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What is forensic toxicology? Forensic toxicology is an examination of all areas of toxicity to aid medico-legal enquiries. Toxicology is the study of poisons. It takes into account the origins and properties of the poisons; including chemical and physical properties and their effects on living organisms as well as the corrective measures for the treatment of poisoning. A forensic toxicologist is interested largely with exposure and evaluation of poisons in tissues and body fluid acquired at autopsy or a living person in blood, urine or gastric material.
What is a poison?
A poison may be regarded as any substance that impairs health or destroys life when ingested, inhaled or absorbed by the body, when taken in sufficient quantity. It has been suggested that, depending on the dose, all substances are poisons. This may include necessities for life such as water, salt and oxygen. The intake of large amounts of water over an extensive time period can cause critical electrolyte imbalance. Whereas, on the other hand tiny amounts of poisons such as cyanide, if ingested cause no evident toxicity. It is clear that most substances can act as poisons at a high enough dose therefore the question in hand is not just the substance involved but also the dose administered.
Applications of forensic toxicology
There are three main areas in which forensic toxicology is used; postmortem drug testing, workplace drug testing and investigation of contraband material. Postmortem drug testing is important in the investigation of a death to find out whether drugs were a cause or contributing factor. It could come under a number of fatalities and it is the toxicologists undertaking to determine the manner of death. This can include accidental poisoning death from drug abuse, suicidal poisoning and rarely homicidal poisoning. Workplace drug testing consists of assessment of usually blood or urine from employees for drug content. This is typically found in occupations such as police officers and custom agents. Occupations such as these allow the law to place the publics need for security above the employees need for privacy. Lastly investigation of contraband material is important for police agencies to stop drug abuse. The seized material has to be proven to be an illegal substance, this is where the forensic toxicologist is important if not a conviction cannot be made without sufficient evidence.
Postmortem specimens for toxicological analysis
During a postmortem investigation the responsibilities of a forensic toxicologist include the quantitative and qualitative analysis of biological specimens collected at autopsy and the explanation of the analytic results in relation to the effects of the identified chemicals on the deceased at the time of death.
At autopsy the pathologist typically carries out the collection of postmortem specimens for analysis. It is necessary to collect specimensâ€™ blood fluids and organs; this is due to the fact that drugs and poisons display varying affinities for body tissues as shown in figure 1 below. As a result of this, discovery of a poison is more probable in a tissue in which it accumulates in.
Figure 1: Cocaine tissue distribution in a fatal poisoning.
In order to complete a successful, thorough toxicologic analysis a large quantity of each specimen is required, as a method that removes and indentifies one compound or class of compounds may be unsuccessful in removing and identifying others. (Table 1)
Table 1: List of specimens and amounts to be collected at autopsy.
The toxicologist must consider several factors before the analysis begins; the amount of specimen available, the nature of the poison sought and the possible biotransformation of the poison. It is important for the toxicologist to have an understanding of biotransformation reactions as in some occasions the metabolites are the only evidence a drug or poison has been administered. Figure 3 shows the biotransformation of cocaine; biotransformation of a drug/poison frequently results in formation of a physiologically inactive substance which is more readily extracted from the body than the parent compounds.
Metabolites can be physiologically active or inactive and more toxic, less toxic or non toxic than the parent compound. Cocaine is an example of this as norcocaine is physiologically active whereas benzoylecgonine and methylecgonine are physiologically inactive.
Figure 3: Biotransformation of cocaine
Clinical samples can be separated into:
blood and related fluids
body fluids other than blood
excretory fluids residues
other clinical samples e.g. hair, stomach contents
A range of further specimens maybe collected for toxicological purposes.
This is the fluid that circulates through the arteries, veins and capillaries. The adult human body contains some 5-6 liters of blood. Blood is seen as the most important specimen in postmortem toxicology. Given that the concentration of toxin in blood often shows a relationship more closely with lethal outcomes than concentrations in other specimens.
Cardiac blood is usually more abundant than peripheral blood as a result initial toxicological tests may be preferentially preformed on a heart blood sample. Drug levels in the heart blood are generally higher than in femoral venous blood. Consequently, a heart blood sample would be favored over a urine sample for screening of drugs that are extensively metabolized.
Approximately 5mL of blood for quantitative analysis should be obtained from two distinct peripheral sites, preferably left and right femoral veins. This should be carried out with care so as not to draw a large volume containing blood from more central vessels. The blood should be placed in a fluoride/oxalate tube containing 1% (w/v) sodium fluoride as a preservative.
An additional larger specimen of blood of approximately 20mL for qualitative screening should be taken from the heart preferable from the right atrium or inferior vena cava.
