fails to detect the presence of a drug when it is present
For those samples that give positive screening results, confirmation tests should be performed, preferably using mass spectrometry (MS) detection. Specific immunoassay tests are available for many drug classes including all the drugs of abuse as well as a range of novel compounds such as buprenorphine, “Bath Salts” and “Spice” and other new synthetic drugs.
Chromatographic techniques - Chromatographic detection is an analytical procedure used for separation of compounds/drugs and is frequently based on either high performance liquid chromatography (HPLC) or gas chromatography (GC) coupled with MS. This is the definitive technique used in toxicology to establish proof of structure of unknown substances. With the extensive development of commercial MS technology at an affordable cost, GC/MS and HPLC-MS (commonly known as LC/MS) have become increasingly popular tools in the modern toxicology laboratory. Learn more about drug chemistry ▸
Systematic toxicological analysis - The usual practice in toxicological examination begins with the preliminary identification of alcohol and screening of a wide spectrum of acidic, neutral and basic organic drugs or poisons. If a toxin is detected, confirmatory and, if necessary, quantitative testing has to be performed. Quantitative testing determines how much of the substance is involved. In general, a positive identification is achieved using at least two independent analyses and preferably using different testing methods. For example, using an immunoassay for initial testing and then GC-MS or LC-MS to confirm results would suffice. A chromatographic confirmation test could also determine quantification, simplifying the testing process.
1. Acetone can derive from abnormal body metabolism in diabetic ketoacidotic states.