A Primer for Lawyers
For thousands of years, accidental or intentional poisoning has been associated with countless deaths and near misses. Poisonous gases were used in large-scale gas chambers during the Holocaust and lethal injections have been used in capital punishment.
The sudden deaths of well-known artistes Michael Jackson (manslaughter by acute propofol intoxication), Amy Winehouse (acute alcohol poisoning) and Heath Ledger (toxic combination of prescription drugs), as well as the doping scandal related to tennis star Maria Sharapova have all made the headlines in recent times.
Substances which are foreign to a living organism are known as xenobiotics. They include therapeutic drugs, alcohol, solvents, drugs of abuse, doping substances, traditional medicines, pesticides, household and industrial chemicals, toxins and other poisons.1 2 Poisons (toxicants or toxic agents) are chemical substances which when administered by accident or design, produce adverse or deleterious responses in biological organisms. Without metabolism and elimination, many xenobiotics would reach harmful or toxic concentrations in the organism.
What is Toxicity?
Toxicity in the human body may be acute or chronic, and may vary from one organ to another. The organs most frequently affected by toxicants are the liver, kidney, brain, lungs and gastro-intestinal tract. Common harmful effects are direct damage of tissue, adverse effects on function, and genetic defects. Common routes of entry or exposure into the human body are orally, through the skin, lungs or eyes.
All substances, including therapeutic drugs, have the potential to be toxic above a threshold dose. Besides the dosage, toxicity also depends on the portal of entry and frequency of administration, the individual’s age or developmental stage, gender, weight, diet, genetic factors, individual sensitivity, kidney and liver functions and health condition.
All substances are poisons: there is none which is not a poison. The right dose differentiates a poison and a remedy.
The period of detection of a xenobiotic or its metabolite is the time interval from last exposure to the time that it is still detectable in the biological specimen. It depends on the biological specimen analysed, and is generally longer in hair than in other specimens. It also varies with the xenobiotic: for example, in urine specimens, the period of detection is 7-12 hours for alcohol, and 1-30 days for benzodiazepines and cannabinoids.
What are Toxicants?
Common toxicants that are identified and quantitated include alcohols and volatile solvents, and drugs such as central stimulants (amphetamines, cocaine), barbiturates, benzodiazepines, antidepressants, opiates, hallucinogens, analgesics, antihistamines, anti-parkinsonian drugs and anti-psychotics.
Toxicants can be classified into six groups based on their physical and chemical characteristics and the manner by which they are extracted (isolated) from biological fluids and tissues for analysis. Refer to Table 1.
Table 1: Classification of toxicants based on physicochemical properties
What is Forensic Toxicology?
Toxicology is the study of adverse effects of chemicals and drugs on humans and animals. This broad discipline has many interrelated branches, such as forensic, clinical, occupational (industrial), environmental and emergency toxicology.
Forensic toxicology is associated with the medico-legal aspects of toxicology; it plays an essential role in criminal and coroner investigations of poisoning, drug use and death, as well as in suspected cases of doping, inhalant or drug abuse, and driving under the influence of alcohol or drugs. This forensic discipline applies analytical chemistry to isolate and chemically identify toxic substances, drugs (prescription and illicit), alcohol, volatile substances and industrial, household or environmental chemicals that adversely affect the human body.
Closely related to forensic toxicology is clinical toxicology which is concerned with the toxic effect of therapeutic drugs. Therapeutic drug monitoring (“TDM”) determines whether drug levels in plasma, serum or blood in patients are in the therapeutic, sub-therapeutic, or toxic ranges, to more accurately tailor dosage to the individual in a clinical setting.
Table 2: Applications of forensic toxicology
What Answers Does it Provide?
