Benefits and Limitations Associated with the Use of Alternative Biological Matrices in Forensic Toxicology, as Well as Genetic Markers of Poisoning and Psychoactive Substance Abuse

In forensic toxicology, the analysis of blood and urine is regarded as the so-called “gold standard.” However, when forensic experts are confronted with advanced postmortem changes, it becomes necessary to secure and examine alternative matrices. Suitable specimens in cases of advanced putrefactive decomposition may include: skin appendages (hair and nails), bones, bone marrow, cartilage tissue, teeth, antemortem fingerprints, cerebrospinal fluid, vitreous humor, breast milk, meconium, placenta and umbilical cord tissue, oral fluid, sweat, and other evidentiary materials. These tissues, owing to their structure and anatomical location, are more resistant to putrefactive decomposition than body fluids and soft tissues. However, several limitations complicate the reliable analysis of these matrices. These include incomplete drug incorporation, depending on physicochemical properties, the inability to correlate analyte concentrations with pharmacological effects, low xenobiotic levels, and the need for highly sensitive analytical methods. Collectively, these factors contribute to interpretative challenges during forensic expert evaluation and reporting. To address key forensic questions, epigenetic variability analyses (forensic epigenetics) may also be employed, particularly when standard DNA profiling is uninformative. DNA methylation patterns in specific tissues and in individual subjects can be used, among other purposes, to identify the tissue of origin of a human biological trace, to differentiate monozygotic twins, and to predict the age of an unidentified trace donor. Over the past few years, this approach has gained increasing importance; in the context of forensic trace analysis, it offers both advantages and limitations. The aim of this study is to present current scientific evidence regarding the use of alternative biological matrices as potential sources of information of forensic relevance. The paper discusses the characteristics of individual biological materials, with particular emphasis on their analytical properties, potential applications, limitations, and possible advantages in forensic toxicological investigations. Furthermore, the key aspects associated with the application of epigenetic methods in forensic science are presented, with particular focus on their role in individual identification and the reconstruction of circumstances surrounding forensic events. Alternative biological matrices and modern analytical approaches may constitute valuable complementary tools to conventional evidentiary materials used in forensic medicine and criminalistics. Due to their physicochemical properties, stability, and ability to preserve specific biological information, these matrices may provide significant data that enable substance identification, assessment of exposure to psychoactive compounds, reconstruction of event circumstances, and support for identification procedures. However, their application requires consideration of analytical limitations, the specific characteristics of the examined matrix, and the necessity for standardization and validation of diagnostic procedures