Forensic toxicology combines principles from chemistry, biology, and pharmacology to detect and quantify foreign substances in the human body. This multidisciplinary field plays a crucial role in criminal investigations, legal cases, workplace safety, and public health.
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Introduction
Toxicological analysis can determine a wide range of substances related to an individual’s health, impairment, or cause of death, making significant contributions to the criminal justice system. Drug identifications (both prescription and illicit) are the most frequently submitted evidence requests in a typical forensic laboratory.1
Other applications include the characterization of toxic chemicals and heavy metals in cases of suspected poisoning or environmental exposure, alcohol and its metabolites, commonly used in driving under the influence (DUI) cases and workplace incidents, and gases like carbon monoxide to determine exposure levels in cases of smoke inhalation, accidental poisoning, or suicide.
With rapid advancements in technology and the emergence of new substances, forensic toxicology is continuously evolving. This article provides an update on its core principles, recent advancements, and emerging trends.
The Evolution of Forensic Toxicology
Core Principles of Forensic Toxicology
Forensic toxicology involves detecting drugs, alcohol, poisons, and other toxic substances, as well as analyzing their effects on humans. The most commonly analyzed samples include blood, urine, and hair, as these can provide insights into the past and present influence of various substances.
Blood analysis provides real-time concentration levels of drugs, alcohol, and other toxins. It is particularly useful in cases involving impaired driving and overdoses. Urine analysis allows the detection of substances long after their effects have worn off and is widely used in workplace and rehabilitation screenings.
Hair analysis offers a historical record of substance use over weeks or even months, making it particularly useful in establishing long-term drug use. Saliva is increasingly being adopted for roadside and workplace drug testing due to its non-invasive collection method.
In postmortem toxicology, samples from the gastrointestinal tract, liver, spleen, and other tissues are examined to determine the cause of death or to assess whether drugs have been consumed.
An example is the quantification of diclofenac, whose high levels are attributable to acute intoxications and anaphylactic shock, as well as the induction of other conditions.
Various methods to quantify diclofenac in biological fluids and tissues have been developed, including liquid chromatography, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and more recently, ultra-high performance liquid chromatography-tandem mass spectrometry with triple quadrupole (UHPLC-QqQ-MS/MS).2
What is Forensic Toxicology?
Advances in Analytical Techniques
Technological advancements have significantly improved the sensitivity, accuracy, and efficiency of forensic toxicological analysis. The latest methodologies enable the precision detection and quantification of trace amounts.
By determining the precise mass of a precursor ion and its fragmentation pattern, high-resolution mass spectrometry (HRMS) is particularly useful for identifying novel psychoactive substances (NPS) that standard testing methods may miss.
Liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS) has become an optimal technique for untargeted screening and presumptive identification of drugs of abuse.3
Other technology improvements have allowed for faster, more efficient analyte separation with liquid chromatography-tandem mass spectrometry (LC-MS/MS), which is the gold standard for forensic toxicology.
LC-MS/MS techniques enable the analysis of complex mixtures and have emerged as the preferred toxicological screening method for urine and other biological matrices.
Urine multi-drug screening via LC-MS/MS allowed the identification of substances like zolpidem, acetaminophen, and citalopram as the most frequently encountered drugs in emergency room patients.4
Advances in UHPLC-QqQ-MS/MS allow the investigation of misuse of substances (i.e., smart drugs), which is a growing cause of concern. A method was developed to quantify modafinil in evidentiary samples (concentration range = 0.1-10.0 µg/mL), providing a valuable tool for forensic toxicology applications.5
In recent years, artificial intelligence (AI) has revolutionized forensic toxicology. Machine learning algorithms can quickly process large datasets, identify trends, and improve the accuracy of toxicological interpretations.
Recent approaches have used AI to study illicit drug analogs for the identification of novel synthetic drugs. Deep learning methods have been able to predict NPS mass spectra from their chemical structures.
However, poor-quality training data can limit accuracy, posing challenges to the implementation of AI-based technologies.6
Advancements in portable mass spectrometers and handheld immunoassay devices are facilitating on-site testing, particularly beneficial for DUI investigations, allowing law enforcement to conduct rapid drug screenings in the field.