The three main objectives of forensic toxicology are to establish the presence and identity of:
1. Toxicants and ascertain whether they contributed to or caused harm or death;
2. Substances that may affect a person’s performance or behaviour and ability to make rational judgement; and
3. Substances that are not compliant with employment regulations or classified as substances of abuse.
Based on its wide range of applications, this forensic discipline can be broadly classified under four sub-disciplines: postmortem (death investigation), human performance, doping control, and forensic drug testing.3 4 Table 2 summarises the various scenarios, purpose and toxicants commonly encountered in each sub-discipline. In cases related to death investigations, forensic toxicology seeks to answer these questions:
1. Was this person poisoned?
2. If so, what was the poison and how much of it was detected?
3. How was it administered?
4. What are its effects?
5. Was it a dangerous or lethal amount?
Suspected drug overdose cases require answers as to whether an excessive intake of the drug occurred, and if so, whether it contributed to death. In many cases, a detected poison may not have caused death but its presence is relevant to the circumstances of death. For example, alcohol consumption increases the probability of traffic accidents.
How is Toxicological Analysis Conducted?5 6
Specimens: Antemortem specimens include blood (whole blood, plasma or serum), urine and gastric contents. Postmortem specimens from autopsies include body tissues such as liver, kidney, lung, and fluids such as blood, vitreous humour, gastric contents and bile. In some death investigations, bone marrow, skeletal muscle, hair and even maggots can be useful. Pills, capsules, tablets, powders, liquids and suspicious bottles found with the patient may also be submitted as exhibits. Specimens should be promptly collected, and properly labelled, sealed, and stored as soon as practicable, to ensure their integrity and to minimise degradation.
Toxicological analysis may involve target analysis which confirms or excludes an expected target substance or a few common drugs. The circumstances of a case may require a systematic analysis for a longer list of potential toxicants and their metabolites. Owing to the wide variations in physical and chemical properties of xenobiotics and their matrices (blood, urine, etc), there is no universal drug or chemical screen. Qualitative analysis detects the presence of a substance in a sample, whereas quantitative analysis determines the concentration of the substance.
Sampling and extraction: Homogeneity and proper sampling procedure are essential in toxicological analysis. What is sampled must be representative of the total biological specimen. Sample preparation is a critical step in the analysis, as instrumental analysis usually cannot be performed directly on biological specimens.
1. Headspace extraction is commonly used to identify and quantitate alcohol and other volatile compounds.
2. Liquid-liquid extraction and solid-phase extraction are commonly used for the isolation of acidic, neutral and basic drugs and other toxicants from blood, urine, and other biological specimens.
3. Sample preparation and analyte clean-up usually involve a combination of processes such as mixing, digestion, precipitation, filtration, dilution, dissolution, pH adjustment, solvent extraction and evaporation.
For most drugs and toxicants, specimens undergo a two-stage analytical process7 involving screening tests, followed by confirmatory tests.
Immunoassay screening uses antibodies to detect a reaction with specific substances, ie whether a specimen is positive or negative for the targeted drug.
Screening tests such as colour tests, immunoassays, spectrophotometry and thin layer chromatography (“TLC”) are useful for detecting the presence or absence of a particular class of compounds in a biological specimen. Breath alcohol tests are another form of screening which allow law enforcement officers to determine action steps for persons who appear to be intoxicated. These routine tests are generally rapid, require minimal sample preparation, have high throughput, and can filter out negative results quickly. Positive results from these tests suffer from the drawback that they are usually not specific or conclusive; they are considered presumptive and must be confirmed before reporting.
Confirmatory tests: The detection of a chemical substance by non-specific tests must be confirmed by a second more specific technique based on a different chemical principle. These definitive techniques provide full or complementary information enabling the unequivocal identification and if necessary, quantification of the chemical substance. Most confirmatory methods are based on mass spectrometric detectors.
A toxicology report may contain factual information on toxicants present and their concentrations, with or without interpretation. “Positive” or “detected” indicates that a particular substance was identified using laboratory protocols, whereas “not detected” indicates that the analytes were absent within the limitations of the tests performed. The list of common drugs which are examined in routine testing is typically included as a standard attachment in toxicology reports. The presence of a toxicant in a biological specimen demonstrates exposure to that agent.