Advantages of using blood as a sample specimen include detection of the parent compound and also easy interpretation of qualitative data. For many drugs, postmortem diffusion of drugs from solid organs into blood has been demonstrated. This has been verified in methamphetamine-associated deaths. It has been found that methamphetamine and amphetamine were both higher in concentration in heart blood than random myocardial tissue samples, it was established than methamphetamine diffusion between heart muscle and blood is evident after death.
However there are limitations of using blood samples such as limited volume and low concentrations of basic drugs and some other poisons.
Detection of drug use or exposure is possible in urine samples as there is accumulation of drugs and metabolites. It is particularly valuable in screening for illicit drugs and is frequently used for quantitative ethanol analysis to support the finding of a blood sample. Unlike blood, urine contains a scare amount of proteins and lipids, as a result it can be analysed by spot tests or directly by immunoassays as well as after removal with a suitable solvent.
Urine maybe taken before an autopsy takes pace by puncture of the abdominal wall or it can be collected under visualisation directly from the bladder. If possible 2 x 25mL urine samples should be collected in sterile plastic containers, one of which should contain preservative (2% w/v fluoride).
Urine has a wide range in pH which is important as it influences the excretion of some drugs. In acidic urine, weakly basic drugs such as methadone are more efficiently excreted. On the other hand, in basic urine, weakly acidic drugs are more efficiently excreted.
Advantages of using urine as a sample is that often large samples can be obtained and there are high concentrations of many poisons. However, it is not always available, the quantitative data is not always useful and correlation between drug concentration in urine and drug effects is usually poor.
Stomach/ gastric contents
This sample includes gastric aspirate, vomit and gastric lavage together with the contents of the stomach at post-mortem. The nature of this specimen can be very inconsistent due to this homogenization and filtration maybe necessary to form a liquid to analyse. Gastric contents are especially needed when information on the drug/poison used is not available. It is common to find whole tablets or plant material.
Testing of gastric contents maybe beneficial in the case of sudden death of a decedent who had large quantities of a lethal agent in his stomach. Ion gastric contents the total amount of a drug is usually far more significant than its concentration. To decide whether an analytical finding is rather more consistent with an overdose or a therapeutic dosage taken just before death an estimation of the amount of drug/poison present in the gastric volume is helpful. If possible approximately 25-50mL of gastric contents should be taken. The use of gastric content is limited as there is no use for it if the drug/poison was inhaled or injected.
Cerebrospinal Fluid and Vitreous Humor
Cerebrospinal fluid and vitreous humor are useful to screen for a variety of drugs as they are thought to be closer to the site of action of drugs than blood. They are aqueous and transparent fluids. Their protected environment inside the brain, spinal column or eye means they are less subject to contamination and bacterial invasion. Both have only slight amounts of enzymes and proteins, thus drugs which are lipophilic or those that are highly protein bound have a tendency to be found in smaller concentrations in these fluids than in blood. The use of cerebrospinal fluid is limited for the measurement of drugs and interpretation of analytical analysis due to the small database of reference values that exist.
Evaluation of post-mortem concentrations of digitalis-glycosides has been predominately useful when using vitreous humor. Digoxin is to some extent hydrophilic at physiological pH, consequently the amount of drug in vitreous humor is likely to have concentrations close to those in blood.
Vitreous humor is aspirated from the eyes by gentle suction, it is replaced by saline. Samples should be collected from each eye separately, and sodium fluoride preservative (2% w/v) added. To collect cerebrospinal fluid at post-mortem, suboccipital puncture is preferred.
Compared to urine or blood testing, the major practical advantage of hair testing for drugs is that it has a larger detection window. Urine and blood analysis provide short term information of an individuals drug use, whereas hair analysis can access long term histories. It is generally proposed that drugs can enter into hair by three sources; (1) from the bloodstream during hair growth, (2) following excretion by sweat and sebum bathing the hair; (3) from passive exposure from hair to drug e.g. smoke.
Hair samples should be collected form the back of the head, known as the vertex posterior. One of the most important pitfalls in hair analysis is environmental contamination, to minimise this effect a washing step is encouraged. Today, gas chromatography coupled with mass spectroscopy is the method most frequently used for hair analysis.
Liver, kidney, lung, brain and skeletal muscle specimen including adipose tissue are the tissues generally collected for post-mortem toxicological investigations. Whenever drugs are involved that are highly lipophilic in nature and are preferably bound to tissue, drug detection is tissue specimens maybe considered. Many toxic substances are present in higher concentrations in liver than in blood.