Machine Learning in Predictive Toxicology
Emerging Trends and Future Directions
NPS, including synthetic opioids, pose significant challenges to forensic toxicology. These constantly evolving substances – designed to circumvent existing laws – lack standardized references. Further research is needed to improve testing methods, as well as continuous education and training of professionals.7
Researchers are working to identify new biomarkers that can provide more accurate indicators of drug use, poisoning, and chronic exposure to hazardous substances and aid in forensic interpretations. The analysis of microRNAs seems to be promising in narrowing down the interval within which to place the time of death.8
As forensic toxicology advances, there are ethical concerns regarding privacy, consent, and the misuse of genetic information. In England and Wales, the Forensic Science Regulator’s Code of Practice sets quality standards for forensic science activities.
Governing bodies, such as the American Board of Forensic Toxicology (ABFT) and the International Association of Forensic Toxicologists (TIAFT), continuously update guidelines to ensure best practices and maintain standards of qualification for laboratories.
Conclusion
Forensic toxicology is a critical branch of forensic science, continuously adapting to scientific advancements and societal changes.
The integration of high-resolution analytical techniques, AI-driven data analysis, and novel detection methods have enhanced toxicological investigations.
However, challenges such as the rise of novel psychoactive substances, ethical concerns, and regulatory complexities require ongoing research and adaptation. Continued efforts in forensic research, technology, and education are essential for maintaining accuracy, reliability, and effectiveness in the field.
References
- Jackson, G. P. & Barkett, M. A. (2023). Forensic Mass Spectrometry: Scientific and Legal Precedents. J Am Soc Mass Spectrom, 34, 1210-1224.10.1021/jasms.3c00124.
- Szpot, P., Wachełko, O. & Zawadzki, M. (2022). Diclofenac Concentrations in Post-Mortem Specimens—Distribution, Case Reports, and Validated Method (UHPLC-QqQ-MS/MS) for Its Determination. Toxics, 10, 421. Available: https://www.mdpi.com/2305-6304/10/8/421
- Bates, M. N., Helm, A. E. & Barkholtz, H. M. (2024). Screening for Forensically Relevant Drugs Using Data-Independent High-Resolution Mass Spectrometry. Chem Res Toxicol, 37, 571-579.10.1021/acs.chemrestox.3c00379.
- Lee, J., Park, J., Go, A., Moon, H., Kim, S., Jung, S., Jeong, W. & Chung, H. (2018). Urine Multi-drug Screening with GC-MS or LC-MS-MS Using SALLE-hybrid PPT/SPE. J Anal Toxicol, 42, 617-624.10.1093/jat/bky032.
- Nowak, K., Chłopaś-Konowałek, A., Szpot, P. & Zawadzki, M. (2025). The Issue of "Smart Drugs" on the Example of Modafinil: Toxicological Analysis of Evidences and Biological Samples. J Xenobiot, 15.10.3390/jox15010015.
- Wang, F., Pasin, D., Skinnider, M. A., Liigand, J., Kleis, J.-N., Brown, D., Oler, E., Sajed, T., Gautam, V., Harrison, S., Greiner, R., Foster, L. J., Dalsgaard, P. W. & Wishart, D. S. (2023). Deep Learning-Enabled MS/MS Spectrum Prediction Facilitates Automated Identification Of Novel Psychoactive Substances. Analytical Chemistry, 95, 18326-18334.10.1021/acs.analchem.3c02413. Available: https://doi.org/10.1021/acs.analchem.3c02413
- Boscolo-Berto, R. (2024). Challenges and future trends of forensic toxicology to keep a cut above the rest. Adv Clin Exp Med, 33, 423-425.10.17219/acem/185730.
- Cianci, V., Mondello, C., Sapienza, D., Guerrera, M. C., Cianci, A., Cracò, A., Luppino, F., Gioffrè, V., Gualniera, P., Asmundo, A. & Germanà, A. (2024). microRNAs as New Biomolecular Markers to Estimate Time since Death: A Systematic Review. International Journal of Molecular Sciences, 25, 9207. Available: https://www.mdpi.com/1422-0067/25/17/9207
Further Reading