An antemortem toxicology report may provide information on whether the blood concentration of the drug is consistent with therapeutic dosage, or at a level that harms or impairs human performance. Antemortem or postmortem blood alcohol concentration indicate compliance or violation of the legal limit regulating drink driving.
Quantitative toxicology results by themselves have little meaning, unless the concentrations are interpreted holistically with case specific factors and reviewed with appropriate toxicological data on the particular drug. The effects of the concentration vary across individuals, and other factors such as the medical history of the deceased, circumstances of the death, autopsy findings and histological findings should be considered when determining whether a substance possibly caused or contributed to behavioural changes or death.
Challenges in Toxicological Analysis and Interpretation
The key challenges in toxicological analysis are:
1. Widely ranging physico-chemical properties of numerous potential toxicants;
2. Possible presence of analytically similar compounds in the specimen and interference effects from the matrix;
3. Availability of specimens: small amounts of specimens and low analyte concentration;
4. Degraded biological samples and toxicants due to decomposition and alteration; and
5. Interpretation of antemortem and postmortem results.
Interpretation of results can be difficult due to one or more factors such as:
1. the nature of the poison(s) present;
2. sample collection, transport and storage;
3. analytical methods used;
4. circumstances of exposure;
5. mechanical factors such as trauma;
6. pharmacological factors such as tolerance, interactions or synergy; and
7. genetic differences which result in large inter-individual variation in response to a toxicant.
Postmortem toxicology has been described as a “toxicological nightmare”, owing to postmortem drug redistribution (“PMR”).8 PMR refers to the processes by which drugs, toxicants and metabolites move between tissues, organs and body fluids after death. This well-known phenomenon, which can significantly change drug concentrations after death, was first reported in the mid-1970s. Large errors and wrong conclusions can arise when postmortem measurements are used to estimate antemortem drug concentrations and the ingested dose.
1. Postmortem drug levels can be greatly affected by drug properties, cadaver handling and storage conditions, cardiopulmonary resuscitation attempts, the chosen site (eg heart, femoral vein, liver, etc), time interval after death, and the technique for postmortem blood sampling. Drugs, if present in high concentrations in the lung, heart, liver and stomach can diffuse into the surrounding tissues, resulting in higher drug concentrations in blood there.
2. There is often no reliable or obvious relation between concentrations measured before and after death. Thus, comparison of postmortem levels with compilations of therapeutic and toxic drug levels in living persons in order to determine overdose can be misleading, and can lead to miscarriages of justice.9 Ratios of postmortem blood levels to plasma therapeutic levels in living persons are specious and unreliable. Unsurprisingly, the use of the lower limit of the therapeutic range for calculating the ratio often yields large, alarming ratios.
3. The case history and circumstances of any fatality suspected to be caused by overdose must be carefully evaluated. Leading forensic practitioners repeatedly warn that postmortem drug concentrations have been over-interpreted in the past.10
4. The awaited solution to this conundrum has been the development in recent years of large databases of post-mortem drug levels in drug-induced deaths (fatal poisonings), and deaths not caused by drugs present, as proposed by Ferner.11 Earlier postmortem studies suffered from small sample sizes, limited drug types, varied sampling procedures and disparate analytical techniques. In 2009, Swedish toxicologists Jones and Holmgren12 reported concentration distributions (median, 90th, 95th and 97.5th percentiles) of 25 common drug in femoral blood from 24,876 autopsies. In 2014, Finnish analytical chemists Launiainen and Ojanpera13 followed up with the largest and most comprehensive study ever undertaken on drug levels in postmortem blood; this landmark study involved 129 drugs and 57,903 autopsy cases over an 11-year period. The sheer size of this database makes it a reliable and valuable source of objective reference data for the forensic practitioner when interpreting postmortem toxicology results, instead of comparing with therapeutic drug levels in living persons, a method questioned by members in the scientific and medical community, or relying solely on the experience of the practitioner.
