Toxicology is one of the oldest branches of science. Toxicology has evolved throughout history, with early challenges such as identifying poisons or tainted foods.

The toxicologist's responsibility is twofold: define and identify substances in the body and provide an interpretation of what their presence (or, in certain situations, absence) implies in terms of an individual's behaviour and/or whether the identified drug or poison was a cause of death.

Postmortem toxicology is a specialized branch of forensic toxicology that looks at whether medicines or poisons played a role in the cause and manner of death.

Although virtually all body parts of the deceased can be used for toxicological investigation, postmortem deterioration or autolysis can impact the quality and availability of specimens.

The selection, collection, storage, and security of specimens place special requirements on the forensic toxicologist after an autopsy.

The standard of the results to look forward to from the laboratory after the autopsy today is high and reflects continuous advancement in instrumentation and analytical methods seen from the beginning of modern forensic toxicology in the early 20th century.

Selection of Postmortem Specimen

Depending on the case history and preferences of the submitter, the specimens available for examination in post mortem cases may be many or restricted to blood or a single tissue. After a recent death, blood, vitreous humour, and at least one organ tissue are frequently retrieved. Muscle, hair, and bone may be the few specimens available in severely deteriorated cases discovered outside.

Blood

Following any person's death, blood has been utilized as the principal sample for forensic toxicological investigation.

Drug and other toxicant concentrations in the blood can be used to evaluate recent drug intake and the effect of a drug on the dead at the time of death or when the blood was drawn.

When a person has been using prescription drugs for a long time, the analysis may be more complicated. Because of its sometimes fluctuating state and variations in concentrations from one part of the body to another after death, post-mortem blood poses a challenge.

Urine

Urine from an autopsy has the best chance of giving a toxicologist qualitative ante-mortem drug exposure data of any specimen. 

Drugs and their metabolites accumulate in urine, resulting in relatively high drug concentrations, making it easier to detect a potential poisoning. Immunoassays and non-instrumental spot tests can be performed directly on urine samples to examine certain drug classes. 

Vitreous Humor 

In a postmortem examination, vitreous humor is crucial in resolving a wide range of issues.

Vitreous humor is less susceptible to contamination and bacterial breakdown because of its protective environment inside the eye.

As a result, it may be used to differentiate antemortem alcohol ingestion from postmortem alcohol production, and it may be the only way to tell the difference.

This is exceptionally beneficial in cases of vehicle accidents, industrial accidents, suicides, and homicides.

Liver/Bile

The presence of drugs and/or metabolites in this specimen is crucial for establishing historical exposures to specific agents as well as a history of chronic drug use.

Bile has also been used as an alternative specimen for alcohol analysis when urine is not available.

Because the liver is where the body metabolizes most medicines and toxicants, it is a key solid tissue for use in post-mortem toxicology.

Many drugs concentrate in the liver and can be identified even at undetectable blood levels.

Hair and Nails

Hair samples can be tested for heavy metal and drug exposure over the course of several weeks or months.

Hair is commonly used to test for substances including amphetamines, cocaine, marijuana (THC), and heroin, and more recently, tests to identify if the deceased drank heavily in the months leading up to death. Drug analysis can also be performed on fingers and toenails.

There are also many other specimens which are collected such as Gastric contents, skeletal muscle and bone.

Sample Preparation

In forensic toxicological analysis, sample preparation is very crucial. With technical improvements and the availability of mass spectrometers with increasing sensitivity, the necessity to remove any interferences, such as matrix components or non-relevant analytes to the analysis, has never been more important. 

Liquid-Liquid Extraction

Sample extraction is determined by the solubility of the components present in the solvents. Protein precipitation, which uses inorganic acids or solvents such as acetonitrile, chloroform, or methanol to physically separate proteins from the matrix, is a typical LLE extraction. The extraction might be done in a single container, with all solvents and compounds of interest introduced, combined, centrifuged, and the organic layer removed.