In cases of intravenous poisoning, high concentrations can be found in lung tissue. Another useful tissue specimen is from the brain for the measurement of drugs as it is the primary site of action for many drugs, and lipophilic substances (antidepressants), narcotics and halogenated hydrocarbons accumulate in central nervous tissue. Kidney is another useful tissue sample as most metabolites are excreted into the urine and will pass though the kidney. To help interpret post-mortem blood data analysis of tissue samples maybe valuable.
In the development of an analytical method, sample preparation is a significant step particularly for isolating required components from complex matrices. Trends in sample preparation in analytical toxicology include automation, miniaturization and high throughput. Traditional approaches such as liquid-liquid extraction and conventional solid-phase extraction have been taken over by microextraction techniques for example liquid-phase microextraction and solid-phase microextraction.
Three - phase
Single-drop microextraction (SDME
Liquid-phase microextraction (LPME)
Solid-phase microextraction (SPME)
Membrane assisted LPME
Flat sheet membrane
Sample preparation can remove potentially interfering matrix compounds and can offer highly concentrated extracts, guaranteeing improved selectivity and a more reproducible method free of variation in the sample matrix.
Liquid-phase microextraction (LPME)
LPME uses minimal amounts of solvents and is a newly developed sample preparation technique. Analytes are extracted from biological specimens such as whole blood or plasma (0.1-4mL) into an acceptor solution in the lumen of a disposable porous hollow fibre supported in a sealed glass vial. LPME is usually carried out between a small amount of water-immiscible solvent and an aqueous phase containing the analytes, LPME is categorized into single-drop microextraction (SDME) and membrane-assisted LPME. LPME is rapid and inexpensive, with a small exposure to toxic organic solvents.
Both these microextraction techniques are regarded as beneficial for the pre-treatment of complex sample matrices prior to chromatographic and capillary electrophoresis processes because they enable rapid analysis at low operating costs and with no environmental pollution.
Solid-phase microextraction (SPME)
SPME was originally used for the investigation of environmental samples; it is now progressively more used for the extraction of drugs and other lipophilic analytes from biological matrices. SPME can be classified into static in-vessel microextraction and dynamic in-flow microextraction (Fig 1). In its simplest form, a spring loaded solid probe coated with a polymer film is positioned through a septum into a vial containing the sample. After an appropriate period the fibre is withdrawn. As SPME is an equilibrium extraction process, the aim is not to extract all the analyte from the sample.
Advantages of SPME are that no extraction solvent is necessary and all the material that is extracted by the probe maybe analysed directly. This solventless sample preparation technique simplifies the extraction protocol considerably.
Colour tests and spectrophotometric and luminescence techniques
A colour test is chemical processes which creates an evident colour or colour change when the substance tested for is acted on by a reagent. Colour tests establish the existence of specific compounds or a general class of compounds. In the absence of interfering compounds many drugs and poisons give characteristic colours with appropriate reagents if present in sufficient amounts. The rapid screening of urine specimens is one of the greatest features of colour tests, as the urine maybe analysed directly without extraction procedures. The â€œTrinders Testâ€ is an example of colour test used for the detection of salicylates in blood or urine. A mixture of ferric nitrate and mercuric chloride is added to 1mL of blood or urine, If salicylates are present, a violet colour is seen.
Colour tests are very quick and cheap; it does not require sample pre-treatment. There are limitations because colour descriptions are very subjective, as the colour produced vary in intensity. The toxicologist must be aware of false-positive reactions; for example a positive Trinderâ€™s test is observed for salicylic acid, a false positive, which is the development of a colour when no salicylates are present maybe observed in urine of diabetic patients releasing acetoacetic acid.
UV/ visible spectrophotometry
A compound will absorb energy which it is irradiated with electromagnetic radiation of an appropriate wavelength. This absorbed energy can possibly be emitted as radiation at a less energetic wavelength, degenerated as heat, or produce a photochemical reaction. The short wavelength region of the spectrum is occupied by gamma and X-rays. Ultraviolet (UV), visible, infrared and microwave radiations come next, followed by radio waves.
Different effects are produced by different types of radiation. UV and visible light excite electrons from their ground state to higher energy (excited) states. Visible spectroscopy was the first reliable quantitative method used for drug/metabolite analysis in blood. The aim of is it to perform a chromogenic reaction directly in a sample with minimum treatment. Problems with visible spectrophotometry include relatively few analytes undergo convenient chromogenic reactions and there is poor selectivity/sensitivity. In UV spectrophotometry double-beam spectrophotometers or linear diode array instruments give the most reliable results. UV spectrometry can be performed directly on sample extracts. However many poisons have either a poorly defined UV absorption spectrum or do not absorb in the UV, although compounds that have characteristic spectra can be readily identified.