Local Cases Involving Toxicology
Some examples of local cases involving forensic toxicology are highlighted below. Besides examining the biological specimens for toxicants, the item which is suspected to cause the toxicity is also examined. For some cases, especially those involving gas poisoning, simulation experiments on build-up can be conducted to determine the likely events that led to the incident.
Glue sniffing remains a problem among the troubled young in Singapore. In 2011, a boy fell into a pond and drowned. Plastic bags containing sticky yellowish substances, and opened tubes of adhesives were found at the walkway near the pond. The yellowish substances were identified to be a contact cement adhesive containing toluene, an intoxicating substance under the Intoxicating Substance Act. They were chemically similar to residues found in the tubes of adhesives recovered from the walkway.
Gas poisoning15 16
Fatal gas poisoning cases often involve seemingly innocuous activities such as scuba diving, cooking with a gas stove, or being in a stationary vehicle with its engine running. Besides the identification and quantitation of carboxyhaemoglobin in blood, diving cylinders are examined for traces of poisonous gases, and simulation experiments are conducted to determine how carbon monoxide concentrations could build up in enclosed spaces. Our findings have assisted in coroner inquiries on the likely cause of death.
Chemical poisoning17 18 19
Three chemical poisoning cases involving adulteration are highlighted. The first case involved a retired toxicology technician who laced a bottle of drinking water with pesticide (methomyl) in an attempt to poison the chairman of a residents’ committee, but caused the death of another woman and hospitalisation of two men. The second case involved a disgruntled maid who injected liquid soap into the feeding tube of a bedridden elderly woman under her care. In the third case, an unemployed man bought a mercury thermometer and injected the toxic mercury into his grandmother’s leg after she had fallen asleep. Similar to cases involving unknown chemical substances,20 our forensic experts have identified various toxicants from a wide range of matrices (eg water, food) in addition to those from biological specimens.
Drug overdose vs mixed drug intoxications21
The deceased’s sister took the insurance firm to Court to seek payouts on two personal accident policies. Our forensic expert was requested to review toxicology and medical reports to provide independent opinions on potential adverse drug interactions, and on the allegedly “elevated” concentrations of four prescribed drugs identified in postmortem blood. Our expert reported that two drugs (bromazepam and mirtazapine) were actually below toxic levels in living persons, and could not be rightly described as “elevated”. In the recent Finnish study,22 duloxetine, mirtazapine and olanzapine were encountered in respectively, 109, 2179 and 1127 of postmortem cases. This large database is particularly useful for relatively new drugs: duloxetine, mirtazapine and olanzapine were approved between 1996 to 2002 in the US. The Finnish database indicated that the concentrations of duloxetine, mirtazapine and olanzapine in the deceased’s blood were below the postmortem blood concentrations of fatal poisoning cases. Thus, the levels of the four drugs in the deceased were not excessive, and did not provide evidence of overdose.
Toxicological analysis is dependent on analytical chemistry techniques. Target analysis of an expected substance is more straight-forward compared to systematic analysis of a longer list of potential toxicants and their metabolites. Presumptive tests such as colour tests, immunoassays and breath alcohol analysis provide a rapid indication of the presence of a particular substance or class of substances. Confirmatory tests often based on mass spectrometry are required to confirm the presence of a toxicant in a biological specimen. The key challenges in forensic toxicology lie in the interpretation of antemortem and postmortem results. Due to postmortem drug redistribution, drug levels in postmortem blood must be cautiously interpreted.
The general concepts and principles behind the analysis of unknown chemicals and materials and forensic toxicology are also applicable to the analysis of drugs. Look out for the next article “Controlled Drugs” in the Forensic Science Series. The article addresses the science behind the discipline, and the methodology commonly adopted for examining and reporting on cases involving controlled drugs.