Solid Phase Extraction

Solid phase extractions may be more effective when analytes of interest have comparable polarity and LLE is not suitable for their recovery. The idea behind SPE is to separate undesirable components and extract analytes of interest by absorbing them onto a solid phase or sorbent. In SPE, the specimen is placed on a solid packing medium, which is commonly silica gel-based but is not always. The separation is achieved by partitioning the sample between the matrix and the solid phase. 

Analytical Methods for Postmortem Toxicology 

Immunoassays 

Immunoassays operate on the idea that antibodies can be made that recognise and bind to specific compounds by interacting with their molecules' unique structural properties.

Some antibodies are so specific that they exclusively bind to one type of drug, like methamphetamine. Others interact with similar-structured chemicals like amphetamine, methamphetamine, phentermine, ephedrine, pseudoephedrine, and others, but not with structurally distinct molecules like morphine. Antibodies with broad selectivity for drugs within a certain class, such as sympathomimetic amines, are recommended for postmortem screening over antibodies sensitive to a single substance, such as methamphetamine.

Beneficial  Immunoassays for Post Mortem Toxicology Screening

• Amphetamines (Class): For postmortem toxicology, polyclonal immunoassays for sympathomimetic amines are recommended over monoclonal immunoassays.
• Barbiturates (Class): The majority of assays detect butalbital, pentobarbital and many more.
• Benzodiazepines (Class): Antibodies directed towards oxazepam are used in the majority of benzodiazepine immunoassays.
• Opiates (Class): These assays detect morphine, heroin, codeine, hydrocodone and their metabolites
• Cannabinoids: At least ten of tetrahydrocannabinol's non-active metabolites interact with the cannabinoid assays (THC).
• Cyclic Antidepressants (Class): Imipramine, amitriptyline and their hydroxy metabolites are detected by these assays.

Chromatography

Separating components of mixtures using chromatographic drug screening procedures involves partitioning them between a stationary phase, which is commonly a solid or viscous liquid, and a mobile phase, which is a gas or liquid.

The time taken by a compound to pass through the chromatographic column is called Retention time(Rf). TLC, GC, and HPLC are three chromatographic techniques that are currently being utilized for postmortem toxicology screening.

Thin Layer Chromatography

Thin Layer Chromatography (or TLC) has been beneficial in drug identification laboratories for a long period of time. The "thin layer" in this method is a sheet made of plastic with a permeable silica coating on it.

The insensitivity of this approach is its biggest flaw. When the known substances are examined carefully, the identification of parent drugs can be possible. The data will be difficult to understand for a colourblind technician.

In toxicology examinations, TLC can be used as a screening or confirmation process.

Gas Chromatography 

This method has been widely used for drug screening, mostly on samples from people who have experienced an acute drug overdose.

The approach has also been used to identify dangerous chemicals such as 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid that have been consumed in poisoning cases.

For both qualitative and quantitative drug analysis, GC is commonly used. It is quite fast and can identify a wide range of chemicals.  Sensitivity of the GC is poor for tricyclic antidepressants and benzodiazepines.

Gas Chromatography and Mass Spectrometry

GC/MS is a valuable analytical method for identifying semivolatile chemical substances. It combines the separation efficiency of gas chromatography with the structural interpretation capabilities of mass spectrometry.

Some molecules will travel quicker than others in chromatography, and at the end of the study, the analyst will spray the plate with a dye, resulting in a number of spots with a distinct pattern. If no good match is identified, the spectrum can be visually compared to printed mass spectral data compilations. It serves as a final confirmation test.

Conclusion

A number of metabolic and biological processes occur after death that may have a significant impact on post-mortem drug concentrations.

Despite the use of many methodologies, these procedures may make drug quantification problematic, or even result in drugs being undetected in some cases.

Changes in drug concentration caused by bacterial breakdown, residual tissue enzymatic activity, or post-mortem redistribution between higher to lower concentration tissues can cause problems.

Analytical processes for toxicological studies should be carried out according to quality assurance. Problems are most likely to occur, during the isolation and identification of a drug. The insufficient information presented in a given case frequently limits the interpretation of analytical results.