* The Forensic Experts Group (“TFEG”) is Singapore’s first one-stop private and independent provider of forensic consultancy, analysis, research, training and education for legal and law enforcement agencies, forensic and tertiary institutions, and private organisations. It comprises a team of accomplished and innovative forensic scientists, who are combining 75 years of specialised knowledge, unique experience and skillsets to deliver high quality forensic services both locally and overseas. While leading the Criminalistics Laboratory and the Forensic Chemistry & Physics Laboratory at the Health Sciences Authority from the mid-1990s, TFEG’s forensic scientists developed advanced instrumental techniques for the detection and identification of a wide spectrum of chemical substances in diverse forms and matrices.
1 Poisons Information Centre – The Singapore experience. Oral presentation, Michael M K Tay, B C Bloodworth, K H Lim, Seminar on Global Experiences of Poisons Information Services, NUS-ISFM/WHO/IPCS Seminar (1991).
2 Michael M K Tay, T C Chao, B C Bloodworth, “Poisons information in Singapore” (1993) 22(1) Annals, Academy of Medicine Singapore 37-42.
3 Scientific Working Group for Toxicology (SWGTOX), The Forensic Toxicology Council, “What is Forensic Toxicology?” (July 2010).
4 A Negrusz & G Cooper, Clarke’s Analytical Forensic Toxicology (Second edition, 2013).
5 T C Chao, D S T Lo, R Gunasegaram, W F Tan-Siew, Michael M K Tay, “Toxicological screen on gastric aspirates – The Singapore experience” (1992) 54 Forens. Sci. Int. 141-151.
6 D S T Lo, T C Chao, R Gunasegaram, M K Tay, “Toxicology services in Singapore” (1993) 22(1) Annals, Singapore Academy of Medicine 37-42.
7 The Forensic Experts Group, “Unknown chemicals and materials – A primer for lawyers”, Singapore Law Gazette (March 2016), pp 41-45.
8 D S Cook, R A Braithwaite & K A Hale, “Estimating antemortem drug concentrations from postmortem blood samples: the influence of postmortem redistribution (2000) 53 J Clin Pathol 282–285; available online at: http://jcp.bmj.com/content/53/4/282.full.pdf+html.
9 Olaf Drummer, A Robert W Forrest, Bruce Goldberger & Steven B Karch on behalf of the International Toxicology Advisory Group (ITAG), “Forensic science in the dock. Postmortem measurements of drug concentration in blood have little meaning” (18 September 2004) 329 BMJ. .
10 Supra (note 8 above).
11 R.E Ferner, “Post-mortem clinical pharmacology” (2008) 66(4) Brit. J. Clin. Pharmacol. 443.
12 A W Jones & A Holmgren, “Concentration distributions of the drugs most frequently identified in post-mortem femoral blood representing all causes of death” (2009) 49(4) Med. Sci. Law .257-273.
13 T Launiainen, I Ojanpera, “Drug concentrations in postmortem femoral blood compared with therapeutic concentrations in plasma” (2014) 6 Drug Testing & Analysis 308-316.
14 “Teen was sniffing glue when he fell”, Asia One . (21 Mar 2011).
15 “Couple found dead in car”, The Straits Times (17 Mar 2006).
16 “Toxic gases in diver’s tank”, Asia One (7 Nov 2008).
17 “Poisoner’s jail term increased to 15 years”, The Straits Times (22 Oct 2002). See also PP v Quek Loo Ming  SGHC 171 and PP v Quek Loo Ming  1 SLR 315;  SGCA 48
18 “Maid gets 4 years for liquid soap injection”, The Straits Times (23 Sep 2005).
19 “Granny poisoning: Long jail term sought for accused”, The Straits Times (19 Jan 2007).
20 Supra (note 7 above).
21 “Insurance suit: Man’s death ‘not accident’”, The Straits Times (2 Apr 2016).
22 Supra (note 13